WO2022150662A1

WO2022150662A1 - Methods for generating antigen-binding molecules from single cells

Info

Publication number: WO2022150662A1
Application number: PCT/US2022/011723
Authority: WO
Inventors: Wyatt James MCDONNELL; Bruce Alexander ADAMS; Michael John Terry STUBBINGTON; David Benjamin JAFFE; Michael Schnall-Levin
Original assignee: 10X Genomics, Inc.
Priority date: 2021-01-08
Filing date: 2022-01-07
Publication date: 2022-07-14
Also published as: EP4275051A1

Abstract

The present disclosure relates generally to the field of immunology, and particularly relates to methods for the identification and characterization of antigen-binding molecules (e.g., antibodies) produced by immune cells (e.g., B cells), using single-cell immune profiling methodologies.

Description

METHODS FOR GENERATING ANTIGEN-BINDING MOLECULES FROM

SINGLE CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial Nos. 63/135,514, filed on January 8, 2021; 63/164,465, filed on March 22, 2021; 63/135,504, filed on January 8, 2021; 63/235,670, filed on August 20, 2021; and 63/253,665, filed on October 8, 2021. The disclosures of the above-referenced applications are herein expressly incorporated by reference it their entireties, including any drawings.

FIELD

[0002] The present disclosure relates generally to the field of immunology, and particularly relates to methods for the identification and characterization of antigen-binding molecules ( e.g ., antibodies) produced by immune cells (e.g., B cells).

BACKGROUND

[0003] A number of approaches and systems are currently available for the isolation and characterization of antigen-binding molecules (e.g, antibodies). However, these existing approaches and methods involve laborious processes of isolating antibodies from activated human B-cells. Furthermore, these approaches are cumbersome, cost-prohibitive, time- consuming, not adaptable to high-throughput and inefficient at retrieving rare antibodies that are produced by a minor fraction of the total repertoire of immune cells, such as B cells. Limitations of current approaches include, e.g, (i) a lack of heavy-light chain pairing (bulk approaches), (ii) inability to efficiently amplify B cell receptor sequences due to poor RNA quality or sample preparation conditions, (iii) low-throughput due to inability to combine and analyze samples from multiple individuals, or such low input that single cell analysis is not possible, and (iv) generation of antibodies that are not fully humanized.

[0004] Therefore, there is a need for alternative approaches that couple high-throughput single-cell phenotypic screening with high-throughput sequencing of antibody-producing primary cells that produce antigen-binding molecules, such as B cells, in a flexible format that enables direct screening for functional activities.

[0005] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly identified emerging coronavirus causing an acute respiratory distress syndrome known as COVID-19 that is similar to severe acute respiratory syndrome (SARS) caused by the closely related SARS-CoV. To date, SARS-CoV-2 is continuing its spread across the world with more than 80 million confirmed cases in 190 countries and nearly two million deaths. In view of the continuing threat to human health, there is an urgent need for preventive and therapeutic antiviral therapies for SARS-CoV-2 control. Because this virus uses its spike glycoprotein for interaction with the cellular receptor ACE2 and the serine protease TMPRSS2 for entry into a target cell, this spike protein represents a target for antibody therapeutics. In particular, fully human antibodies that specifically bind to the SARS-CoV-2 spike protein (SARS-CoV-2-S) with high affinity and that inhibit virus infectivity could be important in the prevention and treatment of COVID-19.

SUMMARY

[0006] The present disclosure provides, inter alia , methods and kits for the identification and characterization of antibodies produced by B cells obtained from biological samples, using single-cell immune profiling methodologies, so as to produce recombinant antibodies with desired properties. The present disclosure thus can be useful for various downstream applications, including identification and/or isolation of antibodies having specificity for an antigen or a biological sample ( e.g ., tumor sample), for an individual, or for population of individuals. Also provided in some embodiments of the disclosure are kits for antibody discovery and/or characterization.

[0007] In one aspect, provided herein are methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, the method including: (a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens includes (i) a target antigen coupled to a first reporter oligonucleotide and (ii) a non target antigen coupled to a second reporter oligonucleotide, and wherein the contacting provides a B cell bound to the target antigen; (b) partitioning the B cell bound to the target antigen into a partition of a plurality of partitions, wherein the partitioning provides a partition including (i) the B cell bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence (e.g., a partition-specific barcode sequence); (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen; (d) assessing the binding affinity of the antibody or antigen binding fragment to the target antigen; and (e) identifying the antibody or antigen-binding fragment as having a binding specificity for the target antigen if the antibody or antigen-binding fragment specifically binds to the target antigen.

[0008] In another aspect, provided herein are methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, the method including: (a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a target antigen coupled to a first reporter oligonucleotide and (ii) a non target antigen coupled to a second reporter oligonucleotide, and wherein the contacting provides a B cell bound to the target antigen; (b) partitioning the B cell bound to the target antigen into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the B cell bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen; and (d) identifying the antibody or antigen-binding fragment as having a binding specificity for the target antigen if the antibody or antigen-binding fragment binds to the target antigen and does not significantly bind the non-target antigen.

[0009] In another aspect, provided herein are methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, the method including: (a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a target antigen coupled to a first reporter oligonucleotide and (ii) a non target antigen coupled to a second reporter oligonucleotide, and wherein the contacting provides a B cell bound to the target antigen; (b) partitioning the B cell bound to the target antigen into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the B cell bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen; and (d) identifying the antibody or antigen-binding fragment as non-specific for the target antigen if the antibody or antigen-binding fragment binds to the non-target antigen.

[0010] Non-limiting exemplary embodiments of the methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, as described herein can include one or more of the following features. In some embodiments, the non-target antigen has been selected such that it is not expected to bind the antibody or antigen-binding fragment. In some embodiments, non-target antigen is an antigen to which the B cell is not expected to bind. In some embodiments, the methods further include coupling a barcode moiety to the antibody or antigen-binding fragment described herein to generate a barcoded antibody or antigen-binding fragment. In some embodiments, the first and the second reporter oligonucleotide include (i) a first and a second reporter barcode sequence that identify the target antigen and the non-target antigen, respectively and (ii) a capture handle sequence, optionally wherein the first and/or second reporter barcode sequence identifies a treatment condition that the target antigen and/or non-target antigen are subjected to.

[0011] In some embodiments, provided herein are methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, the method including:

[0012] (a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a first antigen coupled to a first reporter oligonucleotide comprising a first reporter barcode sequence and (ii) the first antigen coupled to a second reporter oligonucleotide comprising a second reporter barcode sequence, wherein the first antigen coupled to the first reporter oligonucleotide is subjected to a first treatment condition and the first antigen coupled to the second reporter oligonucleotide is subjected to a second treatment condition, and wherein the contacting provides a labeled B cell bound to the first antigen coupled to the first reporter oligonucleotide and/or the first antigen coupled to the second reporter oligonucleotide;

[0013] (b) partitioning the labeled B cell into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the labeled B cell and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence;

[0014] (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the labeled B cell; and

[0015] (d) identifying the antibody or antigen-binding fragment as binding to the first antigen subjected to the first and/or second treatment condition,

[0016] wherein optionally the plurality of antigens further comprises (iii) a second antigen coupled to a third reporter oligonucleotide comprising a third reporter barcode sequence and (iv) the second antigen coupled to a fourth reporter barcode sequence, wherein (iii) and (iv) are subjected to the first treatment condition and second treatment conditions, respectively, and wherein the labeled B cell is optionally bound to (iii) and/or (iv), optionally wherein the first antigen is a target antigen and the second antigen is a non-target control antigen, and/or wherein the method further comprises the prior step of subjecting (i) and (ii) to the first and second treatment conditions, respectively, prior to (a), and/or wherein the method further comprises the prior step of subjecting (iii) and (iv) to the first and second treatment conditions, respectively, prior to (a).

[0017] In some embodiments, a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further includes a capture sequence configured to couple to the capture handle sequence of the first and/or second reporter oligonucleotide, and a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further includes a capture sequence configured to couple to an mRNA analyte or DNA analyte. In some embodiments, the capture sequence is configured to couple to the capture handle sequence of the reporter oligonucleotide by complementarity base pairing. In some embodiments, the capture sequence is configured to couple to the mRNA analyte, and wherein the capture sequence configured to couple to the mRNA analyte includes a polyT sequence.

[0018] In some embodiments, the capture sequence is configured to couple to the mRNA analyte, and wherein the capture sequence configured to couple to the mRNA analyte comprises a targeted priming sequence, optionally wherein the targeted priming sequence targets an antibody or BCR region of the mRNA analyte, e.g., a constant sequence of said antibody or BCR region of the mRNA analyte. In some embodiments, a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to the capture handle sequence, and wherein a second nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to non-templated nucleotides appended to a cDNA reverse transcribed from an mRNA analyte. In some embodiments, the mRNA analyte is reverse transcribed to the cDNA utilizing a primer comprising a polyT sequence. In some embodiments, the non-templated nucleotides appended to the cDNA comprise a cytosine. In some embodiments, the capture sequence configured to couple to the cDNA comprise a guanine. In some embodiments, the coupling of the capture sequence to the non-templated cytosine extends reverse transcription of the cDNA into the second nucleic acid barcode to generate the second barcoded nucleic molecule. In some embodiments, the second nucleic acid barcode molecule further comprises a template switch oligonucleotide (TSO).

[0019] In some embodiments, the first and second nucleic acid barcode molecules each include a unique molecule identifier (UMI).

[0020] In some embodiments, the methods of the disclosure further include generating, in the partition, a first barcoded nucleic acid molecule or a plurality of first barcoded nucleic acid molecules including (i) a sequence of the first reporter oligonucleotide or a reverse complement thereof and the common barcode sequence or reserve complement thereof, and (ii) optionally thereby identifying (i) the target antigen coupled to the first reporter oligonucleotide and/or (ii) the non-target antigen coupled to the second reporter oligonucleotide. In some embodiments, the methods of the disclosure further include generating, in the partition, a first barcoded nucleic acid molecule or a plurality of first barcoded nucleic acid molecules including a sequence of the first reporter oligonucleotide, or a reverse complement thereof, and the common barcode sequence or a reserve complement thereof, and optionally using the first barcoded nucleic acid molecule to identify the B cell as having bound to the target antigen coupled to the first reporter oligonucleotide. In some embodiments, the methods of the disclosure further include generating a first barcoded nucleic acid molecule or a plurality of first barcoded nucleic acid molecules including a sequence of the first reporter oligonucleotide, or a reverse complement thereof, and the common barcode sequence or a reserve complement thereof, and optionally using the first barcoded nucleic acid molecule to identify the B cell as having bound to the target antigen coupled to the first reporter oligonucleotide. In some embodiments, the binding affinity is assessed based on the number of first barcoded nucleic acid molecules comprising (i) a sequence of the first reporter oligonucleotide or reverse complement thereof and (ii) the common barcode sequence or reverse complement thereof. In some embodiments, the methods of the disclosure further include generating, in the partition, a second barcoded nucleic acid molecule including (i) a nucleic acid sequence encoding the antibody, or antigen-binding fragment thereof, produced by the B cell, and (ii) the common barcode sequence or reverse complement thereof, and optionally using the second barcoded nucleic acid molecule to identify a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen. In some embodiments, the methods further include generating a second barcoded nucleic acid molecule including (i) a nucleic acid sequence encoding the antibody, or antigen-binding fragment thereof, produced by the B cell, and (ii) the common barcode sequence or reverse complement thereof, and optionally using the second barcoded nucleic acid molecule to identify a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen. In some embodiments, the methods further include generating a third barcoded nucleic acid molecule or plurality of third barcoded nucleic acid molecules including (i) a sequence of the second reporter oligonucleotide, or reverse complement thereof, and (ii) the common barcode sequence or reverse complement thereof, and optionally using the third barcoded nucleic acid molecule or plurality of third barcoded nucleic acid molecules to identify the B cell as having bound to the non-target antigen coupled to the second reporter oligonucleotide, optionally wherein the generating of the third barcoded nucleic acid molecule or plurality thereof occurs in the partition.

[0021] In some embodiments, the methods of the disclosure further include determining sequences of the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule, and optionally determining a sequence of the third barcoded nucleic acid molecule, optionally wherein the determining is performed by sequencing. In some embodiments, the methods further include identifying the antibody or antigen-binding fragment thereof based on the determined sequence of the second barcoded nucleic acid molecule. In some embodiments, the determined sequence comprises a nucleotide sequence. In some embodiments, the determined sequence comprises an amino acid sequence encoded by the nucleotide sequence.

[0022] In some embodiments, the binding affinity of the antibody or antigen-binding fragment to the target antigen is assessed based on the determined sequence of the first barcoded nucleic acid molecule.

[0023] In some embodiments of the methods disclosed herein, the plurality of B cells are obtained from biological sample derived from a vertebrate subject. In some embodiments, the vertebrate subject is a non-mammalian subject. In some embodiments, the non-mammalian subject is an avian species. In some embodiments, the vertebrate subject is a mammalian subject. In some embodiments, the mammalian subject is a human.

[0024] In some embodiments, the first and/or the second reporter oligonucleotide is conjugated to a tag configured for detection or separation. In some embodiments, the tag is configured for magnetic separation. In some embodiments, the tag includes a fluorescent agent.

[0025] In some embodiments, the methods disclosed herein further include, prior to the partitioning at (b), isolating and/or enriching the plurality of single B cells. In some embodiments, the enrichment of the plurality of B cells includes sorting of the B cells bound to the target antigen and/or non-target antigen based on detection of one or more of the labelling agents coupled to the reporter oligonucleotides attached to the antigens. In some embodiments, the enrichment of the plurality of B cells includes sorting cells of the plurality of B cells according to their binding to the target antigen.

[0026] In some embodiments, the single B cell is a plasmablast, a plasma cell, a memory B cell, a regulatory B cell, or a lymphoplasmacytoid cell.

[0027] In some embodiments of the methods disclosed herein, the target antigen and/or the non-target antigen is coupled to a barcode moiety that identifies the target antigen and/or the non-target antigen, respectively.

[0028] In yet another aspect, provided herein are methods for identifying an antibody having binding affinity for a coronavirus spike protein (CoV-S), the methods including: (a) contacting a plurality of B cells obtained from a subject who has been exposed to a coronavirus with a plurality of antigens, wherein the plurality of antigens includes a CoV-S antigen and a non-CoV-S antigen, and wherein each of the antigens include a reporter oligonucleotide, wherein the contacting provides a B cell bound to a CoV-S antigen; (b) partitioning the B cell bound to the CoV-S antigen in a partition of a plurality of partitions, wherein the partitioning provides a partition including (i) the B cell bound to the CoV-S antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence; (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the CoV-S antigen; and d) assessing the binding affinity of the barcoded antibody or antigen-binding fragment to a CoV-S protein; and e) identifying the isolated antibody antigen-binding fragment as an antibody having a binding specificity for the CoV-S protein if the barcoded antibody specifically binds to the CoV-S protein.

[0029] Non-limiting exemplary embodiments of the methods for identifying an antibody having binding affinity for a CoV-S protein as described herein can include one or more of the following features. In some embodiments, the reporter oligonucleotide including (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence. In some embodiments, the methods of the disclosure further include coupling a barcode moiety to the antibody or antigen binding fragment produced by the B cell to generate a barcoded antibody or antigen-binding fragment. In some embodiments, a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further includes a capture sequence configured to couple to the capture handle sequence of the reporter oligonucleotide and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further includes a capture sequence configured to couple to an mRNA analyte. In some embodiments, the capture sequence is configured to couple to the capture handle sequence of the reporter oligonucleotide is complementary to the capture handle sequence of the reporter oligonucleotide. In some embodiments, the capture sequence configured to couple to an mRNA analyte includes a polyT sequence. In some embodiments, the first and second nucleic acid barcode molecules each include a unique molecule identifier (UMI).

[0030] In some embodiments, the antibody or antigen-binding fragment has a binding specificity to an epitope in a target region of the CoV-S protein. In some embodiments, the target region of the CoV-S protein is the SI region. In some embodiments, the target of the CoV-S protein is the S2 region. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a trimeric form of the CoV-S protein. In some embodiments, the CoV-S protein is a spike protein of SARS-CoV-1, SARS-CoV-2, or MERS-CoV. In some embodiments, the subject is suspected of being infected with a coronavirus, has been infected with a coronavirus, has been vaccinated, or has been recovered from a coronavirus infection. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human.

[0031] In some embodiments, the antigens are each coupled to a fluorescent label identifying the antigens. In some embodiments, the methods for identifying an antibody having binding affinity for a CoV-S protein as described herein further include isolating and/or enriching the plurality of B cells prior to (b). In some embodiments, the enriching further including sorting of the B cells bound to the CoV-S antigen and/or non-CoV-S antigen based on detection of one or more of the fluorescent labels coupled to the antigens. In some embodiments, the CoV-S protein is coupled to a barcode moiety.

[0032] In another aspect, some embodiments of the disclosure relate to kits for identifying and/or characterizing an antibody or antigen-binding fragment having binding affinity for an antigen, the kits include: (a) a plurality of target antigens and non-target antigens, and wherein each of the target antigens and non-target antigens include a reporter oligonucleotide including (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence; and (b) instructions for performing a method of the disclosure. In some embodiments, provided herein are kits for identifying an antibody or antigen-binding fragment having binding affinity for a coronavirus spike protein (CoV-S), the kits include: (a) a plurality of CoV-S antigens and non- CoV-S antigens, and wherein each of the antigens comprise a reporter oligonucleotide comprising (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence; and (b) instructions for performing the method of the disclosure.

[0033] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 shows an exemplary microfluidic channel structure for partitioning individual biological particles in accordance with some embodiments of the disclosure.

[0035] FIG. 2 shows an exemplary microfluidic channel structure for the controlled partitioning of beads into discrete droplets.

[0036] FIG. 3 shows an exemplary barcode carrying bead.

[0037] FIG. 4 illustrates another example of a barcode carrying bead.

[0038] FIG. 5 schematically illustrates an example microwell array.

[0039] FIG. 6 schematically illustrates an example workflow for processing nucleic acid molecules.

[0040] FIGS. 7A-7C schematically illustrate examples of labelling agents.

[0041] FIG. 8 shows an exemplary microfluidic channel structure for delivering barcode carrying beads to droplets.

[0042] FIG. 9 depicts an example of a barcode carrying bead.

[0043] FIGS. 10A, 10B and IOC schematically depict an example workflow for processing nucleic acid molecules.

[0044] FIGS. 11A and 11B depict the amino acid sequences of a wild-type SARS-CoV spike protein (FIG. 11 A) and a variant SARS-CoV spike protein (FIG. 11B). Various mutations have been introduced and indicated by the original amino acids above the mutated amino acids. These mutations fall in 3 classes: 1) proline stabilization/S2P mutations (F817P, A892P, A899P, A942P, K986P, V987P), 2) alanine stabilization mutations (R683 A, R685A), and 3) viral variant mutations (D614G). The asterisks in the sequences indicate the start and end of the sequences used to produce the antigens used in the experiments described in the Examples below. The C- terminal end of the antigens (ending at the 2^nd asterisk) is fused to the T4 trimerization domain and the His tag.

[0045] FIG. 12 shows an exemplary scheme for antigen-specific enrichment of B cells by using fluorescence-activated cell sorting (FACS) technique. In these experiments, cells were initially gated on being single, live (7AAD^negatlve) and PE-Cy7-CD19+ and then sorted on their PE and/or APC status directly into master mix and water. In this figure, Y axis represents PE signal (pre-fusion trimerized SARS-2 glycoprotein S antigen+ and/or HSA+ control antigen binding cells). X axis represents APC signal (trimerized SARS-2 S glycoprotein D614G antigen+ and/or HSA+ control antigen-binding cells). Numbers adjacent to each gate name represent the fraction of events relative to the parent population (single, live, CD 19+ cells) for that gate.

[0046] FIG. 13 schematically illustrates that the new scoring system described herein allowed for determining relative KD values which in turn facilitate identification of binding antibodies with good dynamic range of reporter oligonucleotides.

[0047] FIGS. 14A-14B schematically depict the results of representative analysis performed to illustrate that the new scoring system described herein allow for selection of high- affinity antibodies with a data set. In this analysis, BEAM scores are approximately normally distributed, increase exponentially as target antigen-binding relative to expressed antibody and control antigen increases, are correlated with generation probability of the HCDR3 junction, e.g ., following the known general relationship of somatic hypermutation (SHM) and increasing affinity, and also reveal that class switching increases predicted relative affinity in concordance with the literature.

[0048] FIG. 15 schematically summarizes the results of representative analysis performed to illustrate clonotype enrichment based on relative KD. BEAM scores were found to be also generally higher within sublineages that contain more daughter antibodies than narrow sublineages.

[0049] FIGS. 16A-16B schematically summarize the results of representative SPR analyses performed to evaluate binding affinity of exemplary antibodies of the disclosure to the following antigens: (1) a trimerized wild-type SARS-CoV-2 S protein (FIG. 16A), (2) a SARS- CoV-2 S protein variant with D614G substitution (FIG. 16B).

[0050] FIG. 17A summarizes the results of experiments performed to assess RBD binding kinetics. A triple mutant RBD containing triple amino acid substitutions K417N, E484K, and N501 Y was used. FIG. 17B: RBD kinetics in comparison to FDA-approved therapeutic antibodies.

[0051] FIG. 18A depicts binding kinetics of hypothetical antibodies having the same KD value 10 nM, with varying kon and koff rates. Dashed and solid curves (kd=10⁵ and 10⁴, respectively) depict optimal binding kinetics of antibodies having high therapeutic potential due to binding stability, while dash-dot and heavy solid curves (kd=10³ and 10², respectively) depict less optimal binding kinetics.

[0052] FIG. 18B depicts binding kinetics of exemplary TXG antibodies and FDA- approved or late clinical development stage spike antibodies (data from each antibody shown in triplicate). Antibodies having optimal binding kinetics are depicted in FIG. 18B as boxes with asterisk symbols (*). Antibodies having less optimal binding kinetics are depicted in FIG. 18B as boxes with solid circle (·). FDA-approved or late clinical development stage spike antibodies used as positive controls are depicted as boxes with solid triangle (A).

[0053] FIG. 18C depicts the relationship between Koff of a given TXG antibody to the pre-fusion trimeric spike and its binding kinetics. Koff is shown here for the purpose of brevity as half-life and mean-life kinetics of a receptor-ligand pair are determined by the Koff of the interaction and not the Kon or the ratio of Koff to Kon (KD). Box plots are shown for each kinetic profile described above and shown in FIG. 18C. Twenty-seven (27) antibodies are shown as having a Koff rate of le-05, indicating they have surpassed the lower limit of detection from the SPR data and therefore have lower estimated KD values than those reported in the provided data.

[0054] FIG. 18D depicts the relationship between Koff and Kon of given TBS-antibodies to the pre-fusion trimeric spike, color-coded by kinetic profile.

[0055] FIG. 19 schematically depicts binding affinity of exemplary antibodies to wild-type SARS-CoV-2 S protein, illustrating that the majority of tested antibodies could bind to wild-type S protein in picomolar and nanomolar range. Remarkably, several antibodies described herein were found to have binding affinities as good as or superior to FDA-approved antibodies or antibodies in late clinical development.

[0056] FIG. 20 schematically depicts the general procedure of live virus neutralization assay employed to determine the anti-SARS-CoV-2 activity of various antibodies described herein.

[0057] FIG. 21 depicts representative raw data from neutralization assay described in FIG. 20. CTRL-0004: Casirivimab; CTRL-0006: Bamlanivimab; CTRL-0007: Etesevimab; CTRL- 0008: Sotrovimab; CS478 pi_vac_pfl, positive plasma control of Pfizer vaccine.

[0058] FIG. 22 depicts representative neutralization percentage from neutralization assay described in FIG. 20. CTRL-0004: Casirivimab; CTRL-0006: Bamlanivimab; CTRL-0007: Etesevimab; CTRL-0008: Sotrovimab; CS478 pi_vac_pfl, positive plasma control of Pfizer vaccine.

[0059] FIG. 23 schematically depicts representative neutralization curves (ID50) of four FDA-approved antibodies or antibodies in late clinical development (controls). CTRL-0004: Casirivimab; CTRL-0006: Bamlanivimab; CTRL-0007: Etesevimab; CTRL-0008: Sotrovimab; CS478 pi_vac_pfl, positive plasma control of Pfizer vaccine.

[0060] FIG. 24 schematically depicts representative neutralization curves (ID50) of six exemplary antibodies in accordance with some embodiments of the disclosure.

[0061] FIG. 25 schematically depicts an UpSet plot wherein antibodies are binned into antigen bins based on two rounds of SPR binding affinity data. For an antibody to be placed into a bin a detectable kinetic fit at all concentrations of antigen was required from at least one of the SPR experiments described in Examples 9 and 12, or orthogonal neutralization data.

[0062] FIG. 26 schematically depicts an UpSet plot of antibodies identified as having neutralization activity against live SARS-COV-2, wherein the antibodies are binned into antigen bins as described in FIG. 25.

[0063] FIGS. 27A, 27B, and 27C depict UpSet plots of the potently (IC50 <= 1000 ng / ml) neutralizing antibodies retrieved in this BEAM-seq workflow. FIG. 27A is an Upset plot of the potently neutralizing antibodies selected from 239 antibodies identified in Example 6. FIGS. 27B and 27C are two Upset plots of potently neutralizing antibodies selected from the antibodies of Table 3. In these figures, rows represent the binding of these neutralizing antibodies to pre fusion spike trimers from major SARS-CoV-2 variants of concern, the endemic HKU1 coronavirus spike protein and the SARS-CoV-2 N terminal domain.

[0064] FIGS. 28A and 28B schematically summarize of the neutralization potency of the antibodies described in Table 8 as determined in testing against SARS-CoV-2. FIG. 28A represents neutralization data from antibodies of Table 1A and FIG. 28B represents neutralization data from antibodies of Table IB.

[0065] FIG. 29 is a scatter plot depicting the relationship between antigen reporter UMIs per antibody and the ON rate of the antibody for WT trimer.

[0066] FIG. 30 is a scatter plot depicting the relationship between antigen reporter UMIs per antibody and the KD of the antibody for WT trimer.

[0067] FIG. 31 depicts IC50 values for antibodies tested for neutralization activity against live SARS-CoV-2 and the technology used to originally isolate each antibody tested in this experiment. Comparator technologies include phage display, mouse hybridoma, and humanized mouse isolation.

[0068] FIG. 32 depicts a heat map summarizing results of the epitope binning assays described in Example 14, wherein antibodies were tested against one another in a pairwise and combinatorial fashion for binding to a specific target antigen,

pre-fusion trimerized spike protein from SARS-CoV-2 USA-WA1/2020 isolate.

[0069] FIG. 33 depicts a heat map summarizing the results of the epitope binning assays described in Example 14, wherein antibodies were tested against one another in a pairwise and combinatorial fashion for binding to spike protein from SARS-CoV-2 delta variant.

[0070] FIG. 34 schematically illustrates a non-limiting example of a BEAM-seq workflow for the analysis of antibodies for their ability to bind to antigens under a variety of treatment conditions.

[0071] FIG. 36 depicts results from an experiment testing the impact of Fc block and antigen storage conditions on BEAM-seq analysis.

DETAILED DESCRIPTION OF THE DISCLOSURE [0072] The present disclosure generally relates to, inter alia , methods for the identification and characterization of antigen-binding molecules ( e.g ., antibodies) produced by immune cells ( e.g ., B cells), using single-cell immune profiling methodologies, so as to produce recombinant antigen-binding molecules with desired properties.

[0073] As described in greater detail below, antibodies binding the S glycoprotein, including neutralizing antibodies, are one of the central determinants of effective immunity against human-infecting coronaviruses such as SARS-1, SARS-2, MERS, and endemic coronaviruses 229E, NL63, OC43, and HKU 1. Antibodies isolated from subjects that had been exposed to target antigens of interest, e.g ., human survivors of SARS-2 and other coronavirus infection, are ideal therapeutics as the natural selective pressure of somatic hypermutation drives the production of strongly neutralizing antibodies which are inherently low in immunogenicity. Successful isolation of such antibodies can be achieved using other methods including phage display, immunization of humanized mice or other mammals, structural homology search, and more. However, the actual process of selecting antibodies from such approaches can be daunting, time consuming, and often excludes biologically relevant information. As illustrated in greater detail below, the approaches described in the present disclosure allow for the identification and isolation of potent neutralizing antibodies from a naturally infected human survivor of SARS-2 within a short time period, e.g. , within one week, using “barcode-enabled antigen mapping by sequencing” (BEAM-seq). In particular, by staining survivor peripheral blood mononuclear cells (PBMCs) with antigens labeled with 1) Feature Barcode Technology-compatible DNA oligonucleotides, 2) the PE or APC fluorophores, and 3) biotin and tetramerizing these antigens, it was demonstrated herein the ability to successfully identify antibodies that specifically bound the S glycoprotein of SARS-2, while excluding antibodies that bound biotin, the PE or APC fluorophores, or an irrelevant control antigen (human serum albumin). As discussed in greater detail in the experiments described in the Examples, the final antigens tested were tetramers of trimers (e.g, four trimers coupled on each streptavidin). Additionally, it was demonstrated herein the ability to account for biological covariates of antigen binding that are missed in other experimental approaches, including the expression of the antibody / B cell receptor itself.

[0074] As illustrated in greater detail below, the BEAM-seq workflows disclosed herein identified higher numbers of antibody hits that neutralize live SARS-CoV-2 at greater potency, and within a much shorter timeframe than traditional discovery approaches. The antibody hits identified via BEAM-seq workflows are likely to have lower developability burden than those identified using display methodologies. In addition, the BEAM-seq workflows disclosed herein allows directly capture full-length antibody sequences, enabling rapid expression of the native antibody, including somatic hypermutations. Furthermore, as illustrated below, the BEAM-seq workflows disclosed herein is also found to be highly reproducible. Collectively, the figures and data disclosed here demonstrate that the affinity and functional profiles of BEAM-seq-derived antibodies are typically superior or non-inferior to those of antibodies derived using slower and lower-throughput approaches.

[0075] For example, as illustrated in the Examples, analysis of the IC50 values associated with external control antibodies previously characterized as having binding affinity for SARS- CoV-2 spike protein, including FDA authorized and/or approved external controls, as well as several control antibodies discovered via traditional means ( e.g ., originating from phage display, hybridoma, humanized mouse), shows that the BEAM-seq workflows disclosed herein yield greater numbers of antibodies with superior properties as compared to traditional antibody discovery workflows. For example, the data presented in FIG. 29 demonstrated that the ON rate of antibodies isolated with BEAM is correlated with the detected antigen UMIs. As described in greater detail below, the advantages of the workflow disclosed herein can be generalizable to Ab discovery for other target antigens, e.g., clinically relevant antigens.

[0076] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.

[0077] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0078] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, I, & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (_jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology . New York, NY: Wiley (including supplements through 2014); Bollag, D. M. etal. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. etal. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. etal. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. etal. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY : Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

DEFINITIONS

[0079] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. [0080] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0081] The term “barcode” is used herein to refer to a label, or identifier, that conveys or is capable of conveying information ( e.g ., information about an analyte in a sample, a bead, and/or a nucleic acid barcode molecule). A barcode can be part of an analyte or nucleic acid barcode molecule, or independent of an analyte or nucleic acid barcode molecule. A barcode can be attached to an analyte or nucleic acid barcode molecule in a reversible or irreversible manner. A particular barcode can be unique relative to other barcodes. Barcodes can have a variety of different formats. For example, barcodes can include polynucleotide barcodes, random nucleic acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences. A barcode can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow for or facilitates identification and/or quantification of individual sequencing-reads. In some embodiments, a barcode can be configured for use as a fluorescent barcode. For example, in some embodiments, a barcode can be configured for hybridization to fluorescently labeled oligonucleotide probes. Barcodes can be configured to spatially resolve molecular components found in biological samples, for example, at single-cell resolution (e.g., a barcode can be or can include a “spatial barcode”). In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g, sub-barcodes). In some embodiments, the two or more sub-barcodes are separated by one or more non-barcode sequences. In some embodiments, the two or more sub-barcodes are not separated by non barcode sequences.

[0082] In some embodiments, a barcode can include one or more unique molecular identifiers (UMIs). Generally, a unique molecular identifier is a contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that function as a label or identifier for a particular analyte, or for a nucleic acid barcode molecule that binds a particular analyte (e.g, mRNA) via the capture sequence. A UMI can include one or more specific polynucleotides sequences, one or more random nucleic acid and/or amino acid sequences, and/or one or more synthetic nucleic acid and/or amino acid sequences. In some embodiments, the UMI is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample. In some embodiments, the UMI has less than 80% sequence identity ( e.g ., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences across a substantial part (e.g., 80% or more) of the nucleic acid molecules in the biological sample. These nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by 1 or more nucleotides.

[0083] The term “analyte carrier,” as used herein, generally refers to a discrete biological system derived from a biological sample. The analyte carrier may be or comprise a biological particle. The analyte carrier, e.g. , biological particle, may be a macromolecule. The analyte carrier, e.g. , biological particle, may be a small molecule. The analyte carrier, e.g. , biological particle, may be a virus, e.g. , a phage. The analyte carrier, e.g. , biological particle, may be a cell or derivative of a cell. The analyte carrier, e.g. , biological particle, may be an organelle. The analyte carrier, e.g. , biological particle, may be a rare cell from a population of cells. The analyte carrier, e.g. , biological particle, may be any type of cell, including without limitation prokaryotic cells, eukaryotic cells, bacterial, fungal, plant, mammalian, or other animal cell type, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from single cell or multicellular organisms. The analyte carrier, e.g. , biological particle, may be a constituent of a cell. The analyte carrier, e.g. , biological particle, may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The analyte carrier, e.g. , biological particle, may be or may include a matrix (e.g, a gel or polymer matrix) including a cell or one or more constituents from a cell (e.g, cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof, from the cell. The analyte carrier, e.g, biological particle, may be obtained from a tissue of a subject. The analyte carrier, e.g, biological particle, may be a hardened cell. Such hardened cell may or may not include a cell wall or cell membrane. The analyte carrier, e.g, biological particle, may include one or more constituents of a cell, but may not include other constituents of the cell. An example of such constituents is a nucleus or an organelle. A cell may be a live cell. The live cell may be capable of being cultured, for example, being cultured when enclosed in a gel or polymer matrix, or cultured when including a gel or polymer matrix. [0084] An “equivalent amino acid residue” refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and/or functionality of the polypeptide. Equivalent amino acids thus have similar properties such as bulkiness of the side-chain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic/aliphatic). As such, “equivalent amino acid residues” can be regarded as “conservative amino acid substitutions” to each other.

[0085] Within the meaning of the term “equivalent amino acid substitution” as applied herein, one amino acid may be substituted for another without substantially altering the structure and/or functionality of the polypeptide. Exemplary equivalent or conserved amino acid substitutions are within the groups of amino acids indicated herein below:

[0086] i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys);

[0087] ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met);

[0088] iii) Amino acids having aliphatic side chains (Gly, Ala Val, Leu, lie);

[0089] iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro);

[0090] v) Amino acids having aromatic side chains (Phe, Tyr, Trp);

[0091] vi) Amino acids having acidic side chains (Asp, Glu);

[0092] vii) Amino acids having basic side chains (Lys, Arg, His);

[0093] viii) Amino acids having amide side chains (Asn, Gin);

[0094] ix) Amino acids having hydroxy side chains (Ser, Thr);

[0095] x) Amino acids having sulphur-containing side chains (Cys, Met);

[0096] xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr);

[0097] xii) Hydrophilic, acidic amino acids (Gin, Asn, Glu, Asp); and.

[0098] xiii) Hydrophobic amino acids (Leu, He, Val).

[0099] In some embodiments, a Point Accepted Mutation (PAM) matrix is used to determine equivalent amino acid substitutions. In some embodiments, a BLOck Substitution Matrix (BLOSUM) is used to determine equivalent amino acid substitutions.

[0100] As used herein, “isolated” antigen-binding polypeptides, antibodies or antigen binding fragments thereof, polypeptides, polynucleotides and vectors, are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or antigen-binding fragments.

[0101] The term “recombinant” when used with reference to a cell, a nucleic acid, a protein, or a vector, indicates that the cell, nucleic acid, protein or vector has been altered or produced through human intervention such as, for example, has been modified by or is the result of laboratory methods. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant proteins can include amino acid residues not found within the native (non-recombinant or wild-type) form of the protein or can be include amino acid residues that have been modified, e.g ., labeled. The term can include any modifications to the peptide, protein, or nucleic acid sequence. Such modifications may include the following: any chemical modifications of the peptide, protein or nucleic acid sequence, including of one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and/or substitution of one or more of amino acids in the peptide or protein; creation of a fusion protein, e.g. , a fusion protein comprising an antibody fragment; and addition, deletion, and/or substitution of one or more of nucleic acids in the nucleic acid sequence. The term “recombinant” when used in reference to a cell is not intended to include naturally-occurring cells but encompass cells that have been engineered/modified to include or express a polypeptide or nucleic acid that would not be present in the cell if it was not engineered/modified.

[0102] As used herein, a “subject” or an “individual” includes animals, such as human (e.g, human individuals) and non-human animals. The term “non-human animals” includes all vertebrates, e.g, mammals, e.g, rodents, e.g, mice, non-human primates, and other mammals, such as e.g, rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit; and non-mammals, such as amphibians, reptiles, etc. A subject can be a mammal, preferably a human or humanized animal, e.g. , an animal with humanized VDJC loci. The subject may be non-human animals with humanized VDJC loci and knockouts of a target of interest. The subject may be in need of prevention and/or treatment of a disease or disorder such as viral infection or cancer. The subject may have a viral infection, e.g. , a coronavirus infection, or be predisposed to developing an infection. Subjects predisposed to developing an infection, or subjects who may be at elevated risk for contracting an infection (e.g., of coronavirus), include subjects with compromised immune systems because of autoimmune disease, subjects receiving immunosuppressive therapy (for example, following organ transplant), subjects afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), subjects with forms of anemia that deplete or destroy white blood cells, subjects receiving radiation or chemotherapy, or subjects afflicted with an inflammatory disorder. Additionally, subjects of very young (e.g, 5 years of age or younger) or old age (e.g, 65 years of age or older) are at increased risk.

Moreover, a subject may be at risk of contracting a viral infection due to proximity to an outbreak of the disease, e.g, subject resides in a densely-populated city or in close proximity to subjects having confirmed or suspected infections of a virus, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.

[0103] A “variant” of a polypeptide, such as an immunoglobulin chain (e.g, VH, VL, HC, or LC), refers to a polypeptide comprising an amino acid sequence that has at least about 70- 99.9% (e.g, 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity or similarity to a referenced amino acid sequence that is set forth herein. In some embodiments, the term “percent identity,” as used herein in the context of two or more proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acids that are the same, e.g, about 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9%9, 99.5%, 99.9%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See, e.g, the NCBI web site at ncbi.nlm.nih.gov/BLAST. In some embodiments, this definition also refers to, or may be applied to, the complement of a query sequence. In some embodiments, this definition includes sequence comparison performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. In some embodiments, this definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. In some embodiments, sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul etal. , J Mol Biol (1990) 215:403), IgBLAST, and IMGT/V-QUEST. In some embodiments, sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof. Additional methodologies that can suitably be utilized to determine structural similarity or identity amino acid sequences include those relying on position-specific structure scoring matrix (P3SM) that incorporates structure-prediction scores from Rosetta, as well as those based on a length-normalized edit distance as described previously in, e.g ., Setliff etal. , Cell Host & Microbe 23(6), May 2018.

[0104] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0105] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0106] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

METHODS OF THE DISCLOSURE

Methods for identifying and/or characterizing antibodies with binding affinity to a tar set antisen

[0107] In described in more detail below, one aspect of the disclosure relates to new approaches and methods for the identification and characterization of antigen-binding molecules, e.g ., antibodies and antigen-binding fragments. In some embodiments, the methods of the disclosure may be used to identify and/or characterize antigen-binding molecules that are derived from B cells obtained from biological samples, by using single-cell immune profiling methodologies, so as to identify and/or generate antibodies and antigen-binding fragments having a binding specificity for a target antigen, e.g., having the ability to discriminate the target antigen from non-target antigens. Advantages of the new approaches and methods disclosed herein are numerous. For example, as illustrated herein, workflows disclosed herein identified higher numbers of antibody hits that neutralize target antigen at greater potency, and within a much shorter timeframe than traditional discovery approaches. The antibody hits identified via workflows disclosed herein are likely to have lower developability burden than those identified using display methodologies. Furthermore, as illustrated herein, affinity and functional profiles of antibodies identified via workflows disclosed herein are typically superior or non-inferior to those of antibodies derived using slower and lower-throughput approaches. Accordingly, the workflows disclosed herein yield greater numbers of antibodies with superior properties as compared to traditional antibody discovery workflows. Furthermore, as illustrated herein, the workflows disclosed herein allow for rapid identification of many antibodies with broad and robust binding affinity against several antigens, including target antigens and variants of the target antigens. Such workflows are particularly advantageous in the face of rapidly changing disease landscapes where variants of concern evolve over time. Furthermore, as illustrated herein, the workflows disclosed herein can beneficially and rapidly identify a large set of specific antibodies that bind to a highly diverse range of epitopes for a target antigen of interest.

[0108] In some embodiments, the methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, of the disclosure include: (a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens includes (i) a target antigen coupled to a first reporter oligonucleotide and (ii) a non-target antigen coupled to a second reporter oligonucleotide, and wherein the contacting provides a B cell bound to the target antigen; (b) partitioning the B cell bound to the target antigen into a partition of a plurality of partitions, wherein the partitioning provides a partition including (i) the B cell bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence ( e.g ., a partition-specific barcode sequence);

(c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen; (d) assessing the binding affinity of the antibody or antigen-binding fragment to the target antigen; and (e) identifying the antibody or antigen binding fragment as having a binding specificity for the target antigen if the antibody or antigen binding fragment specifically binds to the target antigen.

[0109] In some embodiments, the methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, of the disclosure include: (a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a target antigen coupled to a first reporter oligonucleotide and (ii) a non-target antigen coupled to a second reporter oligonucleotide, and wherein the contacting provides a B cell bound to the target antigen; (b) partitioning the B cell bound to the target antigen into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the B cell bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen; and (d) identifying the antibody or antigen-binding fragment as having a binding specificity for the target antigen if the antibody or antigen-binding fragment binds to the target antigen and does not significantly bind the non-target antigen.

[0110] In some embodiments, the methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, of the disclosure include: (a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a target antigen coupled to a first reporter oligonucleotide and (ii) a non-target antigen coupled to a second reporter oligonucleotide, and wherein the contacting provides a B cell bound to the target antigen; (b) partitioning the B cell bound to the target antigen into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the B cell bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen; and (d) identifying the antibody or antigen-binding fragment as non-specific for the target antigen if the antibody or antigen-binding fragment binds to the non-target antigen.

Target antisens

[0111] The target antigen may be any antigen for which the characterization and/or identification of antigen-binding molecule such as an antibody, or antigen-binding fragment thereof, capable of binding or as having an affinity thereto is desirable. The target antigen may be an antigen associated with an infectious agent, such as a viral, bacterial, parasitic, protozoal or prion agent. If the target antigen is associated with an infectious agent that is a viral agent, the viral agent may be a human immunodeficiency virus (HIV), an influenza virus, a coronavirus, a retrovirus, a rhinovirus, or a sarcoma virus. The viral agent may be severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), a SARS-CoV-2, a Middle East respiratory syndrome coronavirus (MERS-CoV), influenza, respiratory syncytial virus, or Ebola virus. If the target antigen is associated with an infectious agent that is a viral agent, the target antigen may be corona virus spike (S) protein, an influenza hemagglutinin protein, an HIV envelope protein or any other a viral glycoprotein. Further, the target antigen may be associated with a tumor or a cancer. If the target antigen is associated with tumors or cancers, it may be, for example, epidermal growth factor receptor (EGFR), CD38, platelet-derived growth factor receptor (PDGFR) alpha, insulin growth factor receptor (IGFR), CD20, CD 19, CD47, or human epidermal growth factor receptor 2 (HER2). In addition, the target antigen may be an immune checkpoint molecule that may or may not be associated with tumors or cancers ( e.g ., CD38, PD- 1, CTLA-4, TIGIT, LAG-3, VISTA, TIM-3), or it may be a cytokine, a GPCR, a cell-based co stimulatory molecule, a cell-based co-inhibitory molecule, an ion channel, or a growth factor. Further still, the target antigen may be associated with a degenerative condition or disease (e.g., an amyloid protein).

[0112] In some embodiments, the target antigen, for which the characterization and/or identification of an antigen-binding molecule such as an antibody, or antigen-binding fragment thereof, having affinity thereto may be desirable. In further embodiments, the target antigen may be a protein or peptide as expressed by a cell, e.g, full-length target antigen that may or may not include its leader sequence and may or may not have undergone a similar cell processing step.

Antibody-expressing B cells

[0113] The plurality of B cells expressing antibodies, or antigen-binding fragments thereof, may be a plurality of cells that includes cells of B cell lineage, e.g. memory B cells, which express an antibody as a cell surface receptor. The plurality of B cells expressing antibodies, or antigen-binding fragments thereof, may be obtained from a biological sample, which may be obtained from a subject, e.g, a mammal such as a human. The sample of the subject may be obtained by biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a plasma or serum sample.

[0114] If the plurality of B cells is obtained from a sample of the subject, the sample may have been processed prior contacting it with the antigens. The processing of the sample may include steps such as filtration, selective precipitation, purification, centrifugation, agitation, heating, and/or other processes. For example, a sample may be filtered to remove a contaminant or other materials. In some cases, cells and/or cellular constituents of a sample can be processed to separate and/or sort cells of different types, e.g, to separate B cells from other cell types, including the separation of B cell subpopulations such as memory B cells. A separation process can be a positive selection process, a negative selection process (e.g, removal of one or more cell types and retention of one or more other cell types of interest), and/or a depletion process (e.g, removal of a single cell type from a sample, such as removal of red blood cells from peripheral blood mononuclear cells).

[0115] The methods disclosed herein may be useful for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, having specific binding affinity (e.g, ability to bind, with varying degrees of specificity) to a target antigen relative to a non-target antigen. In some embodiments, the methods include: (a) partitioning a sample including biological particles (e.g, cells) producing antigen-binding molecules and a plurality of antigens, wherein the plurality of antigens includes a target antigen coupled to a first reporter oligonucleotide and a non-target antigen coupled to a second reporter oligonucleotide, and wherein the sample includes at least one biological particle bound to the target antigen, and wherein the partitioning provides a partition including (i) the biological particle bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence (e.g., a partition-specific barcode sequence); (c) identifying a sequence of at least one antigen-binding molecule produced by the biological particle that has been bound to the target antigen; and d) assessing the binding affinity (e.g, ability to bind, with varying degrees of specificity) of the antigen-binding molecule to the target antigen; and e) identifying the isolated antibody antigen-binding fragment as an antibody having a binding specificity for the target antigen if the barcoded antibody specifically binds to the target antigen. As discussed above, a target antigen may be any antigen of interest. It may be associated with an infectious agent, such as a viral, bacterial, parasitic, protozoal or prion agent. The target antigen may be associated with a tumor or cancer. Further, the target antigen may be associated with an inflammatory or an autoimmune disease. Further still, the target antigen may be associated with a degenerative condition or disease. As used herein, an “antigen” is not limited to proteins, fats, and/or sugars that is foreign to the subject but may include self antigens, e.g, amyloid or tau protein. Exemplary target antigens are described herein.

[0116] Non-limiting exemplary embodiments of the methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, as described herein can include one or more of the following features. A reporter oligonucleotide, bound to any of a target antigen, or any fragment of the target antigen, may be or include a nucleotide sequence that is specific for the target antigen to which it is coupled or the fragment of the target antigen to which it is coupled. The reporter oligonucleotide may include nucleotide sequences including (a) a reporter sequence, e.g, which may be useful to identify the target antigen or fragment to which the reporter oligonucleotide is coupled, and (b) a capture handle sequence. Accordingly, in some embodiments, the first and the second reporter oligonucleotide include (i) a first and a second reporter barcode sequence that identifies the target antigen and the non-target antigen, respectively, and (ii) a capture handle sequence. In some embodiments, the first and/or second reporter barcode sequence identifies a treatment condition that the target antigen and/or non target antigen are subjected to.

[0117] In some embodiments, the methods of the disclosure further include coupling a barcode moiety to the antibody or antigen-binding fragment produced by the B cell to generate a barcoded antibody or antigen-binding fragment.

[0118] Nucleic acid barcode molecules of the plurality of nucleic acid barcode molecules, included in the partition with the B cell bound to the target antigen may include a common barcode, e.g ., a partition-specific barcode. A partition-specific barcode sequence may identify the partition in which the nucleic acid barcode molecule is partitioned. Nucleic acid barcode molecules of the plurality of nucleic acid barcode molecules may further include a capture sequence. A capture sequence may be configured to couple to the handle sequence of a reporter oligonucleotide, e.g. , by complementary base pairing. A capture sequence may be configured to couple to an mRNA or a DNA analyte. In instances wherein the capture sequence is configured to couple to an mRNA analyte, it may include a polyT sequence. In some embodiments wherein the capture sequence is configured to couple to an mRNA analyte, it may include a targeted priming sequence. In some embodiments, the targeted priming sequence targets an antibody or BCR region of the mRNA analyte (e.g., targets a region of the mRNA analyte encoding the antibody or BCR), such as a region of the mRNA analyte that encodes a constant sequence or variable sequence of said antibody or BCR. Accordingly, in some embodiments, a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further includes a capture sequence configured to couple to the capture handle sequence of the first and/or second reporter oligonucleotide, and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further includes a capture sequence configured to couple to an mRNA analyte or DNA analyte. In some embodiments, the capture sequence is configured to couple to the capture handle sequence of a reporter oligonucleotide by complementarity base pairing. In some embodiments, the capture sequence configured to couple to an mRNA analyte includes a polyT sequence. In some embodiments, polyadenylated mRNAs are captured using polyT capture sequence, followed by reverse-transcription, e.g., within individual partitions, e.g., droplets. The resulting cDNA libraries can be selectively enriched for BCR sequences, e.g., by targeted bait capture. These enriched sequences can then be efficiently analyzed by using methodologies suitable for long-read sequencing analysis (e.g., using single molecule real-time sequencing (Pacific Biosciences) or nanopore sequenceing (Oxford Nanopore). The unenriched cDNA pool can be separately analyzed by short-read sequencing methodologies, such as Illumina sequencing techniques.

[0119] In some embodiments the capture sequence configured to couple to an mRNA analyte includes a targeted priming sequence. In some embodiments, the targeted priming sequence targets (e.g., is complementary to) an antibody or BCR region of the mRNA analyte, e.g., a constant sequence or variable sequence of said antibody or BCR region of the mRNA analyte. It will be understood that any of the barcoded nucleic acid molecules may further include a UMI. The UMI may be a sequence originating from a reporter oligonucleotide or a nucleic acid barcode molecule. It will be understood that any of the barcoded nucleic acid molecules may further include a functional sequence. Functional sequences are disclosed herein. In some embodiments, the first and second nucleic acid barcode molecules each include a unique molecule identifier (UMI). In any of the methods provided herein, once the reaction is partitioned, barcoded nucleic acid molecules, including a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule, may be generated, e.g., in the partition. In some embodiments, a first nucleic acid barcode molecule couples to the capture handle sequence of the first and/or second reporter oligonucleotide in the partition and a second nucleic acid barcode molecule couples to the mRNA analyte or DNA analyte in the partition. In some embodiments, a first nucleic acid barcode molecule couples to the capture handle sequence of the first reporter oligonucleotide in the partition, an additional first nucleic acid barcode molecule couples to the capture handle sequence of the second reporter oligonucleotide in the partition, and a second nucleic acid barcode molecule couples to the mRNA analyte or DNA analyte in the partition. In some embodiments, a first barcoded nucleic molecule is generated, e.g., in the partition, wherein the first barcoded nucleic acid molecule comprising a sequence of the first or second reporter oligonucleotide, or a reverse complement thereof, and the common barcode sequence or a reserve complement thereof. In some embodiments, the method further comprises using the first barcoded nucleic acid molecules to identify the B cell as having bound to the target antigen coupled to the first reporter oligonucleotide. [0120] The methods disclosed herein may be useful for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, having specific binding affinity for a target antigen relative to a non-target antigen. In some embodiments, the methods include: (a) partitioning a sample including biological particles ( e.g ., cells, e.g. , B cells) producing antigen binding molecules and a plurality of antigens, wherein the plurality of antigens includes a target antigen coupled to a first reporter oligonucleotide and a non-target antigen coupled to a second reporter oligonucleotide, and wherein the sample includes at least one biological particle bound to the target antigen, and wherein the partitioning provides a partition including (i) the biological particle bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence (e.g., a partition-specific barcode sequence); (c) identifying a sequence of at least one antigen-binding molecule produced by the biological particle that has been bound to the target antigen; and d) assessing the binding affinity of the antigen-binding molecule to the target antigen; and e) identifying the isolated antibody antigen binding fragment as an antibody having a binding specificity for the target antigen if the barcoded antibody specifically binds to the target antigen.

[0121] As described in greater detail below, in some embodiments, the methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, as described herein can further include, prior to step (a), contacting the biological sample with a plurality of antigens. In some embodiments, the biological sample is contacted with a plurality of antigens and a plurality of additional labelling agents. In some embodiments, the additional labeling agents are configured to bind or otherwise couple to one or more cell-surface features of an immune cell. In some embodiments, such additional labeling agents can be used to characterize cells and/or cell features. In some embodiments, reporter oligonucleotides of the additional labeling agents have different sequencing primer sequences than reporter oligonucleotides attached to target and/or non-target antigens.

[0122] In some embodiments, the additional labeling agents comprise a panel of barcoded antibodies for profiling immune cells. In some embodiments, the panel of barcoded antibodies includes antibodies from a “T and B Natural Killer” (TBNK) panel with binding affinity for individual immune cell specific antigens. In some embodiments, immune cell specific antigens include one or more of the following antigens: CD38, CD27, CD24, IgD, CD20, CD 19, CD3E, CD4, CD8A, CD14, and CD16. In some embodiments, immune cell specific antigens include one or more of CD3, CD4 and CD8 (for T-cells), CD56 (for NK cells), and CD19 (for B-cells).

In some embodiments, one or more of the additional labeling agents (e.g., barcoded antibodies for profiling immune cells) is coupled to a detectable label. The detectable label can be magnetic or fluorescent.

[0123] The antigen-binding molecule, identified and/or characterized in the methods may be an antibody or an antigen-binding fragment of an antibody. In some instances, the antigen binding molecule is an antibody, and the antibody may be an antibody having a human immunoglobulin (Ig)A (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g, IgGl, IgG2, IgG3 and IgG4) or IgM constant region. In some instances, the antigen-binding molecule is an antibody, and the antibody may be an antibody having a murine immunoglobulin IgA, IgD, IgE (e.g, IgGl or IgG2a), IgG (e.g, IgG2b or IgG3), or IgM constant region. In some other instances, the antigen binding molecule is a fragment of an antibody, and the fragment of the antibody may be any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0124] The plurality of B cells expressing antibodies, or antigen-binding fragments thereof, may be a plurality of cells that includes cells of B cell lineage, e.g. memory B cells, which express an antibody as a cell surface receptor. In some embodiments, the B cells are obtained from a biological sample. The biological sample can be obtained from a subject. In some embodiments, the subject is a vertebrate subject. In some embodiments, the vertebrate subject is a non-mammalian subject. In some embodiments, the non-mammalian subject is an avian species. In some embodiments, the vertebrate subject is a mammalian subject. In some embodiments, the mammalian subject is a human. In some embodiments, the mammalian subject is a mouse. Exemplary subjects are further disclosed herein.

[0125] In principle, there are no particular restrictions in regard to the types of biological samples suitable for use in the methods described herein. For example, samples that can be suitably used include any tissue or fluid sample obtainable from a subject. In some embodiments, the biological sample includes sputum, bronchoalveolar lavage, pleural effusion, tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, circulating tumor cells, circulating nucleic acids, bone marrow, or any combination thereof. In some embodiments, the biological sample includes cells or tissue. For example, the biological sample of the subject may be obtained by biopsy, core biopsy, needle aspirate, or fine needle aspirate. The biological sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The biological sample can be a plasma or serum sample. The sample can be a bone marrow sample. The sample can be a spleen sample. The sample can be a lymph node sample. The sample can be a lymphoid tissue (e.g. tonsil, mucosal-associated lymphoid tissue) sample. In some embodiments, the biological sample can be a skin sample. In some embodiments, the biological sample can be a cheek swab. In some embodiments, the biological sample includes whole blood and blood components.

[0126] The subject can be previously exposed to the target antigen or suspected of having been exposed to the target antigen, or an evolutionarily related antigen. The evolutionarily related antigen can belong to the same family, the same sub-family, the same genus. The evolutionarily related antigen can be a variant of the target antigen and be the same species. The evolutionarily related antigen can have at least 75% identity as the target antigen. The evolutionarily related antigen can be a functional variant. For example, in cases where the target antigen is a disease-associated antigen, the subject can be previously known or suspected of having the disease. In some embodiments, the subject can have been immunized with the target antigen. In some embodiments, the subject can have recovered from the disease.

[0127] In some embodiments, the non-target antigen may be any antigen to which the antibodies or antigen-binding fragments thereof would not be expected to bind. In some embodiments, the non-target antigen does not significantly bind the antibodies or antigen binding fragments thereof.

[0128] In some embodiments, the non-target antigen has been selected such that it is not expected to bind the antibody or antigen-binding fragment thereof. In some embodiments, the non-target antigen has been selected such that it is not expected to significantly bind the antibody or antigen-binding fragment thereof. In some embodiments, the non-target antigen is an off- target antigen for which binding to an antibody or antigen-binding fragment is undesirable. By way of example, the non-target antigen may be any antigen for which a subject, e.g. , a human would not be expected to develop an antibody response to or to have antibodies with a specificity for. Such a non-target antigen may be an antigen endogenous to and abundantly expressed in a subject, e.g. , a human subject, e.g. , human serum albumin. Other suitable non-target antigens (which can be negative control antigens) are further described herein. Exemplary non-target antigens (also referred to herein as negative control antigens) are further disclosed herein. [0129] In further embodiments, the target antigen may be a protein or peptide as expressed in vitro , ex vivo , or in vivo (e.g. , by a cell) or by cell-free expression system. The target antigen may be a full-length target antigen that may or may not include its leader sequence and may or may not have undergone a similar cell processing step. This is because leader peptides are used by many proteins; it lets the cell know that the protein of interest needs to be secreted, and typically the leader peptide is removed from the final (e.g, mature) protein.

[0130] As discussed above, a target antigen may be any antigen of interest. It may be associated with an infectious agent, such as a viral, bacterial, parasitic, protozoal or prion agent. The target antigen may be an exogenous antigen derived from transplantation. The target antigen may be associated with a tumor or cancer. Further, the target antigen may be associated with an inflammatory or an autoimmune disease. Further still, the target antigen may be associated with a degenerative condition or disease. As used herein, an “antigen” is not limited to proteins, fats, and/or sugars that is foreign to the subject but may include self-antigens, e.g, amyloid or tau protein or type I interferons. Exemplary target antigens are described herein.

[0131] In some embodiments, if the plurality of B cells is obtained from a sample of the subject, the sample may have been processed prior to contacting it with the antigens. The processing of the sample may include steps such as filtration, selective precipitation, purification, centrifugation, agitation, heating, and/or other processes. For example, a sample may be filtered to remove a contaminant or other materials. In some cases, cells and/or cellular constituents of a sample can be processed to separate and/or sort cells of different types, e.g, to separate B cells from other cell types, including the separation of B cell subpopulations such as memory B cells. A separation process can be a positive selection process, a negative selection process (e.g, removal of one or more cell types and retention of one or more other cell types of interest), and/or a depletion process (e.g, removal of a single cell type from a sample, such as removal of red blood cells from peripheral blood mononuclear cells).

[0132] Non-limiting exemplary embodiments of the methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, as described herein can include one or more of the following features. A reporter oligonucleotide, bound to any of a target antigen may be or include a nucleotide sequence that identifies the target antigen to which it is coupled or the fragment of the target antigen to which it is coupled. The reporter oligonucleotide may include nucleotide sequences including (a) a reporter sequence, e.g, which may be useful to identify the target antigen or fragment to which the reporter oligonucleotide is bound, and (b) a capture handle sequence. Accordingly, in some embodiments, the first and the second reporter oligonucleotide include (i) a first and a second reporter barcode sequence that identifies the target antigen and the non-target antigen, respectively, and (ii) a capture handle sequence.

[0133] In some embodiments, the methods of the disclosure further include coupling a barcode moiety to the antibody or antigen-binding fragment produced by the B cell to generate a barcoded antibody or antigen-binding fragment.

[0134] Nucleic acid barcode molecules of the plurality of nucleic acid barcode molecules, included in the partition with the B cell bound to the target antigen may include a common barcode, e.g ., a partition-specific barcode. A partition-specific barcode sequence may identify the partition in which the nucleic acid barcode molecule is partitioned. Nucleic acid barcode molecules of the plurality of nucleic acid barcode molecules may further include a capture sequence. A capture sequence may be configured to couple to the handle sequence of a reporter oligonucleotide, e.g. , by complementary base pairing. A capture sequence may be configured to couple to an mRNA or a DNA analyte. In instances wherein the capture sequence is configured to couple to an mRNA analyte, it may include a polyT sequence. A capture sequence may be configured to couple to a cDNA molecule of an mRNA analyte, e.g. , as generated by a reverse transcription reaction. In some embodiments, the generated cDNA molecule can have an additional sequence (e.g, non-templated bases, e.g, a poly-C sequence) appended to the cDNA. In some embodiments, a capture sequence of a nucleic acid barcode molecule includes a sequence complementary to the non-templated bases. In some cases, the generated cDNA molecule can hybridize to the capture sequence of the nucleic acid barcode molecule and a reverse transcriptase can perform a template switching reaction onto the nucleic acid barcode molecule. In some instances, a capture sequence includes a sequence complementary to a region of an immune molecule, such as the constant region of a BCR sequence. Accordingly, in some embodiments, a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further includes a capture sequence configured to couple to the capture handle sequence of the first and/or second reporter oligonucleotide, and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further includes a capture sequence configured to couple to an mRNA analyte or DNA analyte. In some embodiments, the capture sequence is configured to couple to the capture handle sequence of a reporter oligonucleotide by complementarity base pairing.

[0135] In some embodiments, the capture sequence configured to couple to an mRNA analyte includes a polyT sequence. It will be understood that any of the barcoded nucleic acid molecules may further include a UMI. The UMI may be a sequence originating from a reporter oligonucleotide or a nucleic acid barcode molecule. It will be understood that any of the barcoded nucleic acid molecules may further include a functional sequence. Functional sequences are disclosed herein. In some embodiments, the first and second nucleic acid barcode molecules each include a unique molecule identifier (UMI). In any of the methods provided herein, once the reaction is partitioned, barcoded nucleic acid molecules, including a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule, may be generated in the partition. In some embodiments, a first barcode nucleic molecule is generated in the partition, wherein the first barcoded nucleic acid molecule comprising a sequence of the first or second reporter oligonucleotide, or a reverse complement thereof, and the common barcode sequence or a reserve complement thereof. In these instances, the sequence of the first barcoded nucleic molecule may facilitate the identification of (i) the target antigen coupled to the first reporter oligonucleotide and/or (ii) the non-target antigen coupled to the second reporter oligonucleotide.

[0136] In some embodiments, the antibody or antigen-binding fragment has a binding specificity to an epitope on the target antigen. One skilled in the art will understand that the term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site of an antigen-binding polypeptide, e.g ., a variable region of an antibody molecule, known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can be linear or conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have specific three- dimensional structural characteristics, and/or specific charge characteristics. [0137] Methods for determining the epitope of an antigen-binding polypeptide, e.g, antibody or antigen-binding fragment or polypeptide, include alanine scanning mutational analysis, peptide blot analysis, peptide cleavage analysis, crystallographic studies andNMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed. Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding polypeptide (e.g, antibody or fragment or polypeptide) interacts is hydrogen/deuterium exchange detected by mass spectrometry.

[0138] In some embodiments, the methods disclosed herein include assessing the binding affinity (e.g, ability to bind, with varying degrees of specificity) of the antibody or antigen binding fragment to the target antigen. In some embodiments, the methods disclosed herein optionally include identifying the antibody or antigen-binding fragment as having a binding specificity for the target antigen if the antibody or antigen-binding fragment specifically binds to the target antigen. Generally, binding affinity can be used as a measure of the strength of a non- covalent interaction between two molecules, e.g, an antibody or antigen-binding fragment thereof and an antigen. In some cases, binding affinity can be used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules can be quantified by determination of the equilibrium dissociation constant (K_D). In turn, K_D can be determined by measurement of the kinetics of complex formation and dissociation using, e.g, the surface plasmon resonance (SPR) method (Biacore™). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants k_a (or k_on) and dissociation rate constant k_d (or k_0ff), respectively. K_D is related to k_a and k_d through the equation K_D = k_d / k_a. The value of the dissociation constant can be determined directly by various methods, and can be computed even for complex mixtures by methods such as those set forth in Caceci etal. (1984, Byte 9: 340-362). For example, the K_D can be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428- 5432). Other standard assays to evaluate the binding ability of the antibodies and antigen-binding fragments of the present disclosure towards target antigens are known in the art, including for example, ELISAs, Western blots, RIAs, Octet® HTX biosensor, solution-affinity ELISA, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system, or KinExA. In some embodiments, the binding affinity of an antibody or an antigen-binding fragment for a target antigen can be calculated by the Scatchard method described by Frankel et aI.,Moί Immunol , 16: 101-106, 1979. It will be understood that an antibody or antigen-binding fragment that “specifically binds” a target antigen is an antigen-binding fragment that does not significantly bind other antigens (e.g., non-target antigens) but binds the target antigen with high affinity, e.g. , with an equilibrium dissociation constant (K_D) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less. In some embodiments, the antibody or antigen-binding fragment “does not significantly bind” the non-target antigen if the binding affinity of the antibody or antigen binding fragment for the non-target antigen is less than about 10%, e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of the binding affinity for the target antigen.

[0139] In cases wherein the methods determine binding affinity (e.g, ability to bind with varying degrees of specificity) of an antigen-binding molecule (e.g, antibody or antigen-binding fragment) to a target antigen and/or one or more fragments thereof, and the barcoded nucleic acid molecules include a unique molecular identifier (UMI), the binding affinity (e.g, ability to bind with varying degrees of specificity) of an antigen-binding molecule (e.g, antibody or antigen binding fragment) to a target antigen can be determined based on a quantity/number of unique molecular identifiers (UMIs) associated with the antigen-binding molecule (e.g, antibody or antigen-binding fragment thereof) bound to the antigen. For example, the binding affinity (e.g, ability to bind with varying degrees of specificity) of an antigen-binding molecule expressed by a B cell can be determined based on a quantity/number of antigen UMIs associated with the antigen-binding molecule, e.g, quantity/number of antigen UMIs associated with the same partition-specific barcode as the B cell expressing the antigen-binding molecule. In some embodiments, the binding affinity determined in this manner may be confirmed by other techniques that determine affinity of antigen-binding molecules for target antigens including, for example, competition binning and competition enzyme-linked immunosorbent assay (ELISA), NMR, and HDX-MS. In some embodiments, binding affinity of an anti gen -binding molecule for its target antigen can also be assayed using a Carterra LSA SPR biosensor equipped with a HC30M chip.

[0140] In addition or alternatively, the binding affinity and/or binding specificity of an antigen-binding molecule ( e.g ., antibody or antigen-binding fragment) to a target antigen (such as S protein) can be determined based on the counts and/or amounts of target antigens and/or non-target antigens associated with the antigen-binding molecule. In some embodiments, the binding affinity and/or binding specificity of an antigen-binding molecule to a target antigen can be determined based on the proportion of target antigens and optionally non-target antigens that are associated with the antigen-binding molecule. As discussed herein, the target antigens and/or non-target antigens are coupled to their respective reporter oligonucleotides. The counts, amounts, and/or proportion of such antigens can be facilitated by the respective reporter oligonucleotides coupled to the antigens, wherein a reporter oligonucleotide coupled to an antigen comprises a reporter barcode sequence that identifies the antigen coupled thereto.

[0141] The counts, amounts, and or proportion of antigens associated with the antigen binding molecule can be determined based on a quantity/number of antigen sequence reads and/or UMIs associated with the antigen-binding molecule via a process termed “barcode- enabled antigen mapping by sequencing” (BEAM-seq) (see, e.g., Example 6, Example 7, FIGs. 13-15, and Table 3 below). Antigen sequence reads and/or UMIs can be associated bioinformatically with antigen-binding molecule sequences via shared partition barcode sequences. For example, binding affinity and/or binding specificity of an antigen-binding molecule to a target antigen can be determined based on independent observations of quantity/number of UMIs associated with the antigen from one or more partitions, wherein each of the one or more partitions comprise a cell expressing the same antigen-binding molecule. For other example, binding affinity and/or binding specificity of an antigen-binding molecule to a target antigen can be determined based on independent observations of quantity/number of UMIs associated with the antigen from one or more partitions, wherein each of the one or more partitions comprise a cell expressing an antigen-binding molecule belonging to the same clonotype group.

[0142] In some embodiments, an antigen-binding molecule is determined to have binding affinity for the target antigen if at least 40 target antigen UMIs are associated with the antigen binding molecule, e.g., at least 50, 60, 70, 80, 90, or 100 target antigen UMIs are associated with the antigen-binding molecule. [0143] In some embodiments, the antibody or antigen-binding fragment is determined to “not significantly bind” the non-target antigen if 10 or fewer non-target antigen UMIs are associated with the antigen-binding molecule, e.g., 9 or fewer non-target antigen UMIs, 8 or fewer non-target antigen UMIs, 7 or fewer non-target antigen UMIs, 6 or fewer non-target antigen UMIs, 5 or fewer non-target antigen UMIs, 4 or fewer non-target antigen UMIs, 3 or fewer non-target antigen UMIs, 2 or fewer non-target antigen UMIs, 1 non-target antigen UMI, or 0 non-target antigen UMIs are associated with the antigen-binding molecule. In particular embodiments, the antibody or antigen-binding fragment is determined to “not significantly bind” the non-target antigen if 5 or fewer non-target antigen UMIs are associated with the antigen binding molecule.

[0144] In some embodiments, the binding specificity of the antigen-binding molecule is determined based on the ratio of antigen UMIs/non-target antigen UMIs associated with the antigen-binding molecule. For example, the antigen-binding molecule can be determined to specifically bind the target antigen if the ratio of target antigen UMIs/non-target antigen UMIs is greater than 1, e.g., at least about 5: 1, at least about 10:1, at least about 20: 1, at least about 30:1, at least about 40: 1, at least about 50:1, at least about 60: 1, at least about 70: 1, at least about 80: 1, at least about 90: 1, at least about 100: 1, at least about 200: 1, at least about 400: 1, at least about 500: 1, or at least about 1000: 1. The antigen-binding molecule can be determined to specifically bind the target antigen if the ratio of target antigen UMIs/non-target antigen UMIs is between about 1000:1 to about 5:1.

[0145] In some embodiments, the biological sample is from a mammalian subject. In some embodiments, the mammalian subject is a non-human mammal. In some embodiments, the mammalian subject is a non-human primate. In some embodiments, the mammalian subject is a human.

[0146] In addition, a reporter oligonucleotide as described herein may have a further characteristic in that it may be coupled to a labeling agent. The labelling agent may be coupled to the reporter oligonucleotide via a labelling of the target antigen and/or any fragment thereof, or via a labelling of a nucleotide(s) of the reporter oligonucleotide. Accordingly, in some embodiments, the first and/or the second reporter oligonucleotide is coupled to a tag, detectable label, or labelling agent. In some embodiments, the tag, detectable label, or labelling agent is magnetic or fluorescent. In some embodiments, the tag, detectable label, or labelling agent is magnetic. In some embodiments, the tag, detectable label, or labelling agent is fluorescent. In some embodiments, the tag, detectable label, or labelling agent includes a fluorescent label identifying the antigen or the target antigen.

[0147] A challenge in the use of antibodies as research tools is that an antibody that recognizes a protein, often recognize that protein under some but not all conditions. For example, an antibody may recognize an epitope that gets modified by treatment with a certain fixative, e.g., paraformaldehyde (PFA) so that it no longer recognizes the protein in a PFA treated sample. Often times, there is substantial work spent validating antibodies to work under different treatment conditions, e.g., methylation, formalin-fixed and paraffin-embedded (FFPE), etc., after they are already developed.

[0148] BEAM-seq workflows disclosed herein can be deployed to analyze antibodies during the discovery phase for their ability to bind to antigens under a variety of treatment conditions. For instance, antigen-associated reporter oligonucleotides can include a reporter barcode sequence that identifies (i) the antigen that is associated with the reporter oligonucleotide and (ii) the treatment condition to which the antigen is subjected to.

[0149] In some embodiments, provided herein are methods for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, the method including:

(a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a first antigen coupled to a first reporter oligonucleotide comprising a first reporter barcode sequence and (ii) the first antigen coupled to a second reporter oligonucleotide comprising a second reporter barcode sequence, wherein the first antigen coupled to the first reporter oligonucleotide is subjected to a first treatment condition and the first antigen coupled to the second reporter oligonucleotide is subjected to a second treatment condition, and wherein the contacting provides a labeled B cell bound to the first antigen coupled to the first reporter oligonucleotide and/or the first antigen coupled to the second reporter oligonucleotide; (b) partitioning the labeled B cell into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the labeled B cell and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the labeled B cell; and (d) identifying the antibody or antigen-binding fragment as binding to the first antigen subjected to the first and/or second treatment condition, wherein optionally the plurality of antigens further comprises (iii) a second antigen coupled to a third reporter oligonucleotide comprising a third reporter barcode sequence and (iv) the second antigen coupled to a fourth reporter barcode sequence, wherein (iii) and (iv) are subjected to the first treatment condition and second treatment conditions, respectively, and wherein the labeled B cell is optionally bound to (iii) and/or (iv), optionally wherein the first antigen is a target antigen and the second antigen is a non-target control antigen, and/or wherein the method further comprises the prior step of subjecting (i) and (ii) to the first and second treatment conditions, respectively, prior to (a), and/or wherein the method further comprises the prior step of subjecting (iii) and (iv) to the first and second treatment conditions, respectively, prior to (a).

[0150] For example, as illustrated in FIG. 34, a set of reporter oligonucleotide-associated antigens may comprise a first antigen (or epitope) coupled to a first reporter oligonucleotide comprising a first barcode sequence (BC1), and the same first antigen (or epitope) coupled to a second reporter oligonucleotide comprising a second barcode sequence (BC2). In some embodiments, the set of reporter oligonucleotide-associated antigens further includes a second antigen (or epitope) coupled to a third reporter oligonucleotide comprising a third barcode sequence (BC3) and the same second antigen (or epitope) coupled to a fourth reporter oligonucleotide comprising a fourth barcode sequence (BC4). The first antigen may be a target antigen and the second antigen may be a negative control antigen (e.g., as described herein). In some embodiments, the BC1 -associated first antigen (and optionally the BC3 -associated second antigen) is subjected to a first treatment condition and the BC2-associated first antigen (and optionally the BC4-associated second antigen) is subjected to a second treatment condition.

Thus, the reporter barcode sequences (e.g., BC1-BC4) are used to identify both the antigen (or epitope) and the treatment condition that the antigen (or epitope) are subjected to. In some embodiments, a method provided herein may comprise contacting B cells with the set of reporter oligonucleotide-associated antigens, to provide a B cell bound to one or more of the antigens of the set. The method may further comprise partitioning the B cell and generation of barcoded nucleic acid molecules, according to methods described in further detail herein. The binding affinity and/or binding specificity of an antigen-binding molecule expressed by the B cell to an antigen under the different treatment conditions can be determined based on the counts and/or amounts of the antigens associated with the antigen-binding molecule. As described above, such counts, amounts, and/or proportion of such antigens can be facilitated by the respective reporter oligonucleotides (e.g., BC1, BC2, BC3, BC4) coupled to the antigens.

Enrichment

[0151] In some embodiments, the methods described herein further include, prior to the partitioning step, isolating and/or enriching the plurality of B cells. In some embodiments, the enrichment step enriches for B cells bound to the target antigen and/or non-target antigen based on detection of one or more of the labelling agents coupled to the reporter oligonucleotides attached to respective antigens.

[0152] In some embodiments, a biological sample obtained from a subject can be subjected to enrichment for specific populations of cells of interest. For example, biological samples can be enriched for B cells. As discussed in greater detail below, cell separation techniques can be used to enrich for specific populations of cells of interest (e.g., B cells). Non-limiting examples of separation techniques useful for separating (e.g, sorting) one or more different types of cells can include, for example, centrifugation, filtration, microfluidic-based sorting, flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), buoyancy- activated cell sorting (BACS), or any other useful methods. In some embodiments, the biological sample is enriched for cells of interest (e.g, B cells) prior to contacting the cells of interest (e.g, B cells) with the target antigen and non-target antigen. In some embodiments, the enrichment step enriches for B cells, e.g., by depleting T cells from the biological sample.

[0153] In additional embodiments, the methods of the disclosure include identifying a B cell as expressing an antibody or antigen-binding fragment that has binding affinity for an antigen. In one embodiment, the method includes partitioning a plurality of B cells into a plurality of partitions.

[0154] In some embodiments, the B cell or partition comprising the B cell comprises (i) a surface-bound antigen comprising a reporter oligonucleotide having a reporter sequence, (ii) a surface-bound control antigen having a control reporter oligonucleotide having a control reporter sequence, and a plurality of nucleic acid barcode molecules comprising a partition barcode sequence. In some embodiments, a partition of the plurality of partitions comprises: (a) a B cell comprising (i) a surface-bound antigen comprising a reporter oligonucleotide having a reporter sequence and (ii) a surface-bound control antigen having a control reporter oligonucleotide having a control reporter sequence; and (b) a plurality of nucleic acid barcode molecules comprising a partition barcode sequence. In an additional embodiment, the method further comprises generating in said partition a plurality of barcoded nucleic acid molecules from said reporter oligonucleotide, said control reporter oligonucleotide, and said plurality of nucleic acid barcode molecules. In one embodiment, the plurality of barcoded nucleic acid molecules comprises (i) a first barcoded nucleic acid molecule comprising said reporter sequence or complement thereof and said partition barcode sequence or complement thereof, and (ii) a second barcoded nucleic acid molecule comprising said control reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In one other embodiment, the method further comprises determining the sequence of said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule or complements thereof to identify said B cell as expressing an antibody that has binding affinity for said antigen. In some embodiments, the single B cell is a plasmablast, a plasma cell, a memory B cell, a regulatory B cell, or a lymphoplasmacytoid cell. In one embodiment, the B cell is a memory B cell.

[0155] In some embodiments, the target antigen and/or the non-target antigen is coupled to a barcode moiety ( e.g ., reporter oligonucleotide) that identifies the target antigen and/or the non target antigen, respectively.

Non-target control antisens

[0156] As described above, some embodiments of the methods disclosed herein include contacting a plurality of B cells from a biological sample with a plurality of antigens, wherein the plurality of antigens including one or more non-target antigens (e.g., negative control antigens). Non-target antigens (e.g, negative control antigens) may be any antigen to which the antibodies or antigen-binding fragments thereof, would not be expected to bind. In some embodiments, the non-target antigen has been selected such that it is not expected to bind the antibody or antigen-binding fragment thereof. By way of example, the non-target antigen may be any antigen for which a subject (e.g, a human subject) would not be expected to develop an antibody response to or to have antibodies with a specificity for. Such a non-target antigen may be an antigen endogenous to and abundantly expressed in a subject, e.g, a human subject, e.g, human serum albumin (HSA). In some embodiments, negative controls can each be coupled to a fluorophore to allow for identification of non-specific antibodies that bind to labeled antigens by interacting with the fluorophore rather than with the antigen of interest. In some embodiments, a single control antigen, e.g, HSA, can be coupled to two different detectable labels such as phycoerythrin (PE) and allophycocyanin (APC) to generate two control reagents. In some embodiments, control antigens can be different non-target antigens. The number of control antigens can vary dependent on specific experimental parameters and can be about 1 to about 100, for example can be 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the plurality of antigens including from about 5 to about 100, from about 10 to about 50, from about 15 to about 70, from about 20 to about 80, from about 30 to about 90, or from about 40 to about 100 non target antigens. In some embodiments, the plurality of antigens including from about 5 to about 50, from about 10 to about 30, from about 20 to about 40, from about 40 to about 100, from about 15 to about 60, from about 20 to about 80, from about 50 to about 100, from about 30 to about 80, from about 1 to about 10, or from about 5 to about 20 non-target antigens.

[0157] Generally, two conceptually different classes of control antigens can be included: “in-line controls” and “process-reassurance controls.” In-line control antigens can be sample- dependent, which subsequently can aid in data analysis and successful interpretation of data. Alternatively or in addition, “process-reassurance control” antigens can also be included. This type of control antigens ( e.g ., positive control antigens) can be dependent on sample antigen specificities in order to confirm that experiments protocols work properly in experimenters’ hands. Inclusion of process-reassurance controls can be optional.

[0158] In some embodiments, negative control antigens can include a reporter oligonucleotide associated with any one or more of: a detectable label, a support, and a ligand with binding affinity for a binding region of the support. Exemplary detectable labels (e.g., fluorophores) are further disclosed herein. Exemplary supports (e.g, streptavidin, avidin) and their ligands (e.g, biotin) are also further disclosed herein. For example, a negative control antigen can be or include a complex comprising a detectable label, support, and/or ligand with binding affinity for a binding region of the support. For example, a negative control antigen can be or can include a biotin-saturated streptavidin comprising a detectable label and/or a reporter oligonucleotide. Such negative control antigens can be used to distinguish antibodies that specifically bind a target antigen from antibodies that non-specifically bind to any one or more of the detectable label (e.g, fluorophore), support (e.g, streptavidin, avidin), and ligand (e.g, biotin).

[0159] In some embodiments of the methods disclosed herein, negative control antigens can include a streptavidin molecule saturated with biotin, wherein the streptavidin is coupled to a reporter oligonucleotide. In some embodiments, negative control antigens can include (i) biotinylated human serum albumin complexed with a streptavidin, wherein the streptavidin is coupled to a reporter oligonucleotide.

[0160] The sample including biological particles ( e.g ., cells, e.g ., B cells) producing antigen-binding molecules and the plurality of antigens can be prepared by contacting the biological particles (e.g, cells, e.g, B cells) with the plurality of antigens under conditions sufficient for one or more of the antigens to bind to one or more B cells and/or to one or more antigen-binding molecules produced by the B cells.

[0161] In some embodiments, the biological particles (e.g, cells, e.g, B cells) are contacted with one or more blocking reagents. Exemplary blocking reagents can include any one or more of the following: an Fc blocking agent, ssDNA, dsDNA, an animal serum (e.g, horse serum, calf serum, bovine serum, and the like), a serum protein (e.g, BSA), and a nucleic acid binding protein (e.g., E. coli SSB, Sso7d, Dpsl, HMG1 & HGM2, Sac7d). Exemplary nucleic acid binding proteins are described in Dickey TH et al. Single-stranded DNA-binding proteins: multiple domains for multiple functions. Structure. 2013;21(7): 1074-1084, which is hereby incorporated by reference in its entirety. In some embodiments, a blocking agent can be or include a negative control antigen or portion thereof, e.g., a complex comprising a detectable label, support, and/or ligand with binding affinity for a binding region of the support. For example, the blocking agent can be or can include a biotin-saturated streptavidin comprising a detectable label and/or a reporter oligonucleotide. In some embodiments, the biological particles are contacted with the one or more blocking reagents prior to contacting with the plurality of antigens. In some embodiments, the biological particles are contacted with the one or more blocking reagents and the plurality of antigens.

[0162] In some embodiments, the contacting further comprises contacting the biological particles (e.g, cells, e.g, B cells) with one or more binding agents (e.g, antibodies or antigen binding fragments thereof, aptamers, and the like) comprising a detectable label (e.g, a fluorophore or other detectable label disclosed herein), the binding agents having binding specificity for one or more cell markers of interest. In some embodiments, the one or more binding agents specifically bind a B cell marker of interest. Such binding agents can be used to enrich for cell populations of interest such as B cells, prior to partitioning. Exemplary antibodies specific for B cell markers of interest are described herein, e.g, in Example 4. In some embodiments, the contacting further comprises contacting the biological particles (e.g, cells, e.g, B cells) with one or more reagents for labeling live or dead cells. Exemplary reagents for labeling live or dead cells are described herein, e.g. , in Example 4. In some embodiments, reagents for labeling live or dead cells can be used to sort or enrich for live cells, prior to partitioning.

[0163] In an additional embodiment, the methods further include partitioning the plurality of B cells into a plurality of partitions, which can be a plurality of droplets in an emulsion or a plurality of wells. In a further embodiment, a partition of said plurality of partitions includes (i) a B cell from said plurality of B cells, and (ii) a plurality of nucleic acid barcode molecules including a partition-specific barcode sequence.

[0164] In one additional embodiment, the method further includes generating in said partition a plurality of barcoded nucleic acid molecules using the first and/or second reporter oligonucleotide, and the plurality of nucleic acid barcode molecules. In other embodiments, the plurality of barcoded nucleic acid molecules includes a first barcoded nucleic acid molecule or a plurality of first barcoded nucleic acid molecules including a sequence of the first reporter oligonucleotide or a reverse complement thereof and the common barcode sequence or reserve complement thereof, and optionally using the first barcoded nucleic acid molecule to identify (i) the target antigen coupled to the first reporter oligonucleotide and/or (ii) the non-target antigen coupled to the second reporter oligonucleotide. In some embodiments, the methods of the disclosure further include generating a first barcoded nucleic acid molecule or a plurality of first barcoded nucleic acid molecules including a sequence of the first reporter oligonucleotide, or a reverse complement thereof, and the common barcode sequence or a reserve complement thereof, and optionally using the first barcoded nucleic acid molecule to identify the B cell as having bound to the target antigen coupled to the first reporter oligonucleotide. In some embodiments, the binding affinity is assessed based on the number of first barcoded nucleic acid molecules comprising (i) a sequence of the first reporter oligonucleotide or reverse complement thereof and (ii) the common barcode sequence or reverse complement thereof. In some embodiments, the methods of the disclosure further include generating, in the partition, a second barcoded nucleic acid molecule including a nucleic acid sequence encoding the antibody, or antigen-binding fragment thereof, produced by the B cell, and the common barcode sequence or reverse complement thereof, and optionally thereby identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen. In some embodiments, the plurality of barcoded nucleic acid molecules may further include a second barcoded nucleic acid molecule including a nucleic acid sequence encoding the antibody, or antigen-binding fragment thereof, produced by the B cell, and the common barcode sequence or reverse complement thereof. In these instances, the second barcoded nucleic acid molecule may facilitate the identification of a sequence of an antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen.

[0165] In some embodiments, the methods further include generating a third barcoded nucleic acid molecule or plurality of third barcoded nucleic acid molecules comprising a sequence of the second reporter oligonucleotide, or reverse complement thereof, and the common barcode sequence or reverse complement thereof, and optionally using the third barcoded nucleic acid molecule or plurality of third barcoded nucleic acid molecules to identify the B cell as having bound to the non-target antigen coupled to the second reporter oligonucleotide.

[0166] The methods provided herein may, optionally, include subsequent operations following the generation of barcoded nucleic acid molecules in the partition. These subsequent operations may further include amplification of the barcoded nucleic acid molecules. The amplification of the barcoded nucleic acid molecules may optionally be performed using primers that add additional functional sequences to the barcoded nucleic acid molecules. These subsequent operations may include further processing ( e.g ., shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)). These operations can occur in bulk (e.g, outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations. These subsequent operations may include determining sequences of the generated barcoded nucleic acid molecules. In the methods, the determining sequence of the second barcoded nucleic acid molecule may identify the antibody or antigen-binding fragment thereof expressed by the B cell in the partition in which the barcoded nucleic was generated. The determining the sequence of the first barcoded nucleic acid molecule may also assess the affinity of the antibody or antigen-binding fragment produced by the cell in the partition in which the barcoded nucleic was generated. In other embodiments, the method further includes determining a sequence of the third barcoded nucleic acid molecule. In some embodiments, the determining of the sequence of the third barcoded nucleic acid molecule is performed by sequencing. The determining the sequence of the third barcoded nucleic acid molecule may allow to identify the B cell as having bound to the non target antigen coupled to the second reporter oligonucleotide.

[0167] In other embodiments, the method further includes obtaining immune receptor information from the plurality of B cells. In one embodiment, the method includes generating a third barcoded nucleic acid molecule using a nucleic acid molecule encoding for an immune receptor and said plurality of nucleic acid barcode molecules. The nucleic acid molecule encoding for an immune receptor may be a messenger ribonucleic acid (mRNA) molecule. In another embodiment, the third barcoded nucleic acid molecule includes an immune receptor nucleic acid sequence from the B cell or complement thereof and the partition barcode sequence or complement thereof. Suitable methods, compositions, systems, and kits for single cell analysis of immune receptors and/or antigen binding are disclosed in US20180105808A1, US20180179590A1, US20190338353A1, and US20190367969A1.

[0168] In cases wherein the methods determine sequences that identify the antibody or antigen-binding fragment thereof expressed by the B cell of a partition, the sequences may be nucleic acid sequences encoding the antibody of the antigen-binding fragment thereof. The nucleic acid sequences may encode one or more of a complementarity determining region (CDR), a framework (FWR), a variable heavy chain domain (VH), or a variable light chain domain (VL) of the antibody or antigen-binding fragment thereof. Alternatively, if the methods determine sequences that identify the antibody or antigen-binding fragment thereof expressed by the cell of a partition, the sequences may be amino acid sequences of the antibody or antigen binding fragment thereof. The amino acid sequences may include a sequence of one or more of a CDR, FWR, VH or VL of the antibody or antigen-binding fragment thereof.

[0169] Accordingly, in a further embodiment, the method further includes determining the sequence of the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule to identify said B cell as expressing an antibody that has binding affinity for said target antigen, or to identify an antibody that has binding affinity for a target antigen. In these instances, the binding affinity can be determined based at least in part on the count, quantity, and/or proportion of antigen associated with a B cell expressing the antigen-binding molecule. For example, the count, quantity, and/or proportion of antigen associated with a B cell expressing the antigen-binding molecule can be determined based on the count and/or frequency of sequence reads and/or UMIs which correspond to the first barcoded nucleic acid molecule. [0170] In another embodiment, the method further includes determining the sequence of the third barcoded nucleic acid molecule. In some embodiments, the method includes identifying an antibody expressed by said B cell as having binding affinity for a target antigen based on a determination of (a) a sequence including (i) a sequence of the first or second reporter oligonucleotide, or a reverse complement thereof, and (ii) the common barcode sequence ( e.g ., partition-specific barcode sequence) or a reverse complement thereof, and another sequence including (i) a sequence of the second reporter sequence and (ii) the common barcode sequence (e.g., partition-specific barcode sequence), or a reverse complement thereof. In some embodiment, the identification of an antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen may include generating a second barcoded nucleic acid molecule including a nucleic acid sequence encoding the antibody, or antigen-binding fragment thereof, produced by the B cell, and the common barcode sequence (e.g, partition specific barcode sequence) or a reverse complement thereof.

[0171] Sequencing may be by performed by any of a variety of approaches, systems, or techniques, including next-generation sequencing (NGS) methods. Sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g, digital PCR and droplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-based singleplex methods, emulsion PCR), and/or isothermal amplification. Non-limiting examples of nucleic acid sequencing methods include Maxam-Gilbert sequencing and chain-termination methods, de novo sequencing methods including shotgun sequencing and bridge PCR, next- generation methods including Polony sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD™ sequencing, Ion Torrent semiconductor sequencing, Heli Scope single molecule sequencing, and SMRT® sequencing.

[0172] Further, sequence analysis of the nucleic acid molecules can be direct or indirect. Thus, the sequence analysis can be performed on a barcoded nucleic acid molecule or it can be a molecule which is derived therefrom (e.g, a complement thereof).

[0173] Other examples of methods for sequencing include, but are not limited to, DNA hybridization methods, restriction enzyme digestion methods, Sanger sequencing methods, ligation methods, and microarray methods. Additional examples of sequencing methods that can be used include targeted sequencing, single molecule real-time sequencing, exon sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole- genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid- phase sequencing, high-throughput sequencing, massively parallel signature sequencing, co amplification at lower denaturation temperature-PCR (COLD-PCR), sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, Solexa Genome Analyzer sequencing, MS-PET sequencing, whole transcriptome sequencing, and any combinations thereof.

[0174] In one additional embodiment, the method further includes generating in said partition a plurality of barcoded nucleic acid molecules using said first reporter oligonucleotide, said second reporter oligonucleotide, and said plurality of nucleic acid barcode molecules. In other embodiments, the plurality of barcoded nucleic acid molecules includes a first barcoded nucleic acid molecule including said first reporter sequence or complement thereof and said partition barcode sequence or complement thereof. The plurality of barcoded nucleic acid molecules may further include a second barcoded nucleic acid molecule including said second reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In a further embodiment, the plurality of barcoded nucleic acid molecules may further include a third barcoded nucleic acid molecule including said control reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In other embodiments, the method further includes obtaining immune receptor information from the plurality of B cells. In one embodiment, the method includes generating an additional barcoded nucleic acid molecule using a nucleic acid molecule encoding for an immune receptor and said plurality of nucleic acid barcode molecules. The nucleic acid molecule encoding for an immune receptor may be a messenger ribonucleic acid (mRNA) molecule. In another embodiment, the additional barcoded nucleic acid molecule includes an immune receptor nucleic acid sequence from the B cell or complement thereof and the partition barcode sequence or complement thereof.

[0175] In some embodiments, the plurality of barcoded nucleic acid molecules may further include a third barcoded nucleic acid molecule comprising said control reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In other embodiments, the method further comprises obtaining immune receptor information from the plurality of B cells. In one embodiment, the method comprises generating an additional barcoded nucleic acid molecule using a nucleic acid molecule encoding for an immune receptor and said plurality of nucleic acid barcode molecules. The nucleic acid molecule encoding for an immune receptor may be a messenger ribonucleic acid (mRNA) molecule. In another embodiment, the additional barcoded nucleic acid molecule comprises an immune receptor nucleic acid sequence from the B cell or complement thereof and the partition barcode sequence or complement thereof.

[0176] In certain embodiments, the partition-based methods of the disclosure use droplet- based partitions ( e.g ., droplets in an emulsion) or well-based partitions. In one embodiment, the plurality of partitions is a plurality of droplets (e.g., a plurality of droplets in an emulsion) or a plurality of wells. In other embodiments, the plurality of nucleic acid barcode molecules is coupled to a support. The support may be a bead, which is optionally a gel bead. In another embodiment, the plurality of nucleic acid barcode molecules is coupled to a support via a labile moiety. In other embodiments, the plurality of nucleic acid barcode molecules is releasably coupled to said support. The plurality of nucleic acid barcode molecules may be releasable from said support upon application of a stimulus. In one embodiment, the stimulus is selected from the group consisting of a thermal stimulus, an enzymatic stimulus, a photo stimulus, and a chemical stimulus. In another embodiment, the application of said stimulus results in one or more of (i) cleavage of a linkage between nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules and said bead, and (ii) degradation of said bead to release nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules from said bead. In one embodiment, the bead is provided in said partition, and wherein said nucleic acid barcode molecule is released from said bead within said partition.

CORONA VIRUSES

[0177] Coronaviruses (CoVs) are a family of large, enveloped, positive-sense single- stranded RNA viruses. They infect humans, other mammals and avian species, including livestock and companion animals (such as dogs, cats, chicken, cattle, pigs, and birds), and are therefore not only a challenge for public health but also a veterinary and economic concern. Coronaviruses include the genera of alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. The most common coronaviruses in clinical practice are 229E, OC43, NL63, and HKU1, which typically cause common cold symptoms in immunocompetent individuals. Other coronaviruses include severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2, which have emerged in the human population over the past 20 years and are highly pathogenic.

[0178] The initial steps of coronavirus infection involve the specific binding of the coronavirus spike (S) protein to the cellular entry receptors, which have been identified for several coronaviruses and include human aminopeptidase N (APN; HCoV-229E), angiotensin converting enzyme 2 (ACE2; HCoV-NL63, SARS-CoV and SARS-CoV-2) and dipeptidyl peptidase 4 (DPP4; MERS-CoV).

[0179] The sites of receptor binding domains (RBD) within the SI region (often referred to as S 1 subunit) of a coronavirus S protein vary depending on the virus, with some having the RBD at the C-terminus of SI. The S-protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and also governs the tissue tropism of the virus. Additional information regarding coronavirus biology, pathophysiology, diagnosis, and treatment can be found in recent reviews by V’kovski P. etal. (Nature Rev. Microbiol. Oct. 28, 2020) and Wiersinga WJ et al. (JAMA. 2020;324(8):782-793).

[0180] The amino acid sequence of full-length SARS-CoV-2 spike protein is exemplified by the amino acid sequence provided in SEQ ID NO: 1 and FIG. 11A. The term “CoV-S” as used herein includes protein variants of CoV spike protein isolated from different CoV isolates as well as recombinant CoV spike protein or a fragment thereof. CoV spike protein variants include CoV spike proteins with one or more substitutions, as exemplified by the amino acid sequence provided in SEQ ID NO: 2 and FIG. 11B.

Methods for identifying antibodies with binding affinity to a target antisen, e.g., a coronavirus spike protein CoV-S)

[0181] In described in more detail below, one aspect of the disclosure relates to new approaches and methods for the identification and characterization of antigen-binding molecules, e.g., antibodies and antigen-binding fragments. In some embodiments, these methods are used to identify antigen-binding molecules that are derived from B cells obtained from subjects who have been exposed to a coronavirus, by using single-cell immune profiling methodologies, so as to generate antibodies and antigen-binding fragments having a binding specificity for a coronavirus spike protein (CoV-S).

[0182] Some embodiments of the disclosure relate to methods for identifying an antibody having specific binding affinity for a target antigen relative to a non-target antigen, the methods including: (a) partitioning a sample comprising analyte carriers producing antigen-binding molecules and a plurality of antigens, wherein the plurality of antigens includes a target antigen and a non-target antigen, and wherein each of the antigens include a reporter oligonucleotide, and wherein the sample comprises at least one analyte carrier bound to the target antigen, and wherein the partitioning provides a partition including (i) the analyte carrier bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence; (c) identifying a sequence of at least one antigen-binding molecule produced by the analyte carrier that has been bound to the target antigen; and d) assessing the binding affinity of the antigen-binding molecule to the target antigen; and e) identifying the isolated antibody antigen-binding fragment as an antibody having a binding specificity for the target antigen if the barcoded antibody specifically binds to the target antigen .

[0183] Some embodiments of the disclosure relates to methods for identifying an antibody having binding affinity for a coronavirus spike protein (CoV-S), the methods including: (a) contacting a plurality of B cells obtained from a subject who has been exposed to a coronavirus with a plurality of antigens, wherein the plurality of antigens includes a CoV-S antigen and a non-CoV-S antigen, and wherein each of the antigens include a reporter oligonucleotide, wherein the contacting provides a B cell bound to a CoV-S antigen; (b) partitioning the B cell bound to the CoV-S antigen in a partition of a plurality of partitions, wherein the partitioning provides a partition including (i) the B cell bound to the CoV-S antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence; (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the CoV-S antigen; and d) assessing the binding affinity of the barcoded antibody or antigen-binding fragment to a CoV-S protein; and e) identifying the isolated antibody antigen-binding fragment as an antibody having a binding specificity for the CoV-S protein if the barcoded antibody specifically binds to the CoV-S protein.

[0184] Non-limiting exemplary embodiments of the methods for identifying an antibody having binding affinity for a CoV-S protein as described herein can include one or more of the following features. In some embodiments, the reporter oligonucleotide includes (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence. In some embodiments, the methods of the disclosure further include coupling a barcode moiety to the antibody or antigen binding fragment produced by the B cell to generate a barcoded antibody or antigen-binding fragment. In some embodiments, a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further includes a capture sequence configured to couple to the capture handle sequence of the reporter oligonucleotide and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further includes a capture sequence configured to couple to an mRNA analyte. In some embodiments, the capture sequence is configured to couple to the capture handle sequence of the reporter oligonucleotide is complementary to the capture handle sequence of the reporter oligonucleotide. In some embodiments, the capture sequence configured to couple to an mRNA analyte includes a polyT sequence. In some embodiments, the first and second nucleic acid barcode molecules each include a unique molecule identifier (UMI).

[0185] In some embodiments, the antigens are each coupled to a fluorescent label identifying the antigens. In some embodiments, the methods for identifying an antibody having binding affinity for a CoV-S protein as described herein further include isolating and/or enriching the plurality of B cells prior to (b). In some embodiments, the enrichment further includes sorting of the B cells bound to the CoV-S antigen and/or non-CoV-S antigen based on detection of one or more of the fluorescent labels coupled to the antigens. In some embodiments, the CoV-S protein is coupled to a barcode moiety.

[0186] In certain embodiments, the disclosure provides methods for identifying a B cell as expressing an antibody that has binding affinity for a CoV-S antigen or a non-CoV-S antigen, or for identifying an antibody that has binding affinity for a CoV-S antigen or a non-CoV-S antigen. In one embodiment, the method comprises contacting a plurality of B cells with a plurality of antigens. The plurality of antigens may include at least two antigens that are different from one another, e.g. , a first antigen and a second antigen, wherein the first antigen is a different type of antigen than the second antigen. For example, the plurality of antigens may include, without limitation, (i) antigens that are the same, (ii) antigens that are different, (iii) a CoV-S antigen and a control antigen (e.g., a non-CoV-S antigen) or (iv) a first CoV-S antigen and a second CoV-S antigen which are different types of CoV-S antigens. In another embodiment, the plurality of B cells is obtained from a subject (or obtained from a sample that was obtained from a subject) who has been exposed to a coronavirus.

[0187] In an additional embodiment, the method further comprises partitioning the plurality of B cells into a plurality of partitions, which can be a plurality of droplets in an emulsion or a plurality of wells. In a further embodiment, a partition of said plurality of partitions comprises a B cell from said plurality of B cells, and a plurality of nucleic acid barcode molecules comprising a partition barcode sequence. In one embodiment, the B cell in said partition comprises (i) a surface-bound CoV-S antigen comprising a CoV-S reporter oligonucleotide having a CoV-S reporter sequence and (ii) a surface-bound control antigen having a control reporter oligonucleotide that comprises a control reporter sequence.

[0188] In one additional embodiment, the method further comprises generating in said partition a plurality of barcoded nucleic acid molecules using said CoV-S reporter oligonucleotide, said control reporter oligonucleotide, and said plurality of nucleic acid barcode molecules. In other embodiments, the plurality of barcoded nucleic acid molecules comprises a first barcoded nucleic acid molecule comprising said CoV-S reporter sequence or complement thereof and said partition barcode sequence or complement thereof. The plurality of barcoded nucleic acid molecules may further comprise a second barcoded nucleic acid molecule comprising said control reporter sequence or complement thereof and said partition barcode sequence or complement thereof.

[0189] In other embodiments, the method further comprises obtaining immune receptor information from the plurality of B cells. In one embodiment, the method comprises generating a third barcoded nucleic acid molecule using a nucleic acid molecule encoding for an immune receptor and said plurality of nucleic acid barcode molecules. The nucleic acid molecule encoding for an immune receptor may be a messenger ribonucleic acid (mRNA) molecule. In another embodiment, the third barcoded nucleic acid molecule comprises an immune receptor nucleic acid sequence from the B cell or complement thereof and the partition barcode sequence or complement thereof. Suitable methods, compositions, systems, and kits for single cell analysis of immune receptors and/or antigen binding are disclosed in US20180105808A1, US20180179590A1, US20190338353A1, and US20190367969A1, each of which are incorporated by reference herein in their entirety.

[0190] In a further embodiment, the method further comprises determining the sequence of said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule to identify said B cell as expressing an antibody that has binding affinity for said CoV-S, or to identify an antibody that has binding affinity for a CoV-S antigen or a non-CoV-S antigen. In another embodiment, the method further comprises determining the sequence of the third barcoded nucleic acid molecule. In some embodiments, the method comprises identifying an antibody expressed by said B cell as having binding affinity for a CoV-S antigen or a non-CoV-S antigen based on a determination of (a) a sequence comprising the CoV-S reporter sequence and the partition barcode sequence or complement thereof, and another sequence comprising the control reporter sequence and the partition barcode sequence or complement thereof; and (b) a sequence comprising the CoV-S reporter sequence and the partition barcode sequence or complement thereof, another sequence comprising the control reporter sequence and the partition barcode sequence or complement thereof; and an additional sequence corresponding to an immune receptor and the partition barcode sequence or complement thereof.

[0191] In other embodiments, the disclosure provides methods for identifying a B cell as expressing an antibody that is cross-reactive against more than one antigen, or for identifying an antibody that is cross-reactive against more than one antigen. In one embodiment, the method comprises contacting a plurality of B cells with a plurality of antigens. The plurality of antigens may include at least two antigens that are different from one another, e.g. , a first antigen and a second antigen, wherein the first antigen is a different type of antigen than the second antigen.

For example, the plurality of antigens may include, without limitation, (i) antigens that are different or (ii) a first antigen and a second antigen which are different types of antigens. The plurality of antigens may further comprise a control antigen (e.g., an antigen that is unrelated to the first or second antigens). For example, where the first and second antigen are viral protein antigens, the control antigen is a non-viral protein antigen. In another embodiment, the plurality of B cells is obtained from a subject (or obtained from a sample that was obtained from a subject) who has been exposed to a pathogen (e.g, a virus).

[0192] In an additional embodiment, the method further comprises partitioning the plurality of B cells into a plurality of partitions, which can be a plurality of droplets in an emulsion or a plurality of wells. In a further embodiment, a partition of said plurality of partitions comprises a B cell from said plurality of B cells, and a plurality of nucleic acid barcode molecules comprising a partition barcode sequence. In one embodiment, the B cell in said partition comprises (i) a first surface-bound antigen comprising a first reporter oligonucleotide having a first reporter sequence and (ii) a second surface-bound antigen comprising a second reporter oligonucleotide having a second reporter sequence. In one additional embodiment, the B cell in the partition further comprises a surface-bound control antigen having a control reporter oligonucleotide that comprises a control reporter sequence.

[0193] In one additional embodiment, the method further comprises generating in said partition a plurality of barcoded nucleic acid molecules using said first reporter oligonucleotide, said second reporter oligonucleotide, and said plurality of nucleic acid barcode molecules. In other embodiments, the plurality of barcoded nucleic acid molecules comprises a first barcoded nucleic acid molecule comprising said first reporter sequence or complement thereof and said partition barcode sequence or complement thereof. The plurality of barcoded nucleic acid molecules may further comprise a second barcoded nucleic acid molecule comprising said second reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In a further embodiment, the plurality of barcoded nucleic acid molecules may further comprise a third barcoded nucleic acid molecule comprising said control reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In other embodiments, the method further comprises obtaining immune receptor information from the plurality of B cells. In one embodiment, the method comprises generating an additional barcoded nucleic acid molecule using a nucleic acid molecule encoding for an immune receptor and said plurality of nucleic acid barcode molecules. The nucleic acid molecule encoding for an immune receptor may be a messenger ribonucleic acid (mRNA) molecule. In another embodiment, the additional barcoded nucleic acid molecule comprises an immune receptor nucleic acid sequence from the B cell or complement thereof and the partition barcode sequence or complement thereof.

[0194] In a further embodiment, the method further comprises determining the sequence of said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule to identify said B cell as expressing an antibody that is cross-reactive against more than one antigen, or to identify an antibody that is cross-reactive against more than one antigen. In another embodiment, the method further comprises determining the sequence of the third barcoded nucleic acid molecule. In some embodiments, the method comprises identifying an antibody expressed by said B cell as cross-reactive against more than one antigen based on a determination of a sequence comprising the first reporter sequence and the partition barcode sequence or complement thereof, and another sequence comprising the second reporter sequence and the partition barcode sequence or complement thereof. The determination may further comprise determination of (i) a sequence comprising the control reporter sequence and the partition barcode sequence or complement thereof, and/or (ii) an additional sequence corresponding to an immune receptor and the partition barcode sequence or complement thereof.

[0195] In other embodiments, the disclosure provides methods for identifying a B cell as expressing an antibody that is cross-reactive against more than one antigen, or for identifying an antibody that is cross-reactive against more than one antigen. In one embodiment, the method comprises contacting a plurality of B cells with a plurality of antigens. The plurality of antigens may include at least two antigens that are different from one another, e.g. , a first antigen and a second antigen, wherein the first antigen is a different type of antigen than the second antigen.

For example, the plurality of antigens may include, without limitation, (i) antigens that are different or (ii) a first antigen and a second antigen which are different types of antigens. The plurality of antigens may further comprise a control antigen (e.g., a non-CoV-S antigen). In another embodiment, the plurality of B cells is obtained from a subject (or obtained from a sample that was obtained from a subject) who has been exposed to a coronavirus.

[0196] In an additional embodiment, the method further comprises partitioning the plurality of B cells into a plurality of partitions, which can be a plurality of droplets in an emulsion or a plurality of wells. In a further embodiment, a partition of said plurality of partitions comprises a B cell from said plurality of B cells, and a plurality of nucleic acid barcode molecules comprising a partition barcode sequence. In one embodiment, the B cell in said partition comprises (i) a first surface-bound CoV-S antigen comprising a first CoV-S reporter oligonucleotide having a first CoV-S reporter sequence and (ii) a second surface-bound CoV-S antigen comprising a second CoV-S reporter oligonucleotide having a second CoV-S reporter sequence. In one additional embodiment, the B cell in the partition further comprises a surface- bound control antigen having a control reporter oligonucleotide that comprises a control reporter sequence.

[0197] In one additional embodiment, the method further comprises generating in said partition a plurality of barcoded nucleic acid molecules using said first CoV-S reporter oligonucleotide, said second CoV-S reporter oligonucleotide, and said plurality of nucleic acid barcode molecules. In other embodiments, the plurality of barcoded nucleic acid molecules comprises a first barcoded nucleic acid molecule comprising said first CoV-S reporter sequence or complement thereof and said partition barcode sequence or complement thereof. The plurality of barcoded nucleic acid molecules may further comprise a second barcoded nucleic acid molecule comprising said second CoV-S reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In a further embodiment, the plurality of barcoded nucleic acid molecules may further comprise a third barcoded nucleic acid molecule comprising said control reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In other embodiments, the method further comprises obtaining immune receptor information from the plurality of B cells. In one embodiment, the method comprises generating an additional barcoded nucleic acid molecule using a nucleic acid molecule encoding for an immune receptor and said plurality of nucleic acid barcode molecules. The nucleic acid molecule encoding for an immune receptor may be a messenger ribonucleic acid (mRNA) molecule. In another embodiment, the additional barcoded nucleic acid molecule comprises an immune receptor nucleic acid sequence from the B cell or complement thereof and the partition barcode sequence or complement thereof.

[0198] In a further embodiment, the method further comprises determining the sequence of said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule to identify said B cell as expressing an antibody that is cross-reactive against more than one CoV-S antigen, or to identify an antibody that is cross-reactive against more than one CoV-S antigen. In another embodiment, the method further comprises determining the sequence of the third barcoded nucleic acid molecule. In some embodiments, the method comprises identifying an antibody expressed by said B cell as cross-reactive against more than one CoV-S antigen based on a determination of a sequence comprising the first CoV-S reporter sequence and the partition barcode sequence or complement thereof, and another sequence comprising the second CoV-S reporter sequence and the partition barcode sequence or complement thereof. The determination may further comprise determination of (i) a sequence comprising the control reporter sequence and the partition barcode sequence or complement thereof, and/or (ii) an additional sequence corresponding to an immune receptor and the partition barcode sequence or complement thereof.

[0199] In certain embodiments, the partition-based compositions and methods use droplet- based partitions ( e.g ., droplets in an emulsion) or well-based partitions. In one embodiment, the plurality of partitions is a plurality of droplets (e.g., a plurality of droplets in an emulsion) or a plurality of wells. In other embodiments, the plurality of nucleic acid barcode molecules is coupled to a support. The support may be a bead, which is optionally a gel bead. In another embodiment, the plurality of nucleic acid barcode molecules is coupled to a support via a labile moiety. In other embodiments, the plurality of nucleic acid barcode molecules is releasably coupled to said support. The plurality of nucleic acid barcode molecules may be releasable from said support upon application of a stimulus. In one embodiment, the stimulus is selected from the group consisting of a thermal stimulus, an enzymatic stimulus, a photo stimulus, and a chemical stimulus. In another embodiment, the application of said stimulus results in one or more of (i) cleavage of a linkage between nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules and said bead, and (ii) degradation of said bead to release nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules from said bead. In one embodiment, the bead is provided in said partition, and wherein said nucleic acid barcode molecule is released from said bead within said partition.

Systems and methods for partitioning

[0200] In some aspects, such as those that have been described above, the methods provided herein include a step of partitioning, or include a step of generating barcoded nucleic acid molecules, or may include an additional processing step(s). This description sets forth examples, embodiments and characteristics of steps of the methods and of reagents useful in the methods.

[0201] In an aspect, the systems and methods described herein provide for the compartmentalization, depositing, or partitioning of one or more particles (e.g, biological particles, macromolecular constituents of biological particles, beads, reagents, etc.) into discrete compartments or partitions (referred to interchangeably herein as partitions), where each partition maintains separation of its own contents from the contents of other partitions.

[0202] In some embodiments disclosed herein, the partitioned particle is a labelled cell of B-cell lineage, e.g. a plasma cell, which expresses an antibody. In some embodiments, the labelled cell of B-cell lineage is a B cell which expresses an antigen-binding molecule (e.g, an immune receptor, an antibody or a functional fragment thereof) on its surface. In other examples, the partitioned particle can be a labelled cell engineered to express (antigen-binding molecules (e.g, an immune receptors, antibodies or functional fragments thereof).

[0203] The term “partition,” as used herein, generally, refers to a space or volume that can be suitable to contain one or more cells, one or more species of features or compounds, or conduct one or more reactions. A partition can be a physical container, compartment, or vessel, such as a droplet, a flow cell, a reaction chamber, a reaction compartment, a tube, a well, or a microwell. In some embodiments, the compartments or partitions include partitions that are flowable within fluid streams. These partitions can include, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core, or, in some cases, the partitions can include a porous matrix that is capable of entraining and/or retaining materials within its matrix. In some aspects, partitions comprise droplets of aqueous fluid within a non-aqueous continuous phase ( e.g ., oil phase). A variety of different vessels are described in, for example, U.S. Patent Application Publication No. 2014/0155295. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in detail in, e.g., U.S. Patent Application Publication No. 2010/010511.

[0204] In some embodiments, a partition herein includes a space or volume that can be suitable to contain one or more species or conduct one or more reactions. A partition can be a physical compartment, such as a droplet or well. The partition can be an isolated space or volume from another space or volume. The droplet can be a first phase (e.g, aqueous phase) in a second phase (e.g, oil) immiscible with the first phase. The droplet can be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition can include one or more other (inner) partitions. In some cases, a partition can be a virtual compartment that can be defined and identified by an index (e.g, indexed libraries) across multiple and/or remote physical compartments. For example, a physical compartment can include a plurality of virtual compartments.

[0205] In some embodiments, the methods and system described herein provide for the compartmentalization, depositing or partitioning of individual cells from a sample material containing cells after at least one labelling agent or reporter agent molecule has been bound to a cell surface feature of a cell, into discrete partitions, where each partition maintains separation of its own contents from the contents of other partitions. Identifiers including unique identifiers (e.g, UMI) and common or universal tags, e.g, barcodes, can be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to one or more particular compartments. Further, identifiers including unique identifiers and common or universal tags, e.g., barcodes, can be coupled to labelling agents and previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to one or more particular compartments. Identifiers including unique identifiers and common or universal tags, e.g, barcodes, can be delivered, for example on an oligonucleotide, to a partition via any suitable mechanism, for example by coupling the barcoded oligonucleotides to a microcapsule, e.g. , bead. In some embodiments, the barcoded oligonucleotides are reversibly (e.g., releasably) coupled to a microcapsule (e.g, bead). The microcapsule suitable for the compositions and methods of the disclosure can have different surface chemistries and/or physical volumes. In some embodiments, the microcapsule includes a polymer gel. In some embodiments, the polymer gel is a polyacrylamide. Additional non-limiting examples of suitable microcapsule include microparticles, nanoparticles, and beads (e.g, microbeads). In some embodiments, the microcapsule includes a bead. The partition can be a droplet in an emulsion. A partition can include one or more particles. A partition can include one or more types of particles. For example, a partition of the present disclosure can include one or more biological particles, e.g, labelled B cells or plasma cells, and/or macromolecular constituents thereof. A partition can include one or more gel beads. A partition can include one or more cell beads. A partition can include a single gel bead, a single cell bead, or both a single cell bead and single gel bead. A partition can include one or more reagents. Alternatively, a partition can be unoccupied. For example, a partition cannot comprise a bead. Unique identifiers, such as barcodes, can be injected into the droplets previous to, subsequent to, or concurrently with droplet generation, such as via a microcapsule (e.g, bead), as described elsewhere herein. Microfluidic channel networks (e.g, on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.

[0206] The partitions can be flowable within fluid streams. The partitions can include, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core. In some cases, the partitions can include a porous matrix that is capable of entraining and/or retaining materials (e.g, expressed antibodies or antigen binding fragments thereof) within its matrix (e.g, via a capture agent configured to couple to both the matrix and the expressed antibody or antigen binding fragment thereof). The partitions can be droplets of a first phase within a second phase, wherein the first and second phases are immiscible. For example, the partitions can be droplets of aqueous fluid within a non-aqueous continuous phase ( e.g ., oil phase). In another example, the partitions can be droplets of a non-aqueous fluid within an aqueous phase. In some examples, the partitions can be provided in a water-in-oil emulsion or oil-in-water emulsion. A variety of different vessels is described in, for example, U.S. Patent Application Publication No. 2014/0155295. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in, for example, U.S. Patent Application Publication No. 2010/0105112.

[0207] In the case of droplets in an emulsion, allocating individual particles (e.g., labelled B cells or plasma cells) to discrete partitions can, in one non-limiting example, be accomplished by introducing a flowing stream of particles in an aqueous fluid into a flowing stream of a non- aqueous fluid, such that droplets are generated at the junction of the two streams. Fluid properties (e.g, fluid flow rates, fluid viscosities, etc.), particle properties (e.g, volume fraction, particle size, particle concentration, etc.), microfluidic architectures (e.g, channel geometry, etc.), and other parameters can be adjusted to control the occupancy of the resulting partitions (e.g, number of biological particles per partition, number of beads per partition, etc.). For example, partition occupancy can be controlled by providing the aqueous stream at a certain concentration and/or flow rate of particles. To generate single biological particle partitions, the relative flow rates of the immiscible fluids can be selected such that, on average, the partitions can contain less than one biological particle per partition in order to ensure that those partitions that are occupied are primarily singly occupied. In some cases, partitions among a plurality of partitions can contain at most one biological particle (e.g, bead, DNA, cell, such as a labelled B cell or plasma cell, or cellular material). In some embodiments, the various parameters (e.g, fluid properties, particle properties, microfluidic architectures, etc.) can be selected or adjusted such that a majority of partitions are occupied, for example, allowing for only a small percentage of unoccupied partitions. The flows and channel architectures can be controlled as to ensure a given number of singly occupied partitions, less than a certain level of unoccupied partitions and/or less than a certain level of multiply occupied partitions.

[0208] In some embodiments, the method further includes individually partitioning one or more single tumor cells from the second tumor sample in a partition of a second plurality of partitions. In some embodiments, the method further includes individually partitioning one or more single cells from a plurality of cells ( e.g ., from a second sample) in a partition of a second plurality of partitions.

[0209] In some embodiments, at least one of the first and second plurality of partitions includes a microwell, a flow cell, a reaction chamber, a reaction compartment, or a droplet. In some embodiments, at least one of the first and second plurality of partitions includes individual droplets in emulsion. In some embodiments, the partitions of the first plurality and/or the second plurality of partition have the same reaction volume.

[0210] In the case of droplets in emulsion, allocating individual cells to discrete partitions can generally be accomplished by introducing a flowing stream of cells in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams. By providing the aqueous cell-containing stream at a certain concentration of cells, the occupancy of the resulting partitions (e.g., number of cells per partition) can be controlled. For example, where single cell partitions are desired, the relative flow rates of the fluids can be selected such that, on average, the partitions contain less than one cell per partition, in order to ensure that those partitions that are occupied, are primarily singly occupied. In some embodiments, the relative flow rates of the fluids can be selected such that a majority of partitions are occupied, e.g, allowing for only a small percentage of unoccupied partitions. In some embodiments, the flows and channel architectures are controlled as to ensure a desired number of singly occupied partitions, less than a certain level of unoccupied partitions and less than a certain level of multiply occupied partitions.

[0211] In some embodiments, the methods described herein can be performed such that a majority of occupied partitions include no more than one cell per occupied partition. In some embodiments, the partitioning process is performed such that fewer than 25%, fewer than 20%, fewer than 15%, fewer than 10%, fewer than 5%, fewer than 2%, or fewer than 1% the occupied partitions contain more than one cell. In some embodiments, fewer than 20% of the occupied partitions include more than one cell. In some embodiments, fewer than 10% of the occupied partitions include more than one cell per partition. In some embodiments, fewer than 5% of the occupied partitions include more than one cell per partition. In some embodiments, it is desirable to avoid the creation of excessive numbers of empty partitions. For example, from a cost perspective and/or efficiency perspective, it may be desirable to minimize the number of empty partitions. While this can be accomplished by providing sufficient numbers of cells into the partitioning zone, the Poissonian distribution can optionally be used to increase the number of partitions that include multiple cells. As such, in some embodiments described herein, the flow of one or more of the cells, or other fluids directed into the partitioning zone are performed such that no more than 50% of the generated partitions, no more than 25% of the generated partitions, or no more than 10% of the generated partitions are unoccupied. Further, in some aspects, these flows are controlled so as to present non-Poissonian distribution of single occupied partitions while providing lower levels of unoccupied partitions. Restated, in some aspects, the above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in some embodiments, the use of the systems and methods described herein creates resulting partitions that have multiple occupancy rates of less than 25%, less than 20%, less than 15%), less than 10%, and in some embodiments, less than 5%, while having unoccupied partitions of less than 50%), less than 40%, less than 30%, less than 20%, less than 10%, and in some embodiments, less than 5%.

[0212] Although described in terms of providing substantially singly occupied partitions, above, in some embodiments, the methods as described herein include providing multiply occupied partitions, e.g., containing two, three, four or more cells and/or microcapsules (e.g, beads) comprising nucleic acid barcode molecules within a single partition.

[0213] In some embodiments, the reporter oligonucleotides contained within a partition are distinguishable from the reporter oligonucleotides contained within other partitions of the plurality of partitions. This can be accomplished by incorporating one or more partition-specific barcode sequences into the reporter barcode sequence of the reporter oligonucleotides contained within the partition.

[0214] In some embodiments, it may be desirable to incorporate multiple different barcode sequences within a given partition, either attached to a single or multiple beads within the partition. For example, in some cases, a mixed, but known barcode sequences set can provide greater assurance of identification in the subsequent processing, e.g, by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.

Microfluidic channel structures

[0215] Microfluidic channel networks (e.g, on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.

[0216] FIG. 1 shows an example of a microfluidic channel structure 100 for partitioning individual biological particles. The channel structure 100 can include channel segments 102,

104, 106 and 108 communicating at a channel junction 110. In operation, a first aqueous fluid 112 that includes suspended biological particles ( e.g ., cells, for example, labelled B cells, memory B cells, or plasma cells) 114 can be transported along channel segment 102 into junction 110, while a second fluid 116 that is immiscible with the aqueous fluid 112 is delivered to the junction 110 from each of channel segments 104 and 106 to create discrete droplets 118, 120 of the first aqueous fluid 112 flowing into channel segment 108, and flowing away from junction 110. The channel segment 108 can be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested. A discrete droplet generated can include an individual biological particle 114 (such as droplets 118). A discrete droplet generated can include more than one individual biological particle (e.g., labelled B cell, e.g. , memory B cell, or plasma cell) 114 (not shown in FIG. 1). A discrete droplet can contain no biological particle 114 (such as droplet 120). Each discrete partition can maintain separation of its own contents (e.g, individual biological particle 114) from the contents of other partitions.

[0217] The second fluid 116 can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 118, 120. Examples of particularly useful partitioning fluids and fluorosurfactants are described, for example, in U.S. Patent Application Publication No. 2010/0105112.

[0218] As will be appreciated, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structure 100 can have other geometries. For example, a microfluidic channel structure can have more than one channel junction. For example, a microfluidic channel structure can have 2, 3, 4, or 5 channel segments each carrying particles (e.g, biological particles, cell beads, and/or gel beads) that meet at a channel junction. Fluid can be directed to flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can comprise compressors ( e.g ., providing positive pressure), pumps (e.g, providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.

[0219] The generated droplets can include two subsets of droplets: (1) occupied droplets 118, containing one or more biological particles 114, e.g, labelled engineered cells, labelled B cells, memory B cells, or plasma cells, and (2) unoccupied droplets 120, not containing any biological particles 114. Occupied droplets 118 can include singly occupied droplets (having one biological particle, such as one labelled B cell, memory B cells, or plasma cell) and multiply occupied droplets (having more than one biological particle, such as multiple engineered cells, labelled B cells, memory B cells, or plasma cells). As described elsewhere herein, in some cases, the majority of occupied partitions can include no more than one biological particle, e.g, labelled B cell, e.g. , memory B cell, or plasma cell, per occupied partition and some of the generated partitions can be unoccupied (of any biological particle, or labelled engineered cell, labelled B cell, memory B cell, or plasma cell). In some cases, though, some of the occupied partitions can include more than one biological particle, e.g, labelled engineered cell, labelled B cell, memory B cell, or plasma cell. In some cases, the partitioning process can be controlled such that fewer than about 25% of the occupied partitions contain more than one biological particle, and in many cases, fewer than about 20% of the occupied partitions have more than one biological particle, while in some cases, fewer than about 10% or even fewer than about 5% of the occupied partitions include more than one biological particle per partition.

[0220] In some cases, it can be desirable to minimize the creation of excessive numbers of empty partitions, such as to reduce costs and/or increase efficiency. While this minimization can be achieved by providing a sufficient number of biological particles (e.g, biological particles, such as labelled engineered cell, labelled B cells, memory B cells, or plasma cells 114) at the partitioning junction 110, such as to ensure that at least one biological particle is encapsulated in a partition, the Poissonian distribution can expectedly increase the number of partitions that include multiple biological particles. As such, where singly occupied partitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated partitions can be unoccupied.

[0221] In some cases, the flow of one or more of the biological particles, such as B cells, e.g, memory B cells, or plasma cells, ( e.g. , in channel segment 102), or other fluids directed into the partitioning junction (e.g, in channel segments 104, 106) can be controlled such that, in many cases, no more than about 50% of the generated partitions, no more than about 25% of the generated partitions, or no more than about 10% of the generated partitions are unoccupied.

These flows can be controlled so as to present a non-Poissonian distribution of single-occupied partitions while providing lower levels of unoccupied partitions. The above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in many cases, the use of the systems and methods described herein can create resulting partitions that have multiple occupancy rates of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases, less than about 5%, while having unoccupied partitions of less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less.

[0222] As will be appreciated, the above-described occupancy rates are also applicable to partitions that include both biological particles (e.g, labelled B cells or plasma cells) and additional reagents, including, but not limited to, microcapsules or beads (e.g, gel beads) carrying barcoded nucleic acid molecules (e.g, nucleic acid barcode molecules or barcoded oligonucleotides) (described in relation to FIGS. 1 and 2). The occupied partitions (e.g, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied partitions) can include both a microcapsule (e.g, bead) comprising barcoded nucleic acid nucleic acid molecules (e.g, nucleic acid barcode molecules) and a biological particle.

[0223] FIG. 8 shows an example of a microfluidic channel structure 800 for delivering barcode carrying beads to droplets. The channel structure 800 can include channel segments 801, 802, 804, 806 and 808 communicating at a channel junction 810. In operation, the channel segment 801 may transport an aqueous fluid 812 that includes a plurality of beads 814 (e.g, with nucleic acid molecules, e.g, nucleic acid barcode molecules or barcoded oligonucleotides, molecular tags) along the channel segment 801 into junction 810. The plurality of beads 814 may be sourced from a suspension of beads. For example, the channel segment 801 may be connected to a reservoir comprising an aqueous suspension of beads 814. The channel segment 802 may transport the aqueous fluid 812 that includes a plurality of biological particles 816 along the channel segment 802 into junction 810. The plurality of biological particles 816 may be sourced from a suspension of biological particles. For example, the channel segment 802 may be connected to a reservoir comprising an aqueous suspension of biological particles 816. In some instances, the aqueous fluid 812 in either the first channel segment 801 or the second channel segment 802, or in both segments, can include one or more reagents, as further described below. A second fluid 818 that is immiscible with the aqueous fluid 812 ( e.g ., oil) can be delivered to the junction 810 from each of channel segments 804 and 806. Upon meeting of the aqueous fluid 812 from each of channel segments 801 and 802 and the second fluid 818 from each of channel segments 804 and 806 at the channel junction 810, the aqueous fluid 812 can be partitioned as discrete droplets 1420 in the second fluid 818 and flow away from the junction 810 along channel segment 808. The channel segment 808 may deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 808, where they may be harvested. As an alternative, the channel segments 801 and 802 may meet at another junction upstream of the junction 810. At such junction, beads and biological particles may form a mixture that is directed along another channel to the junction 810 to yield droplets 820. The mixture may provide the beads and biological particles in an alternating fashion, such that, for example, a droplet comprises a single bead and a single biological particle.

[0224] In another aspect, in addition to or as an alternative to droplet based partitioning, biological particles (e.g., cells) can be encapsulated within a microcapsule that comprises an outer shell, layer or porous matrix in which is entrained one or more individual biological particles or small groups of biological particles. In another aspect, in addition to or as an alternative to droplet-based partitioning, biological particles (e.g, cells) may be encapsulated within a particulate material to form a “cell bead.” In another aspect, in addition to or as an alternative to droplet-based partitioning, biological particles (e.g, cells) may be comprised within a particulate material to form a “cell bead.”

[0225] The microcapsule or cell bead can include other reagents. Encapsulation of biological particles, e.g, labelled engineered cell, B cells, memory B cells, or plasma cells, can be performed by a variety of processes. Such processes can combine an aqueous fluid containing the biological particles with a polymeric precursor material that can be capable of being formed into a gel or other solid or semi-solid matrix upon application of a particular stimulus to the polymer precursor. Such stimuli can include, for example, thermal stimuli (e.g, either heating or cooling), photo-stimuli (e.g, through photo-curing), chemical stimuli (e.g, through crosslinking, polymerization initiation of the precursor (e.g, through added initiators)), mechanical stimuli, or a combination thereof.

[0226] Preparation of microcapsules comprising biological particles, e.g., labelled engineered cells, B cells, memory B cells, or plasma cells, can be performed by a variety of methods. For example, air knife droplet or aerosol generators can be used to dispense droplets of precursor fluids into gelling solutions in order to form microcapsules or cell beads that include individual biological particles or small groups of biological particles (e.g, labelled B cells or plasma cells). Likewise, membrane based encapsulation systems can be used to generate microcapsules or cell beads comprising encapsulated biological particles (e.g, B cells or plasma cells) as described herein. Microfluidic systems of the present disclosure, such as that shown in FIG. 1, can be readily used in encapsulating biological particles (e.g, cells) as described herein. Exemplary methods for encapsulating biological particles (e.g, cells) are also further described in U.S. Patent Application Pub. No. US 2015/0376609 and PCT Pub. No. WO2018140966A1. In particular, and with reference to FIG. 1, the aqueous fluid 112 comprising (i) the biological particles (e.g, labelled B cells or plasma cells) 114 and (ii) the polymer precursor material (not shown) is flowed into channel junction 110, where it is partitioned into droplets 118, 120 through the flow of non-aqueous fluid 116. In the case of encapsulation methods, non-aqueous fluid 116 can also include an initiator (not shown) to cause polymerization and/or crosslinking of the polymer precursor to form the microcapsule (e.g, bead) that includes the entrained biological particles. Examples of polymer precursor/initiator pairs include those described in U.S. Patent Application Publication No. 2014/0378345.

[0227] For example, in the case where the polymer precursor material comprises a linear polymer material, such as a linear polyacrylamide, PEG, or other linear polymeric material, the activation agent can include a cross-linking agent, or a chemical that activates a cross-linking agent within the formed droplets. Likewise, for polymer precursors that comprise polymerizable monomers, the activation agent can include a polymerization initiator. For example, in certain cases, where the polymer precursor comprises a mixture of acrylamide monomer with a N,N’- bis-(acryloyl)cystamine (BAC) comonomer, an agent such as tetraethylmethylenediamine (TEMED) can be provided within the second fluid streams 116 in channel segments 104 and 106, which can initiate the copolymerization of the acrylamide and BAC into a cross-linked polymer network, or hydrogel.

[0228] Upon contact of the second fluid stream 116 with the first fluid stream 112 at junction 110, during formation of droplets, the TEMED can diffuse from the second fluid 116 into the aqueous fluid 112 comprising the linear polyacrylamide, which will activate the crosslinking of the polyacrylamide within the droplets 118, 120, resulting in the formation of gel (e.g, hydrogel) microcapsules or cell beads, as solid or semi-solid beads or particles entraining the cells (e.g, labelled B cells or plasma cells) 114. Although described in terms of polyacrylamide encapsulation, other “activatable” encapsulation compositions can also be employed in the context of the methods and compositions described herein. For example, formation of alginate droplets followed by exposure to divalent metal ions (e.g, Ca²⁺ ions), can be used as an encapsulation process using the described processes. Likewise, agarose droplets can also be transformed into capsules through temperature based gelling (e.g, upon cooling, etc.).

[0229] In some cases, encapsulated biological particles can be selectively releasable from the microcapsule or cell bead, such as through passage of time or upon application of a particular stimulus, that degrades the encapsulating material (e.g, microcapsule) sufficiently to allow the biological particles (e.g, labelled B cells or plasma cells), or its other contents to be released from the encapsulating material, such as into a partition (e.g, droplet). For example, in the case of the polyacrylamide polymer described above, degradation of the polymer can be accomplished through the introduction of an appropriate reducing agent, such as DTT or the like, to cleave disulfide bonds that cross-link the polymer matrix. See, for example, U.S. Patent Application Publication No. 2014/0378345.

[0230] The biological particle (e.g, labelled B cell, memory B cell, or plasma cell), can be subjected to other conditions sufficient to polymerize or gel the precursors. The conditions sufficient to polymerize or gel the precursors can include exposure to heating, cooling, electromagnetic radiation, and/or light. The conditions sufficient to polymerize or gel the precursors can include any conditions sufficient to polymerize or gel the precursors. Following polymerization or gelling, a polymer or gel can be formed around the biological particle (e.g, labelled B cell or plasma cell). The polymer or gel can be diffusively permeable to chemical or biochemical reagents. The polymer or gel can be diffusively impermeable to macromolecular constituents (e.g, secreted antibodies or antigen binding fragments thereof) of the biological particle (e.g, labelled B cell, memory B cell, or plasma cell). In this manner, the polymer or gel can act to allow the biological particle (e.g, labelled B cell, memory B cell, or plasma cell) to be subjected to chemical or biochemical operations while spatially confining the macromolecular constituents to a region of the droplet defined by the polymer or gel. The polymer or gel can include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG- alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin. The polymer or gel can include any other polymer or gel.

[0231] The polymer or gel can be functionalized ( e.g ., coupled to a capture agent) to bind to targeted analytes (e.g., secreted antibodies or antigen binding fragment thereof), such as nucleic acids, proteins, carbohydrates, lipids or other analytes. The polymer or gel can be polymerized or gelled via a passive mechanism. The polymer or gel can be stable in alkaline conditions or at elevated temperature. The polymer or gel can have mechanical properties similar to the mechanical properties of the bead. For instance, the polymer or gel can be of a similar size to the bead. The polymer or gel can have a mechanical strength (e.g. tensile strength) similar to that of the bead. The polymer or gel can be of a lower density than an oil. The polymer or gel can be of a density that is roughly similar to that of a buffer. The polymer or gel can have a tunable pore size. The pore size can be chosen to, for instance, retain denatured nucleic acids. The pore size can be chosen to maintain diffusive permeability to exogenous chemicals such as sodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors. The polymer or gel can be biocompatible. The polymer or gel can maintain or enhance cell viability. The polymer or gel can be biochemically compatible. The polymer or gel can be polymerized and/or depolymerized thermally, chemically, enzymatically, and/or optically.

[0232] The polymer can include poly(acrylamide-co-acrylic acid) crosslinked with disulfide linkages. The preparation of the polymer can include a two-step reaction. In the first activation step, poly(acrylamide-co-acrylic acid) can be exposed to an acylating agent to convert carboxylic acids to esters. For instance, the poly(acrylamide-co-acrylic acid) can be exposed to 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). The polyacrylamide-co-acrylic acid can be exposed to other salts of 4-(4,6-dimethoxy-l,3,5-triazin-2- yl)-4-methylmorpholinium. In the second cross-linking step, the ester formed in the first step can be exposed to a disulfide crosslinking agent. For instance, the ester can be exposed to cystamine (2,2’-dithiobis(ethylamine)). Following the two steps, the biological particle can be surrounded by polyacrylamide strands linked together by disulfide bridges. In this manner, the biological particle can be encased inside of or comprise a gel or matrix ( e.g ., polymer matrix) to form a “cell bead.” A cell bead can contain biological particles (e.g., labelled B cell, memory B cell, or plasma cell) or macromolecular constituents (e.g, RNA, DNA, proteins, secreted antibodies or antigen binding fragments thereof etc.) of biological particles. A cell bead can include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example, after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads. Systems and methods disclosed herein can be applicable to both (i) cell beads (and/or droplets or other partitions) containing biological particles and (ii) cell beads (and/or droplets or other partitions) containing macromolecular constituents of biological particles.

[0233] Encapsulated biological particles (e.g, labelled B cells, memory B cell, or plasma cells) can provide certain potential advantages of being more storable and more portable than droplet-based partitioned biological particles. Furthermore, in some cases, it can be desirable to allow biological particles (e.g, labelled B cell, memory B cell, or plasma cell) to incubate for a select period of time before analysis, such as in order to characterize changes in such biological particles over time, either in the presence or absence of different stimuli (e.g, cytokines, antigens, etc.). In such cases, encapsulation can allow for longer incubation than partitioning in emulsion droplets, although in some cases, droplet partitioned biological particles can also be incubated for different periods of time, e.g, at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, or at least 10 hours or more. The encapsulation of biological particles (e.g, labelled B cells, memory B cells, or plasma cells) can constitute the partitioning of the biological particles into which other reagents are co-partitioned. Alternatively or in addition, encapsulated biological particles can be readily deposited into other partitions (e.g., droplets) as described above.

Microwells

[0234] As described herein, one or more processes can be performed in a partition, which can be a well. The well can be a well of a plurality of wells of a substrate, such as a microwell of a microwell array or plate, or the well can be a microwell or microchamber of a device (e.g, microfluidic device) comprising a substrate. The well can be a well of a well array or plate, or the well can be a well or chamber of a device (e.g, fluidic device). Accordingly, the wells or microwells can assume an “open” configuration, in which the wells or microwells are exposed to the environment ( e.g ., contain an open surface) and are accessible on one planar face of the substrate, or the wells or microwells can assume a “closed” or “sealed” configuration, in which the microwells are not accessible on a planar face of the substrate. In some instances, the wells or microwells can be configured to toggle between “open” and “closed” configurations. For instance, an “open” microwell or set of microwells can be “closed” or “sealed” using a membrane (e.g., semi-permeable membrane), an oil (e.g, fluorinated oil to cover an aqueous solution), or a lid, as described elsewhere herein. The wells or microwells can be initially provided in a “closed” or “sealed” configuration, wherein they are not accessible on a planar surface of the substrate without an external force. For instance, the “closed” or “sealed” configuration can include a substrate such as a sealing film or foil that is puncturable or pierceable by pipette tip(s). Suitable materials for the substrate include, without limitation, polyester, polypropylene, polyethylene, vinyl, and aluminum foil.

[0235] In some embodiments, the well can have a volume of less than 1 milliliter (mL). For example, the well can be configured to hold a volume of at most 1000 microliters (pL), at most 100 pL, at most 10 pL, at most 1 pL, at most 100 nanoliters (nL), at most 10 nL, at most 1 nL, at most 100 picoliters (pL), at most 10 (pL), or less. The well can be configured to hold a volume of about 1000 pL, about 100 pL, about 10 pL, about 1 pL, about 100 nL, about 10 nL, about 1 nL, about 100 pL, about 10 pL, etc. The well can be configured to hold a volume of at least 10 pL, at least 100 pL, at least 1 nL, at least 10 nL, at least 100 nL, at least 1 pL, at least 10 pL, at least 100 pL, at least 1000 pL, or more. The well can be configured to hold a volume in a range of volumes listed herein, for example, from about 5 nL to about 20 nL, from about 1 nL to about 100 nL, from about 500 pL to about 100 pL, etc. The well can be of a plurality of wells that have varying volumes and can be configured to hold a volume appropriate to accommodate any of the partition volumes described herein.

[0236] In some instances, a microwell array or plate includes a single variety of microwells. In some instances, a microwell array or plate includes a variety of microwells. For instance, the microwell array or plate can include one or more types of microwells within a single microwell array or plate. The types of microwells can have different dimensions (e.g, length, width, diameter, depth, cross-sectional area, etc.), shapes (e.g, circular, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, etc.), aspect ratios, or other physical characteristics. The microwell array or plate can include any number of different types of microwells. For example, the microwell array or plate can include 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800

900, 1000 or more different types of microwells. A well can have any dimension (e.g, length, width, diameter, depth, cross-sectional area, volume, etc.), shape (e.g, circular, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, other polygonal, etc.), aspect ratios, or other physical characteristics described herein with respect to any well.

[0237] In certain instances, the microwell array or plate includes different types of microwells that are located adjacent to one another within the array or plate. For example, a microwell with one set of dimensions can be located adjacent to and in contact with another microwell with a different set of dimensions. Similarly, microwells of different geometries can be placed adjacent to or in contact with one another. The adjacent microwells can be configured to hold different articles; for example, one microwell can be used to contain a cell, cell bead, or other sample (e.g, cellular components, nucleic acid molecules, nucleic acid barcode molecules, etc.) while the adjacent microwell can be used to contain a microcapsule, droplet, bead, or other reagent. In some cases, the adjacent microwells can be configured to merge the contents held within, e.g, upon application of a stimulus, or spontaneously, upon contact of the articles in each microwell.

[0238] As is described elsewhere herein, a plurality of partitions can be used in the systems, compositions, and methods described herein. For example, any suitable number of partitions ( e.g ., wells or droplets) can be generated or otherwise provided. For example, in the case when wells are used, at least about 1,000 wells, at least about 5,000 wells, at least about 10,000 wells, at least about 50,000 wells, at least about 100,000 wells, at least about 500,000 wells, at least about 1,000,000 wells, at least about 5,000,000 wells at least about 10,000,000 wells, at least about 50,000,000 wells, at least about 100,000,000 wells, at least about 500,000,000 wells, at least about 1,000,000,000 wells, or more wells can be generated or otherwise provided. Moreover, the plurality of wells can include both unoccupied wells (e.g., empty wells) and occupied wells.

[0239] A well can include any of the reagents described herein, or combinations thereof. These reagents can include, for example, barcode molecules, enzymes, adapters, and combinations thereof. The reagents can be physically separated from a sample (for example, a cell, cell bead, or cellular components, e.g., proteins, nucleic acid molecules, etc.) that is placed in the well. This physical separation can be accomplished by containing the reagents within, or coupling to, a microcapsule or bead that is placed within a well. The physical separation can also be accomplished by dispensing the reagents in the well and overlaying the reagents with a layer that is, for example, dissolvable, meltable, or permeable prior to introducing the polynucleotide sample into the well. This layer can be, for example, an oil, wax, membrane (e.g, semi- permeable membrane), or the like. The well can be sealed at any point, for example, after addition of the microcapsule or bead, after addition of the reagents, or after addition of either of these components. The sealing of the well can be useful for a variety of purposes, including preventing escape of beads or loaded reagents from the well, permitting select delivery of certain reagents (e.g, via the use of a semi-permeable membrane), for storage of the well prior to or following further processing, etc.

[0240] Once sealed, the well may be subjected to conditions for further processing of a cell (or cells) in the well. For instance, reagents in the well may allow further processing of the cell, e.g., cell lysis, as further described herein. Alternatively, the well (or wells such as those of a well-based array) comprising the cell (or cells) may be subjected to freeze-thaw cycling to process the cell (or cells), e.g., cell lysis. The well containing the cell may be subjected to freezing temperatures (e.g., 0°C, below 0°C, -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, - 40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -80°C, or -85°C). Freezing may be performed in a suitable manner, e.g., sub-zero freezer or a dry ice/ethanol bath. Following an initial freezing, the well (or wells) comprising the cell (or cells) may be subjected to freeze-thaw cycles to lyse the cell (or cells). In one embodiment, the initially frozen well (or wells) are thawed to a temperature above freezing (e.g., 4°C or above, 8°C or above, 12°C or above, 16°C or above, 20°C or above, room temperature, or 25°C or above). In another embodiment, the freezing is performed for less than 10 minutes (e.g., 5 minutes or 7 minutes) followed by thawing at room temperature for less than 10 minutes (e.g., 5 minutes or 7 minutes). This freeze-thaw cycle may be repeated a number of times, e.g., 2, 3, 4 or more times, to obtain lysis of the cell (or cells) in the well (or wells). In one embodiment, the freezing, thawing and/or freeze/thaw cycling is performed in the absence of a lysis buffer. Additional disclosure related to freeze-thaw cycling is provided in WO2019165181A1, which is incorporated herein by reference in its entirety. [0241] A well can include free reagents and/or reagents encapsulated in, or otherwise coupled to or associated with, microcapsules, beads, or droplets. In some embodiments, any of the reagents described in this disclosure can be encapsulated in, or otherwise coupled to, a microcapsule, droplet, or bead, with any chemicals, particles, and elements suitable for sample processing reactions involving biomolecules, such as, but not limited to, nucleic acid molecules and proteins. For example, a bead or droplet used in a sample preparation reaction for DNA sequencing can include one or more of the following reagents: enzymes, restriction enzymes (e.g, multiple cutters), ligase, polymerase, fluorophores, oligonucleotide barcodes, adapters, buffers, nucleotides (e.g, dNTPs, ddNTPs) and the like.

[0242] Additional examples of reagents include, but are not limited to: buffers, acidic solution, basic solution, temperature-sensitive enzymes, pH-sensitive enzymes, light-sensitive enzymes, metals, metal ions, magnesium chloride, sodium chloride, manganese, aqueous buffer, mild buffer, ionic buffer, inhibitor, enzyme, protein, polynucleotide, antibodies, saccharides, lipid, oil, salt, ion, detergents, ionic detergents, non-ionic detergents, oligonucleotides, nucleotides, deoxyribonucleotide triphosphates (dNTPs), dideoxyribonucleotide triphosphates (ddNTPs), DNA, RNA, peptide polynucleotides, complementary DNA (cDNA), double stranded DNA (dsDNA), single stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA, polymerase, ligase, restriction enzymes, proteases, nucleases, protease inhibitors, nuclease inhibitors, chelating agents, reducing agents, oxidizing agents, fluorophores, probes, chromophores, dyes, organics, emulsifiers, surfactants, stabilizers, polymers, water, small molecules, pharmaceuticals, radioactive molecules, preservatives, antibiotics, aptamers, and pharmaceutical drug compounds. As described herein, one or more reagents in the well can be used to perform one or more reactions, including but not limited to: cell lysis, cell fixation, permeabilization, nucleic acid reactions, e.g, nucleic acid extension reactions, amplification, reverse transcription, transposase reactions (e.g, tagmentation), etc.

[0243] The wells disclosed herein can be provided as a part of a kit. For example, a kit can include instructions for use, a microwell array or device, and reagents (e.g, beads). The kit can include any useful reagents for performing the processes described herein, e.g, nucleic acid reactions, barcoding of nucleic acid molecules, sample processing (e.g, for cell lysis, fixation, and/or permeabilization).

[0244] In some cases, a well includes a microcapsule, bead, or droplet that includes a set of reagents that has a similar attribute, for example, a set of enzymes, a set of minerals, a set of oligonucleotides, a mixture of different barcode molecules, a mixture of identical barcode molecules. In other cases, a microcapsule, bead, or droplet includes a heterogeneous mixture of reagents. In some cases, the heterogeneous mixture of reagents can include all components necessary to perform a reaction. In some cases, such mixture can include all components necessary to perform a reaction, except for 1, 2, 3, 4, 5, or more components necessary to perform a reaction. In some cases, such additional components are contained within, or otherwise coupled to, a different microcapsule, droplet, or bead, or within a solution within a partition ( e.g ., microwell) of the system.

[0245] A non-limiting example of a microwell array in accordance with some embodiments of the disclosure is schematically presented in FIG. 5. In this example, the array can be contained within a substrate 500. The substrate 500 includes a plurality of wells 502. The wells 502 can be of any size or shape, and the spacing between the wells, the number of wells per substrate, as well as the density of the wells on the substrate 500 can be modified, depending on the particular application. In one such example application, a sample molecule 506, which can include a cell or cellular components (e.g., nucleic acid molecules) is co-partitioned with a bead 504, which can include a nucleic acid barcode molecule coupled thereto. The wells 502 can be loaded using gravity or other loading technique (e.g, centrifugation, liquid handler, acoustic loading, optoelectronic, etc.). In some instances, at least one of the wells 502 contains a single sample molecule 506 (e.g, cell) and a single bead 504.

[0246] Reagents can be loaded into a well either sequentially or concurrently. In some cases, reagents are introduced to the device either before or after a particular operation. In some cases, reagents (which can be provided, in certain instances, in microcapsules, droplets, or beads) are introduced sequentially such that different reactions or operations occur at different steps.

The reagents (or microcapsules, droplets, or beads) can also be loaded at operations interspersed with a reaction or operation step. For example, microcapsules (or droplets or beads) including reagents for fragmenting polynucleotides (e.g, restriction enzymes) and/or other enzymes (e.g, transposases, ligases, polymerases, etc.) can be loaded into the well or plurality of wells, followed by loading of microcapsules, droplets, or beads including reagents for attaching nucleic acid barcode molecules to a sample nucleic acid molecule. Reagents can be provided concurrently or sequentially with a sample, e.g., a cell or cellular components (e.g, organelles, proteins, nucleic acid molecules, carbohydrates, lipids, etc.). Accordingly, use of wells can be useful in performing multi-step operations or reactions.

[0247] As described elsewhere herein, the nucleic acid barcode molecules and other reagents can be contained within a microcapsule, bead, or droplet. These microcapsules, beads, or droplets can be loaded into a partition (e.g, a microwell) before, after, or concurrently with the loading of a cell, such that each cell is contacted with a different microcapsule, bead, or droplet. This technique can be used to attach a unique nucleic acid barcode molecule to nucleic acid molecules obtained from each cell. Alternatively or in addition, the sample nucleic acid molecules can be attached to a support. For example, the partition (e.g, microwell) can include a bead which has coupled thereto a plurality of nucleic acid barcode molecules. The sample nucleic acid molecules, or derivatives thereof, can couple or attach to the nucleic acid barcode molecules attached on the support. The resulting barcoded nucleic acid molecules can then be removed from the partition, and in some instances, pooled and sequenced. In such cases, the nucleic acid barcode sequences can be used to trace the origin of the sample nucleic acid molecule. For example, polynucleotides with identical barcodes can be determined to originate from the same cell or partition, while polynucleotides with different barcodes can be determined to originate from different cells or partitions.

[0248] The samples or reagents can be loaded in the wells or microwells using a variety of approaches. For example, the samples (e.g, a cell, cell bead, or cellular component) or reagents (as described herein) can be loaded into the well or microwell using an external force, e.g, gravitational force, electrical force, magnetic force, or using mechanisms to drive the sample or reagents into the well, for example, via pressure-driven flow, centrifugation, optoelectronics, acoustic loading, electrokinetic pumping, vacuum, capillary flow, etc. In certain cases, a fluid handling system can be used to load the samples or reagents into the well. The loading of the samples or reagents can follow a Poissonian distribution or a non-Poissonian distribution, e.g, super Poisson or sub-Poisson. The geometry, spacing between wells, density, and size of the microwells can be modified to accommodate a useful sample or reagent distribution; for example, the size and spacing of the microwells can be adjusted such that the sample or reagents can be distributed in a super-Poissonian fashion. [0249] In one non-limiting example, the microwell array or plate includes pairs of microwells, in which each pair of microwells is configured to hold a droplet ( e.g ., including a single cell) and a single bead (such as those described herein, which can, in some instances, also be encapsulated in a droplet). The droplet and the bead (or droplet containing the bead) can be loaded simultaneously or sequentially, and the droplet and the bead can be merged, e.g., upon contact of the droplet and the bead, or upon application of a stimulus (e.g, external force, agitation, heat, light, magnetic or electric force, etc.). In some cases, the loading of the droplet and the bead is super-Poissonian. In other examples of pairs of microwells, the wells are configured to hold two droplets including different reagents and/or samples, which are merged upon contact or upon application of a stimulus. In such instances, the droplet of one microwell of the pair can include reagents that can react with an agent in the droplet of the other microwell of the pair. For example, one droplet can include reagents that are configured to release the nucleic acid barcode molecules of a bead contained in another droplet, located in the adjacent microwell. Upon merging of the droplets, the nucleic acid barcode molecules can be released from the bead into the partition (e.g, the microwell or microwell pair that are in contact), and further processing can be performed (e.g, barcoding, nucleic acid reactions, etc.). In cases where intact or live cells are loaded in the microwells, one of the droplets can include lysis reagents for lysing the cell upon droplet merging.

[0250] In some embodiments, a droplet or microcapsule can be partitioned into a well. The droplets can be selected or subjected to pre-processing prior to loading into a well. For instance, the droplets can include cells, and only certain droplets, such as those containing a single cell (or at least one cell), can be selected for use in loading of the wells. Such a pre-selection process can be useful in efficient loading of single cells, such as to obtain a non-Poissonian distribution, or to pre-filter cells for a selected characteristic prior to further partitioning in the wells. Additionally, the technique can be useful in obtaining or preventing cell doublet or multiplet formation prior to or during loading of the microwell.

[0251] In some embodiments, the wells can include nucleic acid barcode molecules attached thereto. The nucleic acid barcode molecules can be attached to a surface of the well (e.g, a wall of the well). The nucleic acid barcode molecules may be attached to a droplet or bead that has been partitioned into the well. The nucleic acid barcode molecule (e.g, a partition barcode sequence) of one well can differ from the nucleic acid barcode molecule of another well, which can permit identification of the contents contained with a single partition or well. In some embodiments, the nucleic acid barcode molecule can include a spatial barcode sequence that can identify a spatial coordinate of a well, such as within the well array or well plate. In some embodiments, the nucleic acid barcode molecule can include a unique molecular identifier for individual molecule identification. In some instances, the nucleic acid barcode molecules can be configured to attach to or capture a nucleic acid molecule from or within a sample or cell distributed in the well. For example, the nucleic acid barcode molecules can include a capture sequence that can be used to capture or hybridize to a nucleic acid molecule ( e.g ., RNA, DNA) from or within the sample. In some embodiments, the nucleic acid barcode molecules can be releasable from the microwell. In some instances, the nucleic acid barcode molecules may be releasable from the bead or droplet. For example, the nucleic acid barcode molecules can include a chemical cross-linker which can be cleaved upon application of a stimulus (e.g., photo-, magnetic, chemical, biological, stimulus). The released nucleic acid barcode molecules, which can be hybridized or configured to hybridize to a sample nucleic acid molecule, can be collected and pooled for further processing, which can include nucleic acid processing (e.g, amplification, extension, reverse transcription, etc.) and/or characterization (e.g, sequencing). In some instances nucleic acid barcode molecules attached to a bead in a well may be hybridized to sample nucleic acid molecules, and the bead with the sample nucleic acid molecules hybridized thereto may be collected and pooled for further processing, which can include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and/or characterization (e.g., sequencing). In such cases, the unique partition barcode sequences can be used to identify the cell or partition from which a nucleic acid molecule originated.

[0252] Characterization of samples within a well can be performed. Such characterization can include, in non-limiting examples, imaging of the sample (e.g, cell, cell bead, or cellular components) or derivatives thereof. Characterization techniques such as microscopy or imaging can be useful in measuring sample profiles in fixed spatial locations. For example, when cells are partitioned, optionally with beads, imaging of each microwell and the contents contained therein can provide useful information on cell doublet formation (e.g, frequency, spatial locations, etc.), cell-bead pair efficiency, cell viability, cell size, cell morphology, expression level of a biomarker (e.g, a surface marker, a fluorescently labeled molecule therein, etc.), cell or bead loading rate, number of cell-bead pairs, etc. In some instances, imaging can be used to characterize live cells in the wells, including, but not limited to: dynamic live-cell tracking, cell cell interactions (when two or more cells are co-partitioned), cell proliferation, etc. Alternatively or in addition to, imaging can be used to characterize a quantity of amplification products in the well.

[0253] In operation, a well can be loaded with a sample and reagents, simultaneously or sequentially. When cells or cell beads are loaded, the well can be subjected to washing, e.g., to remove excess cells from the well, microwell array, or plate. Similarly, washing can be performed to remove excess beads or other reagents from the well, microwell array, or plate. In the instances where live cells are used, the cells can be lysed in the individual partitions to release the intracellular components or cellular analytes. Alternatively, the cells can be fixed or permeabilized in the individual partitions. The intracellular components or cellular analytes can couple to a support, e.g, on a surface of the microwell, on a solid support (e.g., bead), or they can be collected for further downstream processing. For example, after cell lysis, the intracellular components or cellular analytes can be transferred to individual droplets or other partitions for barcoding. Alternatively, or in addition, the intracellular components or cellular analytes (e.g, nucleic acid molecules) can couple to a bead including a nucleic acid barcode molecule; subsequently, the bead can be collected and further processed, e.g, subjected to nucleic acid reaction such as reverse transcription, amplification, or extension, and the nucleic acid molecules thereon can be further characterized, e.g, via sequencing. Alternatively, or in addition, the intracellular components or cellular analytes can be barcoded in the well (e.g, using a bead including nucleic acid barcode molecules that are releasable or on a surface of the microwell including nucleic acid barcode molecules). The barcoded nucleic acid molecules or analytes can be further processed in the well, or the barcoded nucleic acid molecules or analytes can be collected from the individual partitions and subjected to further processing outside the partition. Further processing can include nucleic acid processing (e.g, performing an amplification, extension) or characterization (e.g, fluorescence monitoring of amplified molecules, sequencing). At any convenient, suitable, and/or useful step, the well (or microwell array or plate) can be sealed (e.g, using an oil, membrane, wax, etc.), which enables storage of the assay or selective introduction of additional reagents.

Beads

[0254] In some embodiments of the disclosure, a partition can include one or more unique identifiers, such as barcodes ( e.g ., a plurality of barcode nucleic acid molecules, also referred to herein as nucleic acid barcode molecules which can be or include, for example, a plurality of partition barcode sequences). Barcodes can be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned biological particle (e.g., labelled B cell, memory B cell, or plasma cell). For example, barcodes can be injected into droplets previous to, subsequent to, or concurrently with droplet generation. In some embodiments, the delivery of the barcodes to a particular partition allows for the later attribution of the characteristics of the individual biological particle (e.g, labelled B cell, memory B cell, or plasma cell) to the particular partition. Barcodes can be delivered, for example on a nucleic acid molecule (e.g, a barcoded oligonucleotide, nucleic acid barcode molecule), to a partition via any suitable mechanism. In some embodiments, barcoded nucleic acid molecules, e.g. , nucleic acid barcode molecules can be delivered to a partition via a microcapsule. A microcapsule, in some instances, can include a bead. Beads are described in further detail below.

[0255] In some embodiments, barcodes (e.g, barcoded nucleic acid molecules, nucleic acid barcode molecules) can be initially associated with the microcapsule and then released from the microcapsule. In some embodiments, release of the barcoded nucleic acid molecules ,e.g, nucleic acid barcode molecules can be passive (e.g, by diffusion out of the microcapsule). In addition or alternatively, release from the microcapsule can be upon application of a stimulus which allows the barcoded nucleic acid nucleic acid molecules to dissociate or to be released from the microcapsule. Such stimulus can disrupt the microcapsule, an interaction that couples the barcoded nucleic acid molecules to or within the microcapsule, or both. Such stimulus can include, for example, a thermal stimulus, photo-stimulus, chemical stimulus (e.g, change in pH or use of a reducing agent), a mechanical stimulus, a radiation stimulus, a biological stimulus (e.g, enzyme), or any combination thereof. Methods and systems for partitioning barcode carrying beads into droplets are provided in US. Patent Publication Nos. 2019/0367997 and 2019/0064173, and PCT Publication Nos. WO2020167862 and WO2020176882.

[0256] Beneficially, a discrete droplet partitioning a biological particle and a barcode carrying bead can effectively allow the attribution of the barcode to macromolecular constituents of the biological particle within the partition. The contents of a partition can remain discrete from the contents of other partitions.

[0257] In operation, the barcoded oligonucleotides can be released (e.g, in a partition), as described elsewhere herein. Alternatively, the nucleic acid molecules bound to the bead ( e.g ., gel bead) can be used to hybridize and capture analytes (e.g., one or more types of analytes) on the solid phase of the bead.

[0258] In some examples, beads, biological particles (e.g., labelled B cells, memory B cells, or plasma cells) and droplets can flow along channels (e.g., the channels of a microfluidic device), in some cases at substantially regular flow profiles (e.g., at regular flow rates). Such regular flow profiles can permit a droplet to include a single bead and a single biological particle. Such regular flow profiles can permit the droplets to have an occupancy (e.g, droplets having beads and biological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Such regular flow profiles and devices that can be used to provide such regular flow profiles are provided in, for example, U.S. Patent Publication No. 2015/0292988.

[0259] A bead can be porous, non-porous, solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof. In some instances, a bead can be dissolvable, disruptable, and/or degradable. Degradable beads, as well as methods for degrading beads, are described in PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety. In some cases, any combination of stimuli, e.g., stimuli described in PCT Publication No. WO2014210353 and US Patent Application Pub. No. 2015/0376609, hereby incorporated by reference in its entirety, may trigger degradation of a bead. For example, a change in pH may enable a chemical agent (e.g., DTT) to become an effective reducing agent.

[0260] In some cases, a bead cannot be degradable. In some cases, the bead can be a gel bead. A gel bead can be a hydrogel bead. A gel bead can be formed from molecular precursors, such as a polymeric or monomeric species. A semi-solid bead can be a liposomal bead. Solid beads can include metals including iron oxide, gold, and silver. In some cases, the bead can be a silica bead. In some cases, the bead can be rigid. In other cases, the bead can be flexible and/or compressible.

[0261] A bead can be of any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.

[0262] Beads can be of uniform size or heterogeneous size. In some cases, the diameter of a bead can be at least about 10 nanometers (nm), 100 nm, 500 nm, 1 micrometer (pm), 5pm, 10pm, 20pm, 30pm, 40pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 250pm, 500pm, 1mm, or greater. In some cases, a bead can have a diameter of less than about 10 nm, 100 nm, 500 nm, lpm, 5pm, IOmih, 20mih, 30mih, 40mih, 50mih, 60mm, 70mm, 80mm, 90mm, IOOmih, 250mm, 500mm, 1mm, or less. In some cases, a bead can have a diameter in the range of about 40-75pm, 30-75pm, 20-75pm, 40-85pm, 40-95pm, 20-100pm, 10-100pm, l-lOOpm, 20-250pm, or 20- 500pm.

[0263] In certain aspects, beads can be provided as a population or plurality of beads having a relatively monodisperse size distribution. Where it may be desirable to provide relatively consistent amounts of reagents within partitions, maintaining relatively consistent bead characteristics, such as size, can contribute to the overall consistency. In some embodiments, the beads described herein can have size distributions that have a coefficient of variation in their cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, less than 5%, or less.

[0264] A bead can include natural and/or synthetic materials. For example, a bead can include a natural polymer, a synthetic polymer or both natural and synthetic polymers. See, e.g., PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety. Beads can also be formed from materials other than polymers, including lipids, micelles, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and others.

[0265] In some embodiments, the bead can include covalent or ionic bonds between polymeric precursors (e.g., monomers, oligomers, linear polymers), nucleic acid molecules (e.g., nucleic acid barcode molecules or barcoded oligonucleotides), primers, and other entities. In some embodiments, the covalent bonds can be carbon-carbon bonds, thioether bonds, or carbon- heteroatom bonds.

[0266] In some embodiments, a bead can include an acrydite moiety, which in certain aspects can be used to attach one or more nucleic acid molecules (e.g., barcode sequence, barcoded nucleic acid molecule, nucleic acid barcode molecule, barcoded oligonucleotide, primer, or other oligonucleotide) to the bead. Acrydite moieties, as well as their uses in attaching nucleic acid molecules to beads, are described in PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety.

[0267] For example, precursors (e.g, monomers, cross-linkers) that are polymerized to form a bead can include acrydite moieties, such that when a bead is generated, the bead also includes acrydite moieties. The acrydite moieties can be attached to a nucleic acid molecule (e.g., oligonucleotide such as nucleic acid barcode molecule), which can include a priming sequence (e.g, a primer for amplifying target nucleic acids, random primer, primer sequence for messenger RNA) and/or one or more barcode sequences. The one or more barcode sequences can include sequences that are the same for all nucleic acid molecules coupled to a given bead (e.g, nucleic acid barcode molecules coupled to a given bead) and/or sequences that are different across all nucleic acid molecules coupled to the given bead (e.g, nucleic acid barcode molecules coupled to a given bead). The nucleic acid molecule (e.g, nucleic acid barcode molecule) can be incorporated into the bead.

[0268] In some embodiments, the nucleic acid molecule (e.g, nucleic acid barcode molecule) can include a functional sequence, e.g, for use in downstream sequencing methodologies, for example, a functional sequence for attachment to a sequencing flow cell, such as, for example, a P5 sequence for Illumina® sequencing. In some embodiments, the nucleic acid barcode molecule can include adapters for compatibility with other sequencing platforms. Non limiting examples of nucleic acid sequencing methods include Maxam-Gilbert sequencing and chain-termination methods, de novo sequencing methods including shotgun sequencing and bridge PCR, next-generation methods including Polony sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD™ sequencing, Ion Torrent semiconductor sequencing, HeliScope single molecule sequencing, and SMRT® sequencing.

[0269] Other examples of methods for sequencing nucleic acids include, but are not limited to, DNA hybridization methods, restriction enzyme digestion methods, Sanger sequencing methods, ligation methods, and microarray methods. Additional examples of sequencing methods that can be used include targeted sequencing, single molecule real-time sequencing, exon sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, co-amplification at lower denaturation temperature-PCR (COLD-PCR), sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, Solexa Genome Analyzer sequencing, MS-PET sequencing, and any combinations thereof.

[0270] Accordingly, a wide variety of different approaches, systems, and techniques for nucleic acid sequencing, including next-generation sequencing (NGS) methods, can be used to determine the nucleic acid sequences encoding the antibodies produced by the partitioned single cells. Generally, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) ( e.g ., digital PCR and droplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-based singleplex methods, emulsion PCR), and/or isothermal amplification. In some embodiments, the nucleic acid barcode molecule can include adapters for compatibility with long read sequencing platforms such as the PacBio SMRT-seq platform and nanopore sequencing.

[0271] In some embodiments, the nucleic acid molecule (e.g., nucleic acid barcode molecule) or derivative thereof (e.g, oligonucleotide or polynucleotide generated from the nucleic acid barcode molecule) can include another functional sequence, such as, for example, a P7 sequence for attachment to a sequencing flow cell for Illumina sequencing. In some cases, the nucleic acid barcode molecule can include a barcode sequence. In some cases, the nucleic acid barcode molecule or primer can further include a unique molecular identifier (UMI). In some cases, the nucleic acid barcode molecule or primer can include an R1 primer sequence for Illumina sequencing. In some cases, the nucleic acid barcode molecule or primer can include an R2 primer sequence for Illumina sequencing. Examples of such nucleic acid molecules (e.g, oligonucleotides, polynucleotides, etc.) and uses thereof, as can be used with compositions, devices, methods and systems of the present disclosure, are provided in U.S. Patent Pub. Nos. 2014/0378345 and 2015/0376609.

[0272] FIG. 3 illustrates an example of a barcode carrying bead. A nucleic acid molecule (e.g, nucleic acid barcode molecule, barcoded nucleic acid molecule) 302, such as an oligonucleotide, can be coupled to a bead 304 by a releasable linkage 306, such as, for example, a disulfide linker. The same bead 304 can be coupled (e.g, via releasable linkage) to one or more other nucleic acid molecules (e.g, other nucleic acid barcode molecules) 318, 320. The nucleic acid molecule 302 can be or include a barcode. As noted elsewhere herein, the structure of the barcode can include a number of sequence elements. The nucleic acid molecule 302 can include a functional sequence 308 that can be used in subsequent processing. For example, the functional sequence 308 can include one or more of a sequencer specific flow cell attachment sequence (e.g, a P5 sequence for Illumina® sequencing systems) and a sequencing primer sequence ( e.g. , a R1 primer for Illumina® sequencing systems). The nucleic acid molecule 302 can include a barcode sequence 310 for use in barcoding the sample (e.g, DNA, RNA, protein, etc.). In some cases, the barcode sequence 310 can be bead-specific such that the barcode sequence 310 is common to all nucleic acid molecules (e.g, including nucleic acid molecule 302 ) coupled to the same bead 304. Alternatively or in addition, the barcode sequence 310 can be partition- specific such that the barcode sequence 310 is common to all nucleic acid molecules coupled to one or more beads that are partitioned into the same partition. The nucleic acid molecule 302 can include a specific priming sequence 312, such as an mRNA specific priming sequence (e.g, poly-T sequence), a targeted priming sequence, and/or a random priming sequence. The nucleic acid molecule 302 can include an anchoring sequence 314 to ensure that the specific priming sequence 312 hybridizes at the sequence end (e.g, of the mRNA). For example, the anchoring sequence 314 can include a random short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longer sequence, which can ensure that a poly-T segment is more likely to hybridize at the sequence end of the poly-A tail of the mRNA.

[0273] The nucleic acid molecule 302 can include a unique molecular identifying sequence 316 (e.g, unique molecular identifier (UMI)). In some cases, the unique molecular identifying sequence 316 can include from about 5 to about 8 nucleotides. Alternatively, the unique molecular identifying sequence 316 can compress less than about 5 or more than about 8 nucleotides. The unique molecular identifying sequence 316 can be a unique sequence that varies across individual nucleic acid molecules (e.g, 302, 318, 320, etc.) coupled to a single bead (e.g, bead 304). In some cases, the unique molecular identifying sequence 316 can be a random sequence (e.g, such as a random N-mer sequence). For example, the UMI can provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA. As will be appreciated, although FIG. 3 shows three nucleic acid molecules 302, 318, 320 coupled to the surface of the bead 304, an individual bead can be coupled to any number of individual nucleic acid molecules, for example, from one to tens to hundreds of thousands, millions, or even a billion of individual nucleic acid molecules. The respective barcodes for the individual nucleic acid molecules can include both common sequence segments or relatively common sequence segments (e.g, 308, 310, 312, etc.) and variable or unique sequence segments ( e.g ., 316) between different individual nucleic acid molecules coupled to the same bead.

[0274] In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can be co-partitioned along with a barcode bearing bead 304. The barcoded nucleic acid molecules 302, 318, 320 can be released from the bead 304 in the partition. By way of example, in the context of analyzing sample RNA, the poly-T segment (e.g, 312) of one of the released nucleic acid molecules (e.g, 302) can hybridize to the poly-A tail of a mRNA molecule. Reverse transcription can result in a cDNA transcript of the mRNA, but which transcript includes each of the sequence segments 308, 310, 316 of the nucleic acid molecule 302. Because the nucleic acid molecule 305 includes an anchoring sequence 314, it will more likely hybridize to and prime reverse transcription at the sequence end of the poly-A tail of the mRNA. Within any given partition, all of the cDNA transcripts of the individual mRNA molecules can include a common barcode sequence segment 310. However, the transcripts made from the different mRNA molecules within a given partition can vary at the unique molecular identifying sequence 312 segment (e.g, UMI segment). Beneficially, even following any subsequent amplification of the contents of a given partition, the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the biological particle (e.g, cell). As noted above, the transcripts can be amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment. While a poly-T primer sequence is described, other targeted or random priming sequences can also be used in priming the reverse transcription reaction. Likewise, although described as releasing the barcoded oligonucleotides into the partition, in some cases, the nucleic acid barcode molecules bound to the bead (e.g, gel bead) can be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents. In such cases, further processing can be performed, in the partitions or outside the partitions (e.g, in bulk). For instance, the RNA molecules on the beads can be subjected to reverse transcription or other nucleic acid processing, additional adapter sequences can be added to the barcoded nucleic acid molecules, or other nucleic acid reactions (e.g, amplification, nucleic acid extension) can be performed. The beads or products thereof (e.g, barcoded nucleic acid molecules) can be collected from the partitions, and/or pooled together and subsequently subjected to clean up and further characterization (e.g, sequencing). [0275] The operations described herein can be performed at any useful or suitable step. For instance, the beads including nucleic acid barcode molecules can be introduced into a partition (e.g, well or droplet) prior to, during, or following introduction of a sample into the partition. The nucleic acid molecules of a sample can be subjected to barcoding, which can occur on the bead (in cases where the nucleic acid molecules remain coupled to the bead) or following release of the nucleic acid barcode molecules into the partition. In cases where the nucleic acid molecules from the sample remain attached to the bead, the beads from various partitions can be collected, pooled, and subjected to further processing (e.g, reverse transcription, adapter attachment, amplification, clean up, and/or sequencing). In other instances, the processing can occur in the partition. For example, conditions sufficient for barcoding, adapter attachment, reverse transcription, or other nucleic acid processing operations can be provided in the partition and performed prior to clean up and sequencing.

[0276] In some instances, a bead can include a capture sequence or binding sequence configured to bind to a corresponding capture sequence or binding sequence. In some instances, a bead can include a plurality of different capture sequences or binding sequences configured to bind to different respective corresponding capture sequences or binding sequences. For example, a bead can include a first subset of one or more capture sequences each configured to bind to a first corresponding capture sequence, a second subset of one or more capture sequences each configured to bind to a second corresponding capture sequence, a third subset of one or more capture sequences each configured to bind to a third corresponding capture sequence, and etc. A bead can include any number of different capture sequences. In some instances, a bead can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences, respectively. Alternatively or in addition, a bead can include at most about 10, 9, 8, 7, 6, 5, 4, 3, or 2 different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences. In some instances, the different capture sequences or binding sequences can be configured to facilitate analysis of a same type of analyte. In some instances, the different capture sequences or binding sequences can be configured to facilitate analysis of different types of analytes (with the same bead). The capture sequence can be designed to attach to a corresponding capture sequence. Beneficially, such corresponding capture sequence can be introduced to, or otherwise induced in, a biological particle (e.g, cell, cell bead, etc.) for performing different assays in various formats ( e.g ., barcoded antibodies including the corresponding capture sequence, barcoded MHC dextramers including the corresponding capture sequence, barcoded guide RNA molecules including the corresponding capture sequence, etc.), such that the corresponding capture sequence can later interact with the capture sequence associated with the bead. In some instances, a capture sequence coupled to a bead (or other support) can be configured to attach to a linker molecule, such as a splint molecule, wherein the linker molecule is configured to couple the bead (or other support) to other molecules through the linker molecule, such as to one or more analytes or one or more other linker molecules.

[0277] FIG. 4 illustrates a non-limiting example of a barcode carrying bead in accordance with some embodiments of the disclosure. A nucleic acid barcode molecule 405, such as an oligonucleotide, can be coupled to a bead 404 by a releasable linkage 406, such as, for example, a disulfide linker. The nucleic acid barcode molecule 405 can include a first capture sequence 460. The same bead 404 can be coupled, e.g., via releasable linkage, to one or more other nucleic acid molecules 403, 407 including other capture sequences. The nucleic acid barcode molecule 405 can be or include a barcode sequence. As described elsewhere herein, the structure of the barcode can include a number of sequence elements, such as a functional sequence 408 (e.g, flow cell attachment sequence, sequencing primer sequence, etc.), a barcode sequence 410 (e.g, bead-specific sequence common to bead, partition-specific sequence common to partition, etc.), and a unique molecular identifier 412 (e.g, unique sequence within different molecules attached to the bead), or partial sequences thereof. The capture sequence 460 can be configured to attach to a corresponding capture sequence 465 (e.g, capture handle). In some instances, the corresponding capture sequence 465 can be coupled to another molecule that can be an analyte or an intermediary carrier. For example, as illustrated in FIG. 4, the corresponding capture sequence 465 is coupled to a guide RNA molecule 462 including a target sequence 464, wherein the target sequence 464 is configured to attach to the analyte. Another oligonucleotide molecule 407 attached to the bead 404 includes a second capture sequence 480 which is configured to attach to a second corresponding capture sequence (e.g, capture handle) 485. As illustrated in FIG. 4, the second corresponding capture sequence 485 is coupled to an antibody 482. In some cases, the antibody 482 can have binding specificity to an analyte (e.g, surface protein). Alternatively, the antibody 482 may not have binding specificity. Another oligonucleotide molecule 403 attached to the bead 404 includes a third capture sequence 470 which is configured to attach to a third corresponding capture sequence 475. As illustrated in FIG. 4, the third corresponding capture sequence ( e.g ., capture handle) 475 is coupled to a molecule 472. The molecule 472 may or may not be configured to target an analyte. The other oligonucleotide molecules 403, 407 can include the other sequences (e.g., functional sequence, barcode sequence, UMI, etc.) described with respect to oligonucleotide molecule 405. While a single oligonucleotide molecule including each capture sequence is illustrated in FIG. 4, it will be appreciated that, for each capture sequence, the bead can include a set of one or more oligonucleotide molecules each including the capture sequence. For example, the bead can include any number of sets of one or more different capture sequences. Alternatively or in addition, the bead 404 can include other capture sequences. Alternatively or in addition, the bead 404 can include fewer types of capture sequences (e.g, two capture sequences). Alternatively or in addition, the bead 404 can include oligonucleotide molecule(s) including a priming sequence, such as a specific priming sequence such as an mRNA specific priming sequence (e.g, poly-T sequence), a targeted priming sequence, and/or a random priming sequence, for example, to facilitate an assay for gene expression.

[0278] The generation of a barcoded sequence, see, e.g, FIG. 3, is described herein.

[0279] A bead injected or otherwise introduced into a partition can include releasably, cleavably, or reversibly attached barcodes (e.g, partition barcode sequences). A bead injected or otherwise introduced into a partition can include activatable barcodes. A bead injected or otherwise introduced into a partition can be degradable, disruptable, or dissolvable beads.

[0280] Barcode containing nucleic acid molecules (e.g, nucleic acid barcode molecules or barcoded oligonucleotides), can be releasably, cleavably or reversibly attached to the beads such that barcodes can be released or be releasable through cleavage of a linkage between the barcode containing nucleic acid molecule and the bead, or released through degradation of the underlying bead itself, allowing the barcodes to be accessed or be accessible by other reagents, or both. In non-limiting examples, cleavage can be achieved through reduction of di-sulfide bonds, use of restriction enzymes, photo-activated cleavage, or cleavage via other types of stimuli (e.g, chemical, thermal, pH, enzymatic, etc.) and/or reactions, such as described elsewhere herein. Releasable barcodes can sometimes be referred to as being activatable, in that they are available for reaction once released. Thus, for example, an activatable barcode can be activated by releasing the barcode from a bead (or other suitable type of partition described herein). Other activatable configurations are also envisioned in the context of the described methods and systems.

[0281] As will be appreciated from the above disclosure, the degradation of a bead can refer to the dissociation of a bound ( e.g ., capture agent configured to couple to a secreted antibody or antigen binding fragment thereof) or entrained species (e.g., labelled B cell, e.g, memory B cell, or plasma cell, or secreted antibody or antigen binding fragment thereof) from a bead, both with and without structurally degrading the physical bead itself. For example, the degradation of the bead can involve cleavage of a cleavable linkage via one or more species and/or methods described elsewhere herein. In another example, entrained species can be released from beads through osmotic pressure differences due to, for example, changing chemical environments. See, e.g., PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety.

[0282] A degradable bead can be introduced into a partition, such as a droplet of an emulsion or a well, such that the bead degrades within the partition and any associated species (e.g, oligonucleotides) are released within the droplet when the appropriate stimulus is applied. The free species (e.g, oligonucleotides, nucleic acid molecules, nucleic acid barcode molecules) can interact with other reagents contained in the partition. See, e.g., PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety.

[0283] Any suitable number of molecular tag molecules (e.g, primer, barcoded oligonucleotide) can be associated with a bead such that, upon release from the bead, the molecular tag molecules (e.g, primer, e.g, barcoded oligonucleotide) are present in the partition at a pre-defmed concentration. Such pre-defmed concentration can be selected to facilitate certain reactions for generating a sequencing library, e.g, amplification, within the partition. In some cases, the pre-defmed concentration of the primer can be limited by the process of producing nucleic acid molecule (e.g, oligonucleotide, e.g, nucleic acid barcode molecule) bearing beads.

[0284] In some cases, beads can be non-covalently loaded with one or more reagents. The beads can be non-covalently loaded by, for instance, subjecting the beads to conditions sufficient to swell the beads, allowing sufficient time for the reagents to diffuse into the interiors of the beads, and subjecting the beads to conditions sufficient to de-swell the beads. The swelling of the beads can be accomplished, for instance, by placing the beads in a thermodynamically favorable solvent, subjecting the beads to a higher or lower temperature, subjecting the beads to a higher or lower ion concentration, and/or subjecting the beads to an electric field. The swelling of the beads can be accomplished by various swelling methods. The de-swelling of the beads can be accomplished, for instance, by transferring the beads in a thermodynamically unfavorable solvent, subjecting the beads to lower or high temperatures, subjecting the beads to a lower or higher ion concentration, and/or removing an electric field. The de-swelling of the beads can be accomplished by various de-swelling methods. Transferring the beads can cause pores in the bead to shrink. The shrinking can then hinder reagents within the beads from diffusing out of the interiors of the beads. The hindrance can be due to steric interactions between the reagents and the interiors of the beads. The transfer can be accomplished microfluidically. For instance, the transfer can be achieved by moving the beads from one co-flowing solvent stream to a different co-flowing solvent stream. The swellability and/or pore size of the beads can be adjusted by changing the polymer composition of the bead.

[0285] In some cases, an acrydite moiety linked to a precursor, another species linked to a precursor, or a precursor itself can include a labile bond, such as chemically, thermally, or photo sensitive bond e.g., disulfide bond, UV sensitive bond, or the like. Once acrydite moieties or other moieties including a labile bond are incorporated into a bead, the bead can also include the labile bond. The labile bond can be, for example, useful in reversibly linking (e.g, covalently linking) species (e.g, barcodes, primers, etc.) to a bead. In some cases, a thermally labile bond can include a nucleic acid hybridization based attachment, e.g, where an oligonucleotide is hybridized to a complementary sequence that is attached to the bead, such that thermal melting of the hybrid releases the oligonucleotide, e.g, a barcode containing sequence, from the bead or microcapsule.

[0286] The addition of multiple types of labile bonds to a gel bead can result in the generation of a bead capable of responding to varied stimuli. Each type of labile bond can be sensitive to an associated stimulus (e.g, chemical stimulus, light, temperature, enzymatic, etc.) such that release of species attached to a bead via each labile bond can be controlled by the application of the appropriate stimulus. Such functionality can be useful in controlled release of species from a gel bead. In some cases, another species including a labile bond can be linked to a gel bead after gel bead formation via, for example, an activated functional group of the gel bead as described above. As will be appreciated, barcodes that are releasably, cleavably or reversibly attached to the beads described herein include barcodes that are released or releasable through cleavage of a linkage between the barcode molecule and the bead, or that are released through degradation of the underlying bead itself, allowing the barcodes to be accessed or accessible by other reagents, or both.

[0287] The barcodes that are releasable as described herein can sometimes be referred to as being activatable, in that they are available for reaction once released. Thus, for example, an activatable barcode can be activated by releasing the barcode from a bead (or other suitable type of partition described herein). Other activatable configurations are also envisioned in the context of the described methods and systems.

[0288] In addition to thermally cleavable bonds, disulfide bonds and UV sensitive bonds, other non-limiting examples of labile bonds that can be coupled to a precursor or bead include an ester linkage ( e.g ., cleavable with an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g., cleavable via sodium periodate), a Diels- Alder linkage (e.g, cleavable via heat), a sulfone linkage (e.g, cleavable via a base), a silyl ether linkage (e.g, cleavable via an acid), a glycosidic linkage (e.g, cleavable via an amylase), a peptide linkage (e.g, cleavable via a protease), or a phosphodiester linkage (e.g, cleavable via a nuclease (e.g, DNAase)). A bond can be cleavable via other nucleic acid molecule targeting enzymes, such as restriction enzymes (e.g, restriction endonucleases), as described further below.

[0289] Species can be encapsulated in beads (e.g, capture agent) during bead generation (e.g, during polymerization of precursors). Such species may or may not participate in polymerization. Such species can be entered into polymerization reaction mixtures such that generated beads include the species upon bead formation. In some cases, such species can be added to the gel beads after formation. Such species can include, for example, nucleic acid molecules (e.g, oligonucleotides, e.g. , nucleic acid barcode molecules), reagents for a nucleic acid amplification reaction (e.g, primers, polymerases, dNTPs, co-factors (e.g, ionic co-factors, buffers) including those described herein, reagents for enzymatic reactions (e.g, enzymes, co factors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation, or digestion, and/or reagents for template preparation (e.g, tagmentation) for one or more sequencing platforms (e.g, Nextera® for Illumina®). Such species can include one or more enzymes described herein, including without limitation, polymerase, reverse transcriptase, restriction enzymes (e.g, endonuclease), transposase, ligase, proteinase K, DNAse, etc. Such species can include one or more reagents described elsewhere herein ( e.g ., lysis agents, inhibitors, inactivating agents, chelating agents, stimulus). Trapping of such species can be controlled by the polymer network density generated during polymerization of precursors, control of ionic charge within the gel bead (e.g., via ionic species linked to polymerized species), or by the release of other species. Encapsulated species can be released from a bead upon bead degradation and/or by application of a stimulus capable of releasing the species from the bead. Alternatively or in addition, species can be partitioned in a partition (e.g, droplet) during or subsequent to partition formation. Such species can include, without limitation, the abovementioned species that can also be encapsulated in a bead.

[0290] Although FIG. 1 and FIG. 2 have been described in terms of providing substantially singly occupied partitions, above, in certain cases, it may be desirable to provide multiply occupied partitions, e.g, containing two, three, four or more cells and/or microcapsules (e.g, beads) including barcoded nucleic acid molecules, e.g. , nucleic acid barcode molecules (e.g, oligonucleotides) within a single partition (e.g, multiomics method described elsewhere, herein). Accordingly, as noted above, the flow characteristics of the biological particle and/or bead containing fluids and partitioning fluids can be controlled to provide for such multiply occupied partitions. In particular, the flow parameters can be controlled to provide a given occupancy rate at greater than about 50% of the partitions, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or higher.

[0291] In some cases, additional microcapsules or beads can be used to deliver additional reagents to a partition. In such cases, it can be advantageous to introduce different beads into a common channel or droplet generation junction, from different bead sources (e.g, containing different associated reagents) through different channel inlets into such common channel or droplet generation junction (e.g, junction 210). In such cases, the flow and frequency of the different beads into the channel or junction can be controlled to provide for a certain ratio of microcapsules from each source, while ensuring a given pairing or combination of such beads into a partition with a given number of biological particles (e.g, one biological particle and one bead per partition).

[0292] The partitions described herein can include small volumes, for example, less than about 10 microliters (pL), 5pL, lpL, 10 nanoliters (nL), 5 nL, 1 nL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

[0293] For example, in the case of droplet based partitions, the droplets can have overall volumes that are less than about 1000 pL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL, 1 pL, or less. Where co-partitioned with microcapsules, it will be appreciated that the sample fluid volume, e.g., including co-partitioned biological particles and/or beads, within the partitions can be less than about 90% of the above described volumes, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the above described volumes.

[0294] As is described elsewhere herein, partitioning species can generate a population or plurality of partitions. In such cases, any suitable number of partitions can be generated or otherwise provided. For example, at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000,000 partitions, at least about 5,000,000 partitions at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, at least about 1,000,000,000 partitions, or more partitions can be generated or otherwise provided. Moreover, the plurality of partitions can include both unoccupied partitions (e.g, empty partitions) and occupied partitions.

Reagents

[0295] In accordance with certain aspects, biological particles can be partitioned along with lysis reagents in order to release the contents of the biological particles within the partition. See, e.g, U.S. Pat. Pub. 2018/0216162 (now U.S. Pat. 10,428,326), U.S. Pat. Pub. 2019/0100632 (nowU.S. Pat. 10,590,244), and U.S. Pat. Pub. 2019/0233878. Biological particles (e.g, cells, cell beads, cell nuclei, organelles, and the like) can be partitioned together with nucleic acid barcode molecules and the nucleic acid molecules of or derived from the biological particle (e.g, mRNA, cDNA, gDNA, etc.,) can be barcoded as described elsewhere herein. In some embodiments, biological particles are co-partitioned with barcode carrying beads (e.g, gel beads) and the nucleic acid molecules of or derived from the biological particle are barcoded as described elsewhere herein. In such cases, the lysis agents can be contacted with the biological particle suspension concurrently with, or immediately prior to, the introduction of the biological particles into the partitioning junction/droplet generation zone (e.g, junction 210), such as through an additional channel or channels upstream of the channel junction. In accordance with other aspects, additionally or alternatively, biological particles can be partitioned along with other reagents, as will be described further below.

[0296] Beneficially, when lysis reagents and biological particles are co-partitioned, the lysis reagents can facilitate the release of the contents of the biological particles within the partition. The contents released in a partition can remain discrete from the contents of other partitions.

[0297] As will be appreciated, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structures can have other geometries and/or configurations. For example, a microfluidic channel structure can have more than two channel junctions. For example, a microfluidic channel structure can have 2, 3, 4, 5 channel segments or more each carrying the same or different types of beads, reagents, and/or biological particles that meet at a channel junction. Fluid flow in each channel segment can be controlled to control the partitioning of the different elements into droplets. Fluid can be directed flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can include compressors ( e.g ., providing positive pressure), pumps (e.g., providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.

[0298] Examples of lysis agents include bioactive reagents, such as lysis enzymes that are used for lysis of different cell types, e.g, gram positive or negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other lysis enzymes available from, e.g, Sigma-Aldrich, Inc. (St Louis, MO), as well as other commercially available lysis enzymes. Other lysis agents can additionally or alternatively be co-partitioned with the biological particles to cause the release of the biological particle’s contents into the partitions. For example, in some cases, surfactant-based lysis solutions can be used to lyse cells (e.g, labelled B cell, memory B cell, or plasma cell), although these can be less desirable for emulsion based systems where the surfactants can interfere with stable emulsions. In some cases, lysis solutions can include non-ionic surfactants such as, for example, TritonX-100 and Tween 20. In some cases, lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanical cellular disruption can also be used in certain cases, e.g., non-emulsion based partitioning such as encapsulation of biological particles that can be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a given size, following cellular disruption.

[0299] Alternatively or in addition to the lysis agents co-partitioned with the biological particles (e.g, labelled B cells, memory B cells, or plasma cells) described above, other reagents can also be co-partitioned with the biological particles, including, for example, DNase and RNase inactivating agents or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids. In addition, in the case of encapsulated biological particles (e.g, labelled B cells, memory B cells, or plasma cells), the biological particles can be exposed to an appropriate stimulus to release the biological particles or their contents from a co-partitioned microcapsule. For example, in some cases, a chemical stimulus can be co-partitioned along with an encapsulated biological particle to allow for the degradation of the microcapsule and release of the cell or its contents into the larger partition. In some cases, this stimulus can be the same as the stimulus described elsewhere herein for release of nucleic acid molecules (e.g, oligonucleotides) from their respective microcapsule (e.g, bead). In alternative aspects, this can be a different and non-overlapping stimulus, in order to allow an encapsulated biological particle to be released into a partition at a different time from the release of nucleic acid molecules into the same partition.

[0300] Additional reagents can also be co-partitioned with the biological particles (e.g, labelled B cell, memory B cell, or plasma cell), such as endonucleases to fragment a biological particle’s DNA, DNA polymerase enzymes and dNTPs used to amplify the biological particle’s nucleic acid fragments and to attach the barcode molecular tags to the amplified fragments.

Other enzymes can be co-partitioned, including without limitation, polymerase, transposase, ligase, proteinase K, DNAse, etc. Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching. In some cases, template switching can be used to increase the length of a cDNA. In some cases, template switching can be used to append a predefined nucleic acid sequence to the cDNA. In an example of template switching, cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g, polyC, to the cDNA in a template independent manner. Switch oligos can include sequences complementary to the additional nucleotides, e.g, polyG. The additional nucleotides (e.g, polyC) on the cDNA can hybridize to the additional nucleotides (e.g, polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA. Template switching oligonucleotides can include a hybridization region and a template region. Template switching oligonucleotides are further described in PCT Pub. No. WO2018119447, which is hereby incorporated by reference in its entirety.

[0301] Once the contents of the cells (e.g, labelled B cells, memory B cell, or plasma cells) are released into their respective partitions, the macromolecular components (e.g, macromolecular constituents of biological particles, such as RNA, DNA, proteins, or secreted antibodies or antigen binding fragments thereof) contained therein can be further processed within the partitions. In accordance with the methods and systems described herein, the macromolecular component contents of individual biological particles (e.g, labelled B cells, memory B cells, or plasma cells) can be provided with unique identifiers such that, upon characterization of those macromolecular components they can be attributed as having been derived from the same biological particle or particles. The ability to attribute characteristics to individual biological particles or groups of biological particles is provided by the assignment of unique identifiers specifically to an individual biological particle or groups of biological particles. Unique identifiers, e.g, in the form of nucleic acid barcodes can be assigned or associated with individual biological particles or populations of biological particles, in order to tag or label the biological particle’s macromolecular components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the biological particle’s components and characteristics to an individual biological particle or group of biological particles.

[0302] In some aspects, this is performed by co-partitioning the individual biological particle (e.g, labelled B cell, memory B cell, or plasma cell) or groups of biological particles (e.g, labelled B cells, memory B cell, or plasma cells) with the unique identifiers, such as described above (with reference to FIGS. 5 and 6). In some aspects, the unique identifiers are provided in the form of nucleic acid molecules ( e.g ., oligonucleotides) that include nucleic acid barcode sequences that can be attached to or otherwise associated with the nucleic acid contents of individual biological particle, or to other components of the biological particle, and particularly to fragments of those nucleic acids. The nucleic acid molecules are partitioned such that as between nucleic acid molecules in a given partition, the nucleic acid barcode sequences contained therein are the same, but as between different partitions, the nucleic acid molecule can, and do have differing barcode sequences, or at least represent a large number of different barcode sequences across all of the partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, although in some cases, two or more different barcode sequences can be present.

[0303] The nucleic acid barcode sequences can include from about 6 to about 20 or more nucleotides within the sequence of the nucleic acid molecules (e.g., oligonucleotides). The nucleic acid barcode sequences can include from about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some cases, the length of a barcode sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence can be at most about 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by one or more nucleotides. In some cases, separated barcode subsequences can be from about 4 to about 16 nucleotides in length. In some cases, the barcode subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at least about 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0304] The co-partitioned nucleic acid molecules can also include other functional sequences useful in the processing of the nucleic acids from the co-partitioned biological particles (e.g, labelled B cells, memory B cells, or plasma cells). These sequences include, e.g, targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences. Other mechanisms of co-partitioning oligonucleotides can also be employed, including, e.g, coalescence of two or more droplets, where one droplet contains oligonucleotides, or microdispensing of oligonucleotides into partitions, e.g, droplets within microfluidic systems.

[0305] In an example, microcapsules, such as beads, are provided that each include large numbers of the above described barcoded nucleic acid molecules (e.g, barcoded oligonucleotides) releasably attached to the beads, where all of the nucleic acid molecules attached to a particular bead will include the same nucleic acid barcode sequence, but where a large number of diverse barcode sequences are represented across the population of beads used. In some embodiments, hydrogel beads, e.g, including polyacrylamide polymer matrices, are used as a solid support and delivery vehicle for the nucleic acid molecules into the partitions, as they are capable of carrying large numbers of nucleic acid molecules, and can be configured to release those nucleic acid molecules upon exposure to a particular stimulus, as described elsewhere herein. In some cases, the population of beads provides a diverse barcode sequence library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences, or more. Additionally, each bead can be provided with large numbers of nucleic acid (e.g, oligonucleotide) molecules attached. In particular, the number of molecules of nucleic acid molecules including the barcode sequence on an individual bead can be at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules, or more. Nucleic acid molecules of a given bead can include identical (or common) barcode sequences, different barcode sequences, or a combination of both. Nucleic acid molecules of a given bead can include multiple sets of nucleic acid molecules. Nucleic acid molecules of a given set can include identical barcode sequences. The identical barcode sequences can be different from barcode sequences of nucleic acid molecules of another set.

[0306] Moreover, when the population of beads is partitioned, the resulting population of partitions can also include a diverse barcode library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences. Additionally, each partition of the population can include at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules.

[0307] In some cases, it may be desirable to incorporate multiple different barcodes within a given partition, either attached to a single or multiple beads within the partition. For example, in some cases, a mixed, but known set of barcode sequences can provide greater assurance of identification in the subsequent processing, e.g., by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.

[0308] The nucleic acid molecules (e.g, oligonucleotides) are releasable from the beads upon the application of a particular stimulus to the beads. In some cases, the stimulus can be a photo-stimulus, e.g, through cleavage of a photo-labile linkage that releases the nucleic acid molecules. In other cases, a thermal stimulus can be used, where elevation of the temperature of the beads environment will result in cleavage of a linkage or other release of the nucleic acid molecules from the beads. In still other cases, a chemical stimulus can be used that cleaves a linkage of the nucleic acid molecules to the beads, or otherwise results in release of the nucleic acid molecules from the beads. In one case, such compositions include the polyacrylamide matrices described above for encapsulation of biological particles, and can be degraded for release of the attached nucleic acid molecules through exposure to a reducing agent, such as DTT.

Systems and methods for controlled partitioning

[0309] In some aspects, provided are systems and methods for controlled partitioning. Droplet size can be controlled by adjusting certain geometric features in channel architecture (e.g, microfluidics channel architecture). For example, an expansion angle, width, and/or length of a channel can be adjusted to control droplet size.

[0310] FIG. 2 shows an example of a microfluidic channel structure for the controlled partitioning of beads into discrete droplets. A channel structure 200 can include a channel segment 202 communicating at a channel junction 206 (or intersection) with a reservoir 204. The reservoir 204 can be a chamber. Any reference to “reservoir,” as used herein, can also refer to a “chamber.” In operation, an aqueous fluid 208 that includes suspended beads 212 can be transported along the channel segment 202 into the junction 206 to meet a second fluid 210 that is immiscible with the aqueous fluid 208 in the reservoir 204 to create droplets 216, 218 of the aqueous fluid 208 flowing into the reservoir 204. At the junction 206 where the aqueous fluid 208 and the second fluid 210 meet, droplets can form based on factors such as the hydrodynamic forces at the junction 206, flow rates of the two fluids 208, 210, fluid properties, and certain geometric parameters (e.g, w, ho, a, etc.) of the channel structure 200. A plurality of droplets can be collected in the reservoir 204 by continuously injecting the aqueous fluid 208 from the channel segment 202 through the junction 206.

[0311] A discrete droplet generated can include a bead (e.g, as in occupied droplets 216). Alternatively, a discrete droplet generated can include more than one bead. Alternatively, a discrete droplet generated cannot include any beads (e.g, as in unoccupied droplet 218). In some instances, a discrete droplet generated can contain one or more biological particles, as described elsewhere herein. In some instances, a discrete droplet generated can include one or more reagents, as described elsewhere herein.

[0312] In some instances, the aqueous fluid 208 can have a substantially uniform concentration or frequency of beads 212. The beads 212 can be introduced into the channel segment 202 from a separate channel (not shown in FIG. 2). The frequency of beads 212 in the channel segment 202 can be controlled by controlling the frequency in which the beads 212 are introduced into the channel segment 202 and/or the relative flow rates of the fluids in the channel segment 202 and the separate channel. In some instances, the beads can be introduced into the channel segment 202 from a plurality of different channels, and the frequency controlled accordingly.

[0313] In some instances, the aqueous fluid 208 in the channel segment 202 can include biological particles ( e.g ., described with reference to FIG. 1). In some instances, the aqueous fluid 208 can have a substantially uniform concentration or frequency of biological particles. As with the beads, the biological particles (e.g., labelled B cells, memory B cells, or plasma cells) can be introduced into the channel segment 202 from a separate channel. The frequency or concentration of the biological particles in the aqueous fluid 208 in the channel segment 202 can be controlled by controlling the frequency in which the biological particles are introduced into the channel segment 202 and/or the relative flow rates of the fluids in the channel segment 202 and the separate channel. In some instances, the biological particles can be introduced into the channel segment 202 from a plurality of different channels, and the frequency controlled accordingly. In some instances, a first separate channel can introduce beads and a second separate channel can introduce biological particles into the channel segment 202. The first separate channel introducing the beads can be upstream or downstream of the second separate channel introducing the biological particles.

[0314] The second fluid 210 can include an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets.

[0315] In some instances, the second fluid 210 cannot be subjected to and/or directed to any flow in or out of the reservoir 204. For example, the second fluid 210 can be substantially stationary in the reservoir 204. In some instances, the second fluid 210 can be subjected to flow within the reservoir 204, but not in or out of the reservoir 204, such as via application of pressure to the reservoir 204 and/or as affected by the incoming flow of the aqueous fluid 208 at the junction 206. Alternatively, the second fluid 210 can be subjected and/or directed to flow in or out of the reservoir 204. For example, the reservoir 204 can be a channel directing the second fluid 210 from upstream to downstream, transporting the generated droplets.

[1] The channel structure 200 at or near the junction 206 can have certain geometric features that at least partly determine the sizes of the droplets formed by the channel structure 200. The channel segment 202 can have a height, ho and width, w, at or near the junction 206. By way of example, the channel segment 202 can include a rectangular cross- section that leads to a reservoir 204 having a wider cross-section (such as in width or diameter). Alternatively, the cross-section of the channel segment 202 can be other shapes, such as a circular shape, trapezoidal shape, polygonal shape, or any other shapes. The top and bottom walls of the reservoir 204 at or near the junction 206 can be inclined at an expansion angle, a. The expansion angle, a, allows the tongue (portion of the aqueous fluid 208 leaving channel segment 202 at junction 206 and entering the reservoir 204 before droplet formation) to increase in depth and facilitate decrease in curvature of the intermediately formed droplet. Droplet size can decrease with increasing expansion angle. The resulting droplet radius, R_d , can be predicted by the following equation for the aforementioned geometric parameters of ho , w, and a:

R_d * 0.44

[0316] Systems and methods for controlled partitioning are described further in W02019040637, which is hereby incorporated by reference in its entirety.

[0317] The methods and systems described herein can be used to greatly increase the efficiency of single cell applications and/or other applications receiving droplet-based input. For example, following the sorting of occupied cells and/or appropriately-sized cells, subsequent operations that can be performed can include generation of amplification products, purification ( e.g ., via solid phase reversible immobilization (SPRI)), further processing ( e.g ., shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)). These operations can occur in bulk (e.g, outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations. Additional reagents that can be co-partitioned along with the barcode bearing bead can include oligonucleotides to block ribosomal RNA (rRNA) and nucleases to digest genomic DNA from cells. Alternatively, rRNA removal agents can be applied during additional processing operations. The configuration of the constructs generated by such a method can help minimize (or avoid) sequencing of the poly-T sequence during sequencing and/or sequence the 5’ end of a polynucleotide sequence. The amplification products, for example, first amplification products and/or second amplification products, can be subject to sequencing for sequence analysis. In some cases, amplification can be performed using the Partial Hairpin Amplification for Sequencing (PHASE) method. [0318] A variety of applications require the evaluation of the presence and quantification of different biological particle or organism types within a population of biological particles, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, e.g., in tracing contamination or the like.

[0319] Partitions including a barcode bead (e.g, a gel bead) associated with barcode molecules and a bead encapsulating cellular constituents (e.g, a cell bead) such as cellular nucleic acids can be useful in constituent analysis as is described in U.S. Patent Publication No. 2018/0216162.

Sample and cell processing

[0320] A sample can be derived from any useful source including any subject, such as a human subject. A sample can include material (e.g, one or more cells) from one or more different sources, such as one or more different subjects. Multiple samples, such as multiple samples from a single subject (e.g, multiple samples obtained in the same or different manners from the same or different bodily locations, and/or obtained at the same or different times (e.g, seconds, minutes, hours, days, weeks, months, or years apparat)), or multiple samples from different subjects, can be obtained for analysis as described herein. For example, a first sample can be obtained from a subject at a first time and a second sample can be obtained from the subject at a second time later than the first time. The first time can be before a subject undergoes a treatment regimen or procedure (e.g, to address a disease or condition), and the second time can be during or after the subject undergoes the treatment regimen or procedure. In another example, a first sample can be obtained from a first bodily location or system of a subject (e.g, using a first collection technique) and a second sample can be obtained from a second bodily location or system of the subject (e.g, using a second collection technique), which second bodily location or system can be different than the first bodily location or system. In another example, multiple samples can be obtained from a subject at a same time from the same or different bodily locations. Different samples, such as different samples collected from different bodily locations of a same subject, at different times, from multiple different subjects, and/or using different collection techniques, can undergo the same or different processing (e.g, as described herein). For example, a first sample can undergo a first processing protocol and a second sample can undergo a second processing protocol.

[0321] A sample can be a biological sample, such as a cell sample (e.g, as described herein). A sample can include one or more analyte carriers, such as one or more cells and/or cellular constituents, such as one or more cell nuclei. For example, a sample can include a plurality of cells and/or cellular constituents. Components ( e.g ., cells or cellular constituents, such as cell nuclei) of a sample can be of a single type or a plurality of different types. For example, cells of a sample can include one or more different types of blood cells.

[0322] A biological sample can include a plurality of cells having different dimensions and features. In some cases, processing of the biological sample, such as cell separation and sorting (e.g., as described herein), can affect the distribution of dimensions and cellular features included in the sample by depleting cells having certain features and dimensions and/or isolating cells having certain features and dimensions.

[0323] A sample may undergo one or more processes in preparation for analysis (e.g, as described herein), including, but not limited to, filtration, selective precipitation, purification, centrifugation, permeabilization, isolation, agitation, heating, and/or other processes. For example, a sample may be filtered to remove a contaminant or other materials. In an example, a filtration process can include the use of microfluidics (e.g, to separate analyte carriers of different sizes, types, charges, or other features).

[0324] In an example, a sample including one or more cells can be processed to separate the one or more cells from other materials in the sample (e.g, using centrifugation and/or another process). In some cases, cells and/or cellular constituents of a sample can be processed to separate and/or sort groups of cells and/or cellular constituents, such as to separate and/or sort cells and/or cellular constituents of different types. Examples of cell separation include, but are not limited to, separation of white blood cells or immune cells from other blood cells and components, separation of circulating tumor cells from blood, and separation of bacteria from bodily cells and/or environmental materials. A separation process can include a positive selection process (e.g, targeting of a cell type of interest for retention for subsequent downstream analysis, such as by use of a monoclonal antibody that targets a surface marker of the cell type of interest), a negative selection process (e.g, removal of one or more cell types and retention of one or more other cell types of interest), and/or a depletion process (e.g, removal of a single cell type from a sample, such as removal of red blood cells from peripheral blood mononuclear cells).

[0325] Separation of one or more different types of cells can include, for example, centrifugation, filtration, microfluidic-based sorting, flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), buoyancy-activated cell sorting (BACS), or any other useful method. For example, a flow cytometry method can be used to detect cells and/or cellular constituents based on a parameter such as a size, morphology, or protein expression. Flow cytometry-based cell sorting can include injecting a sample into a sheath fluid that conveys the cells and/or cellular constituents of the sample into a measurement region one at a time. In the measurement region, a light source such as a laser can interrogate the cells and/or cellular constituents and scattered light and/or fluorescence can be detected and converted into digital signals. A nozzle system ( e.g ., a vibrating nozzle system) can be used to generate droplets (e.g., aqueous droplets) including individual cells and/or cellular constituents. Droplets including cells and/or cellular constituents of interest (e.g., as determined via optical detection) can be labeled with an electric charge (e.g, using an electrical charging ring), which charge can be used to separate such droplets from droplets including other cells and/or cellular constituents. For example, FACS can include labeling cells and/or cellular constituents with fluorescent markers (e.g, using internal and/or external biomarkers). Cells and/or cellular constituents can then be measured and identified one by one and sorted based on the emitted fluorescence of the marker or absence thereof. MACS can use micro- or nano-scale magnetic particles to bind to cells and/or cellular constituents (e.g, via an antibody interaction with cell surface markers) to facilitate magnetic isolation of cells and/or cellular constituents of interest from other components of a sample (e.g, using a column-based analysis). BACS can use microbubbles (e.g, glass microbubbles) labeled with antibodies to target cells of interest. Cells and/or cellular components coupled to microbubbles can float to a surface of a solution, thereby separating target cells and/or cellular components from other components of a sample. Cell separation techniques can be used to enrich for populations of cells of interest (e.g, prior to partitioning, as described herein). For example, a sample including a plurality of cells including a plurality of cells of a given type can be subjected to a positive separation process. The plurality of cells of the given type can be labeled with a fluorescent marker (e.g, based on an expressed cell surface marker or another marker) and subjected to a FACS process to separate these cells from other cells of the plurality of cells. The selected cells can then be subjected to subsequent partition-based analysis (e.g, as described herein) or other downstream analysis. The fluorescent marker can be removed prior to such analysis or can be retained. The fluorescent marker can include an identifying feature, such as a nucleic acid barcode sequence and/or unique molecular identifier.

[0326] In another example, a first sample including a first plurality of cells including a first plurality of cells of a given type ( e.g ., immune cells expressing a particular marker or combination of markers) and a second sample including a second plurality of cells including a second plurality of cells of the given type can be subjected to a positive separation process. The first and second samples can be collected from the same or different subjects, at the same or different types, from the same or different bodily locations or systems, using the same or different collection techniques. For example, the first sample can be from a first subject and the second sample can be from a second subject different than the first subject. The first plurality of cells of the first sample can be provided a first plurality of fluorescent markers configured to label the first plurality of cells of the given type. The second plurality of cells of the second sample can be provided a second plurality of fluorescent markers configured to label the second plurality of cells of the given type. The first plurality of fluorescent markers can include a first identifying feature, such as a first barcode, while the second plurality of fluorescent markers can include a second identifying feature, such as a second barcode, that is different than the first identifying feature. The first plurality of fluorescent markers and the second plurality of fluorescent markers can fluoresce at the same intensities and over the same range of wavelengths upon excitation with a same excitation source (e.g., light source, such as a laser). The first and second samples can then be combined and subjected to a FACS process to separate cells of the given type from other cells based on the first plurality of fluorescent markers labeling the first plurality of cells of the given type and the second plurality of fluorescent markers labeling the second plurality of cells of the given type. Alternatively, the first and second samples can undergo separate FACS processes and the positively selected cells of the given type from the first sample and the positively selected cells of the given type from the second sample can then be combined for subsequent analysis. The encoded identifying features of the different fluorescent markers can be used to identify cells originating from the first sample and cells originating from the second sample. For example, the first and second identifying features can be configured to interact (e.g, in partitions, as described herein) with nucleic acid barcode molecules (e.g, as described herein) to generate barcoded nucleic acid products detectable using, e.g, nucleic acid sequencing.

[0327] FIG. 6 schematically shows an example workflow for processing nucleic acid molecules within a sample. A substrate 600 including a plurality of microwells 602 can be provided. A sample 606 which can include a cell, cell bead, cellular components or analytes (e.g, proteins and/or nucleic acid molecules) can be co-partitioned, in a plurality of microwells 602, with a plurality of beads 604 including nucleic acid barcode molecules. During a partitioning process, the sample 606 can be processed within the partition. For instance, in the case of live cells, the cell can be subjected to conditions sufficient to lyse the cells and release the analytes contained therein. In process 620, the bead 604 can be further processed. By way of example, processes 620a and 620b schematically illustrate different workflows, depending on the properties of the bead 604.

[0328] In 620a, the bead includes nucleic acid barcode molecules that are attached thereto, and sample nucleic acid molecules (e.g, RNA, DNA) can attach, e.g, via hybridization of ligation, to the nucleic acid barcode molecules. Such attachment can occur on the bead. In process 630, the beads 604 from multiple wells 602 can be collected and pooled. Further processing can be performed in process 640. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some instances, adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein. For instance, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 650, further characterization, such as sequencing can be performed to generate sequencing reads. The sequencing reads can yield information on individual cells or populations of cells, which can be represented visually or graphically, e.g, in a plot.

[0329] In 620b, the bead includes nucleic acid barcode molecules that are releasably attached thereto, as described below. The bead can degrade or otherwise release the nucleic acid barcode molecules into the well 602; the nucleic acid barcode molecules can then be used to barcode nucleic acid molecules within the well 602. Further processing can be performed either inside the partition or outside the partition. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some instances, adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein. For instance, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 650, further characterization, such as sequencing can be performed to generate sequencing reads. The sequencing reads can yield information on individual cells or populations of cells, which can be represented visually or graphically, e.g., in a plot.

Multiplexing methods

[0330] In some embodiments of the disclosure, steps (b) and (c) of the methods described herein are performed in multiplex format. For example, in some embodiments, step (a) of the methods disclosed herein can include individually partitioning additional B cells of the plurality of B cells in partitions of the first plurality of partitions, and step (c) can further include determining all or a part of the nucleic acid sequences encoding antibodies produced by the additional B cells.

[0331] Accordingly, in some embodiments, the present disclosure provides methods and systems for multiplexing, and otherwise increasing throughput of samples for analysis. For example, a single or integrated process workflow may permit the processing, identification, and/or analysis of more or multiple analytes, more or multiple types of analytes, and/or more or multiple types of analyte characterizations. For example, in the methods and systems described herein, one or more labelling agents capable of binding to or otherwise coupling to one or more cells or cell features can be used to characterize cells and/or cell features. In some instances, cell features include cell surface features. Cell surface features can include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, aB-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, cell features can include intracellular analytes, such as proteins, protein modifications (e.g, phosphorylation status or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof. A labelling agent can include, but is not limited to, a protein, a peptide, an antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. The labelling agents can include (e.g, are attached to) a reporter oligonucleotide that is indicative of the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide can include a barcode sequence that permits identification of the labelling agent. For example, a labelling agent that is specific to one type of cell feature (e.g, a first cell surface feature) can have a first reporter oligonucleotide coupled thereto, while a labelling agent that is specific to a different cell feature (e.g, a second cell surface feature) can have a different reporter oligonucleotide coupled thereto. For a description of exemplary labelling agents, reporter oligonucleotides, and methods of use, see, e.g, U.S. Pat. 10,550,429; U.S. Pat. Pub. 20190177800; and U.S. Pat. Pub. 20190367969.

[0332] In a particular example, a library of potential cell feature labelling agents can be provided, where the respective cell feature labelling agents are associated with nucleic acid reporter molecules, such that a different reporter oligonucleotide sequence is associated with each labelling agent capable of binding to a specific cell feature. In other aspects, different members of the library can be characterized by the presence of a different oligonucleotide sequence label. For example, an antibody capable of binding to a first protein can have associated with it a first reporter oligonucleotide sequence, while an antibody capable of binding to a second protein can have a different reporter oligonucleotide sequence associated with it. The presence of the particular oligonucleotide sequence can be indicative of the presence of a particular antibody or cell feature which can be recognized or bound by the particular antibody.

[0333] Labelling agents capable of binding to or otherwise coupling to one or more cells can be used to characterize a cell as belonging to a particular set of cells. For example, labeling agents can be used to label a sample of cells or a group of cells. In this way, a group of cells can be labeled as different from another group of cells. In an example, a first group of cells can originate from a first sample and a second group of cells can originate from a second sample. Labelling agents can allow the first group and second group to have a different labeling agent (or reporter oligonucleotide associated with the labeling agent). This can, for example, facilitate multiplexing, where cells of the first group and cells of the second group can be labeled separately and then pooled together for downstream analysis. The downstream detection of a label can indicate analytes as belonging to a particular group.

[0334] In some embodiments, the reporter oligonucleotides of the additional labeling agents include a sample barcode sequence (e.g., sample index) that allows associating the antibodies with their source biological sample. In some embodiments, the reporter oligonucleotides can further include a barcode sequence that permits identification of a pretreatment condition to which the biological sample (or subject from whom the biological sample is obtained) is subjected prior to step (a) obtaining the plurality of B cells from the biological sample. In some embodiments, the pretreatment is performedd prior to the step of contacting the B cells with the antigens.

[0335] For example, a reporter oligonucleotide can be linked to an antibody or an epitope binding fragment thereof, and labeling a cell can include subjecting the antibody-linked barcode molecule or the epitope binding fragment-linked barcode molecule to conditions suitable for binding the antibody to a molecule present on a surface of the cell. The binding affinity between the antibody or the epitope-binding fragment thereof and the molecule present on the surface can be within a desired range to ensure that the antibody or the epitope binding fragment thereof remains bound to the molecule. For example, the binding affinity can be within a desired range to ensure that the antibody or the epitope binding fragment thereof remains bound to the molecule during various sample processing steps, such as partitioning and/or nucleic acid amplification or extension. A dissociation constant (Kd) between the antibody or an epitope binding fragment thereof and the molecule to which it binds can be less than about 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 900 hM, 800 hM, 700 hM, 600 hM, 500 hM, 400 hM, 300 hM, 200 hM, 100 hM, 90 hM, 80 hM, 70 hM, 60 hM, 50 hM, 40 hM, 30 hM, 20 hM, 10 hM, 9 hM, 8 hM, 7 hM, 6 hM, 5 hM, 4 hM, 3 hM, 2 hM, 1 hM, 900 rM, 800 rM, 700 rM, 600 rM, 500 rM, 400 rM, 300 rM, 200 rM, 100 rM, 90 rM, 80 rM, 70 rM, 60 rM, 50 rM, 40 rM, 30 rM, 20 rM, 10 rM, 9 rM, 8 rM, 7 rM, 6 rM, 5 rM, 4 rM, 3 rM, 2 rM, or 1 rM. For example, the dissociation constant can be less than about 10 mM. In some embodiments, the antibody or antigen-binding fragment thereof has a desired dissociation rate constant (koff), such that the antibody or antigen binding fragment thereof remains bound to the target antigen or antigen fragment during various sample processing steps.

[0336] In another example, a reporter oligonucleotide can be coupled to a cell-penetrating peptide (CPP), and labeling cells can include delivering the CPP coupled reporter oligonucleotide into an analyte carrier. Labeling analyte carriers can include delivering the CPP conjugated oligonucleotide into a cell and/or cell bead by the cell-penetrating peptide. A CPP that can be used in the methods provided herein can include at least one non-functional cysteine residue, which can be either free or derivatized to form a disulfide link with an oligonucleotide that has been modified for such linkage. Non-limiting examples of CPPs that can be used in embodiments herein include penetratin, transportan, plsl, TAT(48-60), pVEC, MTS, and MAP. Cell-penetrating peptides useful in the methods provided herein can have the capability of inducing cell penetration for at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of cells of a cell population. The CPP can be an arginine-rich peptide transporter. The CPP can be Penetratin or the Tat peptide. In another example, a reporter oligonucleotide can be coupled to a fluorophore or dye, and labeling cells can include subjecting the fluorophore-linked barcode molecule to conditions suitable for binding the fluorophore to the surface of the cell. In some instances, fluorophores can interact strongly with lipid bilayers and labeling cells can include subjecting the fluorophore-linked barcode molecule to conditions such that the fluorophore binds to or is inserted into a membrane of the cell. In some cases, the fluorophore is a water-soluble, organic fluorophore. In some instances, the fluorophore is Alexa 532 maleimide, tetramethylrhodamine-5-maleimide (TMR maleimide), BODIPY-TMR maleimide, Sulfo-Cy3 maleimide, Alexa 546 carboxylic acid/succinimidyl ester, Atto 550 maleimide, Cy3 carboxylic acid/succinimidyl ester, Cy3B carboxylic acid/succinimidyl ester, Atto 565 biotin, Sulforhodamine B, Alexa 594 maleimide, Texas Red maleimide, Alexa 633 maleimide, Abberior STAR 635P azide, Atto 647N maleimide, Atto 647 SE, or Sulfo-Cy5 maleimide. See, e.g., Hughes L D, etal. PLoS One. 2014 Feb. 4; 9(2):e87649, for a description of organic fluorophores.

[0337] A reporter oligonucleotide can be coupled to a lipophilic molecule, and labeling cells can include delivering the nucleic acid barcode molecule to a membrane of a cell or a nuclear membrane by the lipophilic molecule. Lipophilic molecules can associate with and/or insert into lipid membranes such as cell membranes and nuclear membranes. In some cases, the insertion can be reversible. In some cases, the association between the lipophilic molecule and the cell or nuclear membrane can be such that the membrane retains the lipophilic molecule (e.g, and associated components, such as nucleic acid barcode molecules, thereof) during subsequent processing (e.g, partitioning, cell permeabilization, amplification, pooling, etc.). The reporter nucleotide can enter into the intracellular space and/or a cell nucleus. In some embodiments, a reporter oligonucleotide coupled to a lipophilic molecule will remain associated with and/or inserted into lipid membrane (as described herein) via the lipophilic molecule until lysis of the cell occurs, e.g, inside a partition. Exemplary embodiments of lipophilic molecules coupled to reporter oligonucleotides are described in PCT/US2018/064600.

[0338] A reporter oligonucleotide can be part of a nucleic acid molecule including any number of functional sequences, as described elsewhere herein, such as a target capture sequence, a random primer sequence, and the like, and coupled to another nucleic acid molecule that is, or is derived from, the analyte.

[0339] Prior to partitioning, the cells can be incubated with the library of labelling agents, that can be labelling agents to a broad panel of different cell features, e.g., receptors, proteins, etc., and which include their associated reporter oligonucleotides. Unbound labelling agents can be washed from the cells, and the cells can then be co-partitioned (e.g, into droplets or wells) along with partition-specific barcode oligonucleotides (e.g, attached to a support, such as a bead or gel bead) as described elsewhere herein. As a result, the partitions can include the cell or cells, as well as the bound labelling agents and their known, associated reporter oligonucleotides.

[0340] In other instances, e.g, to facilitate sample multiplexing, a labelling agent that is specific to a particular cell feature can have a first plurality of the labelling agent (e.g, an antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labelling agent coupled to a second reporter oligonucleotide. For example, the first plurality of the labeling agent and second plurality of the labeling agent can interact with different cells, cell populations or samples, allowing a particular report oligonucleotide to indicate a particular cell population (or cell or sample) and cell feature. In this way, different samples or groups can be independently processed and subsequently combined together for pooled analysis (e.g, partition-based barcoding as described elsewhere herein). See, e.g, U.S. Pat. Pub. 20190323088.

[0341] In some embodiments, to facilitate sample multiplexing, individual samples can be stained with lipid tags, such as cholesterol -modified oligonucleotides (CMOs, see, e.g, FIG.

7A), anti-calcium channel antibodies, or anti-ACTB antibodies. Non-limiting examples of anti calcium channel antibodies include anti-KCNN4 antibodies, anti-BK channel beta 3 antibodies, anti-alB calcium channel antibodies, and anti-CACNAl A antibodies. Examples of anti-ACTB antibodies suitable for the methods of the disclosure include, but are not limited to, mAbGEa, ACTN05, AC- 15, 15G5A11/E2, BA3R, and HHF35.

[0342] As described elsewhere herein, libraries of labelling agents can be associated with a particular cell feature as well as be used to identify analytes as originating from a particular cell population, or sample. Cell populations can be incubated with a plurality of libraries such that a cell or cells include multiple labelling agents. For example, a cell can include coupled thereto a lipophilic labeling agent and an antibody. The lipophilic labeling agent can indicate that the cell is a member of a particular cell sample, whereas the antibody can indicate that the cell includes a particular analyte. In this manner, the reporter oligonucleotides and labelling agents can allow multi-analyte, multiplexed analyses to be performed.

[0343] In some instances, these reporter oligonucleotides can include nucleic acid barcode sequences that permit identification of the labelling agent which the reporter oligonucleotide is coupled to. The use of oligonucleotides as the reporter can provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g, using sequencing or array technologies.

[0344] Attachment (coupling) of the reporter oligonucleotides to the labelling agents can be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, oligonucleotides can be covalently attached to a portion of a labelling agent (such a protein, e.g, an antibody or antibody fragment), e.g., via a linker, using chemical conjugation techniques (e.g, Lightning-Link® antibody labelling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g, using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g, Fang, etal, “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g, U.S. Pat. No. 6,265,552. Furthermore, click reaction chemistry such as 5’ Azide oligos and Alkyne- NHS for click chemistry, 4’-Amino oligos for HyNic-4B chemistry, a Methyl tetrazine-PEG5- NHS Ester reaction, a TCO-PEG4-NHS Ester reaction, strain-promoted alkyne-azide cycloaddition (SPAAC), or the like, can be used to couple reporter oligonucleotides to labelling agents. Commercially available kits, such as those from Thunderlink and Abeam, and techniques common in the art can be used to couple reporter oligonucleotides to labelling agents as appropriate. In another example, a labelling agent is indirectly (e.g, via hybridization) coupled to a reporter oligonucleotide including a barcode sequence that identifies the label agent. For instance, the labelling agent can be directly coupled ( e.g ., covalently bound) to a hybridization oligonucleotide that includes a sequence that hybridizes with a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labelling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotides are releasable from the labelling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide can be attached to the labeling agent through a labile bond (e.g., chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein. In some instances, the reporter oligonucleotides described herein can include one or more functional sequences that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, a sequencing primer or primer biding sequence (such as an Rl, R2, or partial R1 or R2 sequence).

[0345] In some cases, the labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a monomer. In some cases, the labelling agent is presented as a multimer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a dimer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a trimer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a tetramer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a pentamer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a hexamer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a heptamer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as an octamer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a nonamer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a decamer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a 10+-mer. [0346] In some cases, the labelling agent can include a reporter oligonucleotide and a label ( e.g ., detectable label). A label (e.g. , detectable label) can be a fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be conjugated to a labelling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labelling agent or reporter oligonucleotide). In some cases, a label is conjugated to an oligonucleotide that is complementary to a sequence of the reporter oligonucleotide, and the oligonucleotide can be allowed to hybridize to the reporter oligonucleotide.

[0347] FIG. 7A describes exemplary labelling agents (710, 720, 730) including reporter oligonucleotides (740) attached thereto. Labelling agent 710 (e.g, any of the labelling agents described herein) is attached (either directly, e.g, covalently attached, or indirectly) to reporter oligonucleotide 740. Reporter oligonucleotide 740 can include barcode sequence 742 that identifies labelling agent 710. Reporter oligonucleotide 740 can also include one or more functional sequences 743 that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, or a sequencing primer or primer biding sequence (such as an Rl, R2, or partial R1 or R2 sequence).

[0348] Referring to FIG. 7A, in some instances, reporter oligonucleotide 740 conjugated to a labelling agent (e.g, 710, 720, 730) includes a functional sequence 741, a reporter barcode sequence 742 that identifies the labelling agent (e.g, 710, 720, 730), and reporter capture handle 743. Reporter capture handle sequence 743 can be configured to hybridize to a complementary sequence, such as a complementary sequence present on a nucleic acid barcode molecule (not shown), such as those described elsewhere herein. In some instances, nucleic acid barcode molecule is attached to a support (e.g, a bead, such as a gel bead), such as those described elsewhere herein (e.g, FIGS. 3, 4, 8 and 9A-9C). For example, nucleic acid barcode molecule can be attached to the support via a releasable linkage (e.g, including a labile bond), such as those described elsewhere herein (e.g, FIGS. 3, 4, 8 and 9A-9C). In some instances, reporter oligonucleotide 740 includes one or more additional functional sequences, such as those described above.

[0349] In some instances, the labelling agent 710 is a protein or polypeptide (e.g, an antigen or prospective antigen) including reporter oligonucleotide 740. Reporter oligonucleotide 740 includes reporter barcode sequence 742 that identifies polypeptide 710 and can be used to infer the presence of an analyte, e.g., a binding partner of polypeptide 710 (i.e., a molecule or compound to which polypeptide 710 can bind). In some instances, the labelling agent 710 is a lipophilic moiety (e.g, cholesterol) including reporter oligonucleotide 740, where the lipophilic moiety is selected such that labelling agent 710 integrates into a membrane of a cell or nucleus. Reporter oligonucleotide 740 includes reporter barcode sequence 742 that identifies lipophilic moiety 710 which in some instances is used to tag cells (e.g, groups of cells, cell samples, etc.) and can be used for multiplex analyses as described elsewhere herein. In some instances, the labelling agent is an antibody 720 (or an epitope binding fragment thereof) including reporter oligonucleotide 740. Reporter oligonucleotide 740 includes reporter barcode sequence 742 that identifies antibody 720 and can be used to infer the presence of, e.g, a target of antibody 720 (i.e., a molecule or compound to which antibody 720 binds). In other embodiments, labelling agent 730 includes an MHC molecule 731 including peptide 732 and reporter oligonucleotide 740 that identifies peptide 732. In some instances, the MHC molecule is coupled to a support 733. In some instances, support 733 can be a polypeptide, such as streptavidin, or a polysaccharide, such as dextran. In some instances, reporter oligonucleotide 740 can be directly or indirectly coupled to MHC labelling agent 730 in any suitable manner. For example, reporter oligonucleotide 740 can be coupled to MHC molecule 731, support 733, or peptide 732. In some embodiments, labelling agent 730 includes a plurality of MHC molecules, (e.g. is an MHC multimer, which can be coupled to a support (e.g, 733)). There are many possible configurations of Class I and/or Class II MHC multimers that can be utilized with the methods and systems disclosed herein, e.g, MHC tetramers, MHC pentamers (MHC assembled via a coiled-coil domain, e.g, Pro5® MHC Class I Pentamers, (Prolmmune, Ltd.), MHC octamers, MHC dodecamers, MHC decorated dextran molecules (e.g, MHC Dextramer® (Immudex)), etc. For a description of exemplary labelling agents, including antibody and MHC -based labelling agents, reporter oligonucleotides, and methods of use, see, e.g, U.S. Pat. 10,550,429 and U.S. Pat. Pub. 20190367969.

[0350] Referring to FIG. 7B, in some instances, reporter oligonucleotide 740 is conjugated to a support 750 that can be used to complex with or bind to an antigen (e.g, an antigen of interest or a non-target antigen). Reporter oligonucleotide 740 includes a functional sequence 741, a reporter barcode sequence 742 that identifies the antigen of interest, and reporter capture handle 743. Reporter capture handle sequence 743 can be configured to hybridize to a complementary sequence, such as a complementary sequence present on a nucleic acid barcode molecule (not shown), such as those described elsewhere herein ( e.g ., FIGS. 3, 4, 8 and 9A-9C). In some instances, nucleic acid barcode molecule is attached to a support (e.g., a bead, such as a gel bead), such as those described elsewhere herein (e.g, FIGS. 3, 4, 8 and 9A-9C). For example, nucleic acid barcode molecule can be attached to the support via a releasable linkage (e.g, including a labile bond), such as those described elsewhere herein. In some instances, reporter oligonucleotide 740 includes one or more additional functional sequences, such as those described above. In one other embodiment, support 750 comprises an anchor sequence 745 that is complementary to functional sequence 741. The reporter oligonucleotide 740 may be attached to support 750 via hybridization to anchor sequence 745. The anchor sequence 745 may further comprise (or may be) a functional sequence (similar to or equivalent to functional sequence 741) as described herein. In some embodiments, the anchor sequence 745 does not comprise a functional sequence. In some embodiments, reporter oligonucleotide 740 includes a functional sequence (not shown). A support 750 may comprise a binding region that can be used to complex with (or bind to) an antigen of interest. In one embodiment, the antigen of interest comprises a ligand that can be bound by the binding region of support 750.

[0351] Referring to FIG. 7B again, labeling agents for antigen receptor analysis are provided. In one embodiment, labelling agent 760 comprises a support 750 that includes an antigen of interest 753 and reporter oligonucleotide 740 that identifies the antigen 753 (e.g, via reporter barcode sequence 742). In some embodiments, the support 750 is coupled to, complexed with, or bound to a ligand 751. In some embodiments, support 750 can be a polypeptide. In some embodiments, the polypeptide can be streptavidin. In some embodiments, the polypeptide can be avidin. In some embodiments, support 750 can be a polysaccharide. In some embodiments, the polysaccharide can be dextran. In some embodiments, the polysaccharide can be a dextran. The ligand 751 can be a molecule with affinity for the binding region of the support 750. For example, the ligand 751 may be biotin and the support 750 may be a streptavidin support. In other embodiments, the ligand 751 is coupled to or conjugated to antigen 753 via a linker 752. Accordingly, in some embodiments of the disclosure, the partitioned cells are contacted with one or more biotinylated antigens. In some embodiments, the antigens can include Avitag biotinylation site and/or a His tag. Protein biotinylation techniques are available. See, e.g., Fang, et al, “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715, and U.S. Pat. No. 6,265,552. In some embodiments, the partitioned cells are contacted with full- length coronavirus spike proteins comprising a trimerization domain. In some embodiments, reporter oligonucleotide 740 can be directly or indirectly coupled to labelling agent 760 in any suitable manner. For example, reporter oligonucleotide 740 can be coupled to the antigen 753, support 750, anchor sequence 745, or ligand 751.

[0352] Referring to FIG. 7C, a labelled cell 755 comprising an antigen receptor of interest 754 is depicted. The labelling agent 760 can be contacted with a plurality of cells comprising antigen receptors of interest. In one example, an antigen receptor of interest 754 is bound by or labeled with the labelling agent 760 via an interaction between the antigen receptor of interest 754 and the antigen 753. Further processing of the labelled cell 755 can be performed in a partition-based methods and system as further described herein.

[0353] Exemplary barcode molecules attached to a support (e.g., a bead) is shown in FIG. 9. In some embodiments, analysis of multiple analytes (e.g, RNA and one or more analytes using labelling agents described herein) can include nucleic acid barcode molecules as generally depicted in FIG. 9. In some embodiments, nucleic acid barcode molecules 910 and 920 are attached to support 930 via a releasable linkage 940 (e.g, including a labile bond) as described elsewhere herein. Nucleic acid barcode molecule 910 can include functional sequence 911, barcode sequence 912 and capture sequence 913. Nucleic acid barcode molecule 920 can include adapter sequence 921, barcode sequence 912, and adapter sequence 923, wherein adapter sequence 923 includes a different sequence than adapter sequence 913. In some instances, adapter 911 and adapter 921 include the same sequence. In some instances, adapter 911 and adapter 921 include different sequences. Although support 930 is shown including nucleic acid barcode molecules 910 and 920, any suitable number of barcode molecules including common barcode sequence 912 are contemplated herein. For example, in some embodiments, support 930 further includes nucleic acid barcode molecule 950. Nucleic acid barcode molecule 950 can include adapter sequence 951, barcode sequence 912 and adapter sequence 953, wherein adapter sequence 953 includes a different sequence than adapter sequence 913 and 923. In some instances, nucleic acid barcode molecules (e.g, 910, 920, 950) include one or more additional functional sequences, such as a UMI or other sequences described herein. The nucleic acid barcode molecules 910, 920 or 950 can interact with analytes as described elsewhere herein, for example, as depicted in FIGS. 10A-10C.

[0354] Referring to FIG. 10A, in an instance where cells are labelled with labeling agents, capture sequence 1023 can be complementary to an adapter sequence of a reporter oligonucleotide. Cells can be contacted with one or more reporter oligonucleotide 1020 conjugated labelling agents 1010 ( e.g ., polypeptide such as an antigen, antibody, or others described elsewhere herein). In some cases, the cells can be further processed prior to barcoding. For example, such processing steps can include one or more washing and/or cell sorting steps. In some instances, a cell that is bound to labelling agent 1010 which is conjugated to reporter oligonucleotide 1020, and a support 1030 (e.g., a bead, such as a gel bead) including nucleic acid barcode molecule 1090 is partitioned into a partition amongst a plurality of partitions (e.g., a droplet of a droplet emulsion or a well of a microwell array). In some instances, the partition includes at most a single cell bound to labelling agent 1010. In some instances, reporter oligonucleotide 1020 conjugated to labelling agent 1010 (e.g, polypeptide such as an antigen, an antibody, pMHC molecule such as an MHC multimer, etc.) includes a first adapter sequence 1011 (e.g, a primer sequence), a barcode sequence 1012 that identifies the labelling agent 1010 (e.g, the polypeptide such as an antigen, antibody, or peptide of a pMHC molecule or complex), and a capture handle sequence 1013. Capture handle sequence 1013 can be configured to hybridize to a complementary sequence, such as capture sequence 1023 present on a nucleic acid barcode molecule 1090 (e.g, partition-specific barcode molecule). In some instances, reporter oligonucleotide 1020 includes one or more additional functional sequences, such as those described elsewhere herein.

[0355] Barcoded nucleic acid molecules can be generated (e.g, via a nucleic acid reaction, such as nucleic acid extension, reverse transcription, or ligation) from the constructs described in FIGS. 10A-10C. For example, capture handle sequence 1013 can then be hybridized to complementary capture sequence 1023 to generate (e.g, via a nucleic acid reaction, such as nucleic acid extension or ligation) a barcoded nucleic acid molecule including cell barcode (for example, common barcode, e.g. , partition-specific barcode) sequence 1022 (or a reverse complement thereof) and reporter barcode sequence 1012 (or a reverse complement thereof). In some embodiments, the nucleic acid barcode molecule 1090 (e.g, partition-specific barcode molecule) further includes a UMI. Barcoded nucleic acid molecules can then be optionally processed as described elsewhere herein, e.g., to amplify the molecules and/or append sequencing platform specific sequences to the fragments. See, e.g, U.S. Pat. Pub. 2018/0105808. Barcoded nucleic acid molecules, or derivatives generated therefrom, can then be sequenced on a suitable sequencing platform.

[0356] In some instances, analysis of multiple analytes (e.g., nucleic acids and one or more analytes using labelling agents described herein) can be performed. For example, the workflow can include a workflow as generally depicted in any of FIGS. 10A-10C, or a combination of workflows for an individual analyte, as described elsewhere herein. For example, by using a combination of the workflows as generally depicted in FIGS. 10A-10C, multiple analytes can be analyzed.

[0357] In some instances, analysis of an analyte (e.g. a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc.) includes a workflow as generally depicted in FIG. 10A. A nucleic acid barcode molecule 1090 can be co-partitioned with the one or more analytes. In some instances, nucleic acid barcode molecule 1090 is attached to a support 1030 (e.g, a bead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 1090 can be attached to support 1030 via a releasable linkage 1040 (e.g, including a labile bond), such as those described elsewhere herein. Nucleic acid barcode molecule 1090 can include a barcode sequence 1021 and optionally include other additional sequences, for example, a barcode sequence 1022 (e.g, common barcode, partition-specific barcode, UMI, or other functional sequences described elsewhere herein). Nucleic acid barcode molecule 1090 can include a functional sequence 1021. In some embodiments, the nucleic acid barcode molecule 1090 can include other additional sequences, for example, a barcode sequence 1022 (e.g, common barcode, partition-specific barcode, or other functional sequences described elsewhere herein), and/or a UMI sequence. The nucleic acid barcode molecule 1090 can include a capture sequence 1023 that can be complementary to another nucleic acid sequence, such that it can hybridize to a particular sequence.

[0358] For example, capture sequence 1023 can include a poly-T sequence and can be used to hybridize to mRNA. Referring to FIG. IOC, in some embodiments, nucleic acid barcode molecule 1090 includes capture sequence 1023 complementary to a sequence of RNA molecule 1060 from a cell. In some instances, capture sequence 1023 includes a sequence specific for an RNA molecule. Capture sequence 1023 can include a known or targeted sequence or a random sequence. In some instances, a nucleic acid extension reaction can be performed, thereby generating a barcoded nucleic acid product including capture sequence 1023, the functional sequence 1021, UMI and/or barcode sequence 1022, any other functional sequence, and a sequence corresponding to the RNA molecule 1060.

[0359] In another example, capture sequence 1023 can be complementary to an overhang sequence or an adapter sequence that has been appended to an analyte. In one embodiment, capture sequence 1023 is complementary to a sequence that has been appended to a nucleic acid molecule derived from an analyte of interest. In another embodiment, the nucleic acid molecule is a cDNA molecule generated in a reverse transcription reaction using an RNA analyte ( e.g ., an mRNA analyte) of interest. In an additional embodiment, capture sequence 1023 is complementary to a sequence that has been appended to the cDNA molecule generated from the mRNA analyte of interest. For example, referring to FIG. 10B, in some embodiments, primer 1050 includes a sequence complementary to a sequence of nucleic acid molecule 1060 (such as an RNA encoding for a BCR sequence) from a biological particle. In some instances, primer 1050 includes one or more sequences 1051 that are not complementary to RNA molecule 1060. Sequence 1051 can be a functional sequence as described elsewhere herein, for example, an adapter sequence, a sequencing primer sequence, or a sequence the facilitates coupling to a flow cell of a sequencer. In some instances, primer 1050 includes a poly-T sequence. In some instances, primer 1050 includes a sequence complementary to a target sequence in an RNA molecule. In some instances, primer 1050 includes a sequence complementary to a region of an immune molecule, such as the constant region of a BCR sequence. Primer 1050 is hybridized to nucleic acid molecule 1060 and complementary molecule 1070 is generated. For example, complementary molecule 1070 can be cDNA generated in a reverse transcription reaction. In some instances, an additional sequence can be appended to complementary molecule 1070. For example, the reverse transcriptase enzyme can be selected such that several non-templated bases 1080 (e.g., a poly-C sequence) are appended to the cDNA. In another example, a terminal transferase can also be used to append the additional sequence. Nucleic acid barcode molecule 1090 includes a sequence 1024 complementary to the non-templated bases, and the reverse transcriptase performs a template switching reaction onto nucleic acid barcode molecule 1090 to generate a barcoded nucleic acid molecule including cell (e.g, partition specific) barcode sequence 1022 (or a reverse complement thereof) and a sequence of complementary molecule 1070 (or a portion thereof). In some instances, capture sequence 1023 includes a sequence complementary to a region of an immune molecule, such as the constant region of a BCR sequence. Capture sequence 1023 is hybridized to nucleic acid molecule 1060 and a complementary molecule 1070 is generated. For example, complementary molecule 1070 can be generated in a reverse transcription reaction generating a barcoded nucleic acid molecule including cell barcode ( e.g ., common barcode or partition-specific barcode) sequence 1022 (or a reverse complement thereof) and a sequence of complementary molecule 1070 (or a portion thereof). Additional methods and compositions suitable for barcoding cDNA generated from mRNA transcripts including those encoding V(D)J regions of an immune cell receptor and/or barcoding methods and composition including a template switch oligonucleotide are described in International Patent Application WO2018/075693, U.S. Patent Publication No. 2018/0105808, U.S. Patent Publication No. 2015/0376609, and U.S. Patent Publication No. 2019/0367969.

[0360] In some embodiments, biological particles (e.g., cells, nuclei) from a plurality of samples (e.g, a plurality of subjects) can be pooled, sequenced, and demultiplexed by identifying mutational profiles associated with individual samples and mapping sequence data from single biological particles to their source based on their mutational profile. See, e.g, Xu J. etal, Genome Biology Vol. 20, 290 (2019); Huang Y. etal., Genome Biology Vol. 20, 273 (2019); and Heaton et al, Nature Methods volume 17, pages 615-620(2020).

[0361] Gene expression data can reflect the underlying genome and mutations and structural variants therein. As a result, the variation inherent in the captured and sequenced RNA molecules can be used to identify genotypes de novo or used to assign molecules to genotypes that were known a priori. In some embodiments, allelic variation that is present due to haplotypic states (including linkage disequilibrium of the human leucocyte antigen loci (HLA), immune receptor loci (BCR), and other highly polymorphic regions of the genome), can also be used for demultiplexing. Expressed B cell receptors can be used to infer germline alleles from unrelated individuals, which information may be used for demultiplexing.

COMPOSITIONS OF THE DISCLOSURE

[0362] As described in greater detail below, one aspect of the present disclosure relates to antigen-binding molecules (e.g., antibodies) or antigen-binding fragments thereof that were identified by a method disclosed herein. There are no particular limitations to the types of antigen-binding molecules or antigen-binding fragments thereof that can be suitably identified by the methods disclosed herein. Examples of suitable antigen-binding molecules include, but are not limited to, those capable of binding or as having an affinity for a target antigen associated with an infectious agent, such as a viral agent, bacterial agent, parasitic agent, protozoal agent, or prion agent. Further, the target antigen may be associated with a tumor or a tumor or cancer. In addition, the target antigen may be an immune checkpoint molecule that may or may not be associated with tumors or cancers, or it may be a cytokine, a GPCR, a cell-based co-stimulatory molecule, a cell-based co-inhibitory molecule, an ion channel, or a growth factor. Further still, the target antigen may be associated with a degenerative condition or disease ( e.g ., an amyloid protein). In some embodiments, provided herein are anti-CoV-S antigen-binding polypeptides, such as antibodies and antigen-binding fragments thereof, that specifically bind to CoV spike protein or an antigenic fragment thereof. Also provided, in other related aspects of the disclosure, are nucleic acids encoding the antibodies and antigen-binding fragments as disclosed herein, recombinant cells and transgenic animals engineered to produce the antibodies and antigen binding fragments as disclosed herein, pharmaceutical compositions containing one or more of the nucleic acids, recombinant cells, and antibodies and antigen-binding fragments as disclosed herein.

Antigen-binding proteins

[0363] One aspect of the present disclosure relates to antigen-binding polypeptides that were identified by a method disclosed herein, such as antibodies and antigen-binding fragments thereof, e.g., that specifically bind to CoV spike protein or an antigenic fragment thereof.

[0364] An antibody is generally understood by the skilled artisan in the art to refer to immunoglobulin molecules including four polypeptide chains, two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds (i.e., “full antibody molecules”), as well as multimers thereof (e.g. IgM). Each heavy chain includes a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (which is comprised of domains CHI, CH2 and CH3). Each light chain is comprised of a light chain variable region (“LCVR or “VL”) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FWR). Each VH and VL includes three CDRs and four FWRs, arranged from amino-terminus to carboxy-terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. Heavy chain CDRs can also be referred to as HCDRs, and numbered as described above ( e.g ., HCDR1, HCDR2, and HCDR3). Likewise, light chain CDRs can be referred to as LCDRs, and numbered LCDR1, LCDR2, and LCDR3. In some embodiments of the disclosure, the FRs of the antibodies or antigen binding fragments thereof are identical to the human germline sequences, or are naturally or artificially modified.

[0365] In some embodiments of the disclosure, the assignment of amino acids to each domain is in accordance with the definitions of Sequences of Proteins of Immunological Interest, Rabat, etal.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Rabat (1978) Adv. Prot. Chem. 32:1-75; Rabat, etal., (1977) J. Biol. Chem. 252:6609- 6616; Chothia, etal., (1987) J Mol. Biol. 196:901-917 or Chothia, etal., (1989) Nature 342:878- 883.

[0366] The term “anti gen -binding fragment” of an antibody or antigen-binding polypeptide, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g, monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.

[0367] An antigen-binding fragment of an antibody, in some embodiment of the disclosure, include at least one variable domain. The variable domain can be of any size or amino acid composition and will generally include at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be situated relative to one another in any suitable arrangement. For example, the variable region can be dimeric and contain VH-VH, VH- VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0368] In some embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that can be found within an antigen binding fragment of an antibody of the present disclosure include: (i) V_H-C_H1; (ii) V_H-C_H2; (iii) V_H-C_H3; (iv) V_H-C_H1-C_H2; (V) V_H-C_H1-C_H2-C_H3; (vi) V_H-C_H2-C_H3; (vii) V_H-C_L; (viii) V_L-C_H1; (ix) V_L-C_H2; (X) V_L-C_H3; (xi) V_L-C_H1-C_H2; (xii) V_L-C_H1-C_H2-C_H3; (xiii) V_L-C_H2-C_H3; and (xiv) V_L-C_L. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 ( e.g ., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present disclosure may include a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). Antigen-binding proteins (e.g, antibodies and anti gen -binding fragments) can be mono-specific or multi-specific (e.g, bi- specific).

[0369] In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a constant region. In some embodiments, the constant region is an IgA, IgD, IgE, IgG, or IgM heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment of the disclosure includes a constant region of the type IgA (e.g, IgAl or IgA2), IgD, IgE, IgG (e.g, IgGl, IgG2, IgG3 and IgG4) or IgM. In some embodiments, the constant region is an IgG constant region.

[0370] In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a kappa type light chain constant region. In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a lambda type light chain constant region.

[0371] In some embodiments, the antibody or antigen-binding fragment of the disclosure is a human antibody or antigen-binding fragment. One of ordinary skill in the art will understand that the term “human” antibody includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non human cell, e.g ., a mouse cell. The human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, such as CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g, mouse) have been grafted onto human FWR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal.

[0372] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody, a chimeric antibody, or a hybrid antibody. The term “humanized antibody” as used herein encompasses antibodies comprising heavy and light chain variable region sequences from a non-human species (e.g, a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Another type of humanized antibody is a FWR- grafted antibody in which human FWR sequences are introduced into non-human VH and VL sequences to replace corresponding non-human FWR sequences. In some embodiments, the antibodies or antigen-binding fragments of the disclosure include a murine antibody, phage display antibody, or nanobody / VHH containing the frameworks and/or CDRs described in this disclosure. As used herein, the term “chimeric antibody” encompasses antibodies having the variable domain from a first antibody and the constant domain from a second antibody, wherein the first and second antibodies are from different species. As used herein, the term “hybrid antibody” encompasses antibodies having the variable domain from a first antibody and the constant domain from a second antibody, wherein the first and second antibodies are from different animals, or wherein the variable domain, but not the constant region, is from a first animal. For example, a variable domain can be taken from an antibody isolated from a human and expressed with a fixed constant region not isolated from that antibody. In some embodiments, hybrid antibodies can be synthetic and/or non-naturally occurring because the variable and constant regions they contain are not isolated from a single natural source. In some embodiments, the hybrid antibodies of the disclosure includes a light chain from a first antibody and a heavy chain from a second antibody, wherein the first and second antibodies are from different species. In some embodiments, the chimeric antibodies of the disclosure includes a non human light chain which is combined with a heavy chain or set of heavy chain CDRs disclosed in this application.

[0373] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment (scFv), a Fab, a Fab', a Fab'-SH, a F(ab')2, or a Fv fragment.

[0374] In some embodiments, the antibody or antigen-binding fragment has a binding affinity ( e.g ., ability to bind, with varying degrees of specificity) to an epitope in a domain of the S protein of SARS-CoV-2.

[0375] Methods for determining the epitope of an antigen-binding polypeptide, e.g., antibody or antigen-binding fragment or polypeptide, include alanine scanning mutational analysis, peptide blot analysis, peptide cleavage analysis, crystallographic studies andNMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed. Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding polypeptide (e.g, antibody or fragment or polypeptide) interacts is hydrogen/deuterium exchange detected by mass spectrometry.

[0376] In some embodiments, the antibodies and antigen-binding fragments of the disclosure bind to a target antigen, such as a CoV-S protein (e.g, SARS-CoV-2 S protein), and compete for binding with another antigen-binding polypeptide (e.g, antibody or antigen-binding fragment thereof) to the target antigen. Accordingly, also provided herein are antibodies or antigen-binding fragments thereof that compete for binding with an antibody described herein.

[0377] The term “competes” as used herein, refers to an antibody or antigen-binding fragment that binds to a target antigen, and inhibits or blocks the binding of another antigen binding polypeptide (e.g, antibody or antigen-binding fragment thereof) to the target antigen.

The term also includes competition between two antigen-binding polypeptides e.g, antibodies, in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice versa. In some embodiments, the first antigen-binding polypeptide (e.g, antibody or antigen- binding fragment) and second antigen-binding polypeptide ( e.g ., antibody or antigen-binding fragment thereof) may bind to the same epitope. Alternatively, the first and second antigen binding polypeptides (e.g., antibodies or antigen-binding fragments) may bind to different, but, for example, overlapping epitopes, wherein binding of one inhibits or blocks the binding of the second antibody, e.g, via steric hindrance. Competition between antigen-binding polypeptides (e.g, antibodies or antigen-binding fragments) may be measured by methods known in the art, for example, by a real-time, label -free bio-layer interferometry assay. Epitope mapping (e.g, via alanine scanning or hydrogen-deuterium exchange (HDX)) can be used to determine whether two or more antibodies are non-competing (e.g, on a spike protein receptor binding domain (RBD) monomer), competing for the same epitope, or competing but with diverse micro-epitopes (e.g, identified through HDX). In some embodiments, competition between a first and second anti-CoV-S antigen-binding polypeptide (e.g, antibody or antigen-binding fragment thereof) is determined by measuring the ability of an immobilized first anti-CoV-S antigen-binding polypeptide (e.g, antibody) (not initially complexed with CoV-S protein) to bind to soluble CoV-S protein complexed with a second anti-CoV-S antigen-binding polypeptide (e.g, antibody or antigen-binding fragment thereof). A reduction in the ability of the first anti-CoV-S antigen binding polypeptide (e.g, antibody or antigen-binding fragment thereof) to bind to the complexed CoV-S protein, relative to uncomplexed CoV-S protein, indicates that the first and second anti-CoV-S antigen-binding polypeptides (e.g, antibodies or antigen-binding fragments thereof) compete. The degree of competition can be expressed as a percentage of the reduction in binding. Such competition can be measured using a real time, label-free bio-layer interferometry assay, e.g, on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance).

[0378] In some embodiments, the antibodies and antigen-binding fragments of the disclosure have a neutralizing activity (e.g, antagonistic activity) against the target antigen (e.g., SARS-CoV-2), e.g, able to bind to and neutralize the activity of the antigen (e.g., SARS-CoV- S), as determined by in vitro or in vivo assays. The ability of the antibodies of the disclosure to bind to, block and/or neutralize the activity of the target antigen (e.g., SARS-CoV-2) may be measured using any standard method known to those skilled in the art, including binding assays, or activity assays, as described herein. For example, the binding affinity and dissociation constants of anti-SARS-CoV-2 antigen-binding polypeptides for SARS-CoV-2 can be determined by surface plasmon resonance (SPR) assay. Alternatively, neutralization assays were used to determine infectivity of SARS-CoV-2 S protein-containing virus-like particles. One of ordinary skill in the art will understand that a neutralizing or antagonistic CoV-S antigen-binding polypeptide, e.g ., antibody or antigen-binding fragment, generally refers to a molecule that inhibits an activity of CoV-S to any detectable degree, e.g. , inhibits or reduces the ability of CoV-S to bind to a receptor such as ACE2, to be cleaved by a protease such as TMPRSS2, or to mediate viral entry into a host cell or mediate viral reproduction in a host cell. In some embodiments, the antibodies and antigen-binding fragments of the disclosure have a neutralization activity IC50 value of less than 150 ng/ml for viral neutralization, as determined a quantitative focus reduction neutralization test (FRNT) described previously by Zost et al. (Nature, 584:443-449, 2020). In some embodiments, the antibodies and antigen-binding fragments of the disclosure have blocking activity IC50 value of less than 150 ng/ml for blocking ACE2. In some embodiments, the antibodies and antigen-binding fragments of the disclosure have blocking activity IC50 value of less than 10 ng/ml for S2P ectodomain binding. In some embodiments, the antibodies and antigen-binding fragments of the disclosure have blocking activity IC50 value of less than 10 ng/ml for RBD ectodomain binding. In some embodiments, the antibody or antigen-binding fragment neutralizes at least 50% of 200 times the tissue culture infectious dose (200><TCID50) of the coronavirus at an antibody concentration of 12.5 pg/ml or less. Here, TCID50 represents the viral load at which 50% of cells are infected when a solution containing the virus is added to cell culture. In some embodiments, neutralizing antibodies are effective at antibody concentrations of <3.125 pg/ml, <.8 pg/ml, <.2 pg/ml, or <.l pg/ml.

Nucleic acids

[0379] As discussed above, one aspect of the disclosure relates to recombinant nucleic acids including a nucleic acid sequence that encodes an antibody of the disclosure or an antigen binding fragment thereof. In some embodiments, the recombinant nucleic acids of the disclosure can be configured as expression cassettes or vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulatory sequences which allow in vivo expression of the receptor in a host cell.

[0380] Nucleic acid molecules of the present disclosure can be of any length, including for example, between about 200 bp to about 2000 bp, e.g. , between about 200 bp to about 1000 bp, between about 300 bp to about 1200 bp, between about 400 bp to about 1400 bp, between about 500 bp to about 1600 bp, between about 600 bp to about 1800 bp, between about 700 bp to about 2000 bp, between about 200 bp to about 500 bp, or between about 400 bp to about 1200 bp, for example between about 400 bp to 800 bp, between about 500 bp to about 1000 bp, between about 600 bp to about 800 bp, about 700 bp to about 1100 bp, or about 800 bp to about 1200 bp. In some embodiments, the nucleic acid molecules of the present disclosure can be about 1 Kb and about 50 Kb, e.g., between about 1.2 Kb and about 10 Kb, between about 2 Kb and about 15 Kb, between about 5 Kb and about 20 Kb, between about 10 Kb and about 20 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0381] Accordingly, in some embodiments, provided herein is a nucleic acid molecule including a nucleotide sequence encoding an antibody of the disclosure or an antigen-binding fragment thereof. In some embodiments, the nucleotide sequence is incorporated into an expression cassette or an expression vector. It will be understood by the skilled artisan that an expression cassette generally includes a construct of genetic material that contains coding sequences of the antibody or antigen-binding fragment thereof and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo. Generally, the expression cassette can be inserted into a vector for targeting to a desired host cell and/or into an individual. As such, in some embodiments, an expression cassette of the disclosure include a coding sequence for an antibody of the disclosure or an antigen-binding fragment thereof, which is operably linked to expression control elements, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0382] An expression cassette can be inserted into a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, as a linear or circular, single-stranded or double- stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g ., operably linked.

[0383] In some embodiments, the nucleic acid molecule of the disclosure is incorporated into an expression vector. It will be understood by one skilled in the art that the term “vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and that can be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted so as to bring about the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.

[0384] In some embodiments, the expression vector can be a viral vector. As will be appreciated by one of skill in the art, the term “viral vector” is widely used to refer either to a nucleic acid molecule (e.g, a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s). The term viral vector can refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements that are primarily derived from a virus. The term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus, which is a genus of retrovirus.

[0385] The nucleic acid sequences encoding the antibodies and antigen-binding fragments as disclosed herein can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to average levels for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon usage optimization are known in the art. Codon usages within the coding sequence of the antibodies and antigen-binding fragment disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0386] Also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acid molecules encoding any antibody or an antigen-binding fragment thereof as disclosed herein. The nucleic acid molecules can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed/transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology . New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. etal. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. etal. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, L (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. etal. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. etal. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference).

[0387] DNA vectors can be introduced into cells, e.g. , eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection.

[0388] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors , CSH Laboratory Press, Cold Spring Harbor, N. Y.).

[0389] For example, an antibody or an antigen-binding fragment thereof as disclosed herein can be produced in a eukaryotic host, such as a mammalian cells ( e.g ., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans can consult P. Jones, “Vectors: Cloning Applications”, John Wiley and Sons, New York, N.Y., 2009).

[0390] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide, e.g., antibody. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (e.g, either a sense or an antisense strand).

[0391] The nucleic acid molecules are not limited to sequences that encode polypeptides (e.g, antibodies); some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g, the coding sequence of an antibody) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription.

Recombinant cell and cell cultures

[0392] The nucleic acid of the present disclosure can be introduced into a host cell, such as, for example, a Chinese hamster ovary (CHO) cell, to produce a recombinant cell containing the nucleic acid molecule. Introduction of the nucleic acid molecules (e.g, DNA or RNA, including mRNA) or vectors of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery. For example, methods for introduction of heterologous nucleic acid molecules into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the nucleic acid molecule(s) in liposomes, lipid nanoparticle technology, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors such as lentivirus or adeno-associated virus. As discussed in greater detail below, in some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure can be introduced to a subject in nucleic acid form ( e.g , DNA or RNA, including mRNA), such that the subject's own cells produce the antibody. The present disclosure further provides modifications to nucleotide sequences encoding the anti-CoV-S antibodies described herein that result in increased antibody expression, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved affinity or specificity of the antibodies for the CoV spike protein.

[0393] Accordingly, in some embodiments, the nucleic acid molecules can be delivered by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR/Cas genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a mini-circle expression vector for transient expression.

[0394] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells can be achieved by viral transduction. In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0395] Lentiviral-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene- delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell -therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0396] In some embodiments, host cells can be genetically engineered ( e.g transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0397] In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the animal cell is a non-human animal cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the recombinant cell is selected from the group consisting of a baby hamster kidney (BHK) cell, a Chinese hamster ovary cell (CHO cell), an African green monkey kidney cell (Vero cell), a human A549 cell, a human cervix cell, a human CHME5 cell, a human PER.C6 cell, a NS0 murine myeloma cell, a human epidermoid larynx cell, a human fibroblast cell, a human HEK-293 cell, a human HeLa cell, a human HepG2 cell, a human HUH- 7 cell, a human MRC-5 cell, a human muscle cell, a mouse 3T3 cell, a mouse connective tissue cell, a mouse muscle cell, and a rabbit kidney cell. In some embodiments, the recombinant cell is a Pichia pastoris cell or a Saccharomyces cerevisiae cell, both of which are also suitable for production of scFv, scFvFc, Fab, and F(ab’)2.

[0398] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0399] Also provided, in another aspect, are animals including a recombinant nucleic acid or a vector as disclosed herein. In some embodiments, the disclosure provides a transgenic animal that is a non-human animal. In some embodiments, the transgenic animal produces an antibody or antigen-binding fragment as disclosed herein.

[0400] The transgenic non-human host animals of the disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal. In some embodiments, the non-human animals of the disclosure are mice. Other animal species suitable for the compositions and methods of the disclosure include animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response. Examples of such species include but are not limited to rats, rabbits, chickens, goats, pigs, sheep and cows. Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector mediated DNA transfer into early embryos and sperm-mediated transgenesis, adenovirus mediated introduction of DNA into animal sperm e.g ., in pig), retroviral vectors (e.g. , avian species), somatic cell nuclear transfer (e.g, in goats). The state of the art in the preparation of transgenic domestic farm animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.

[0401] In some embodiments, the animal is a vertebrate animal or an invertebrate animal.

In some embodiments, the animal is a mammalian subject. In some embodiments, the mammalian animal is a non-human animal. In some embodiments, the transgenic animals of the disclosure can be made using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR/Cas genome editing, or DNA-guided endonuclease genome editing with NgAgo ( Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the transgenic animals of the disclosure can be made using transgenic microinjection technology and do not require the use of homologous recombination technology and thus are considered to be easier to prepare and select than approaches using homologous recombination.

[0402] In another aspect, provided herein are methods for producing an antibody or antigen-binding fragment thereof, wherein the methods include growing (i) a transgenic animal as disclosed herein, or (ii) a recombinant cell as disclosed herein under conditions such that the antibody or antigen-binding fragment is produced.

[0403] In some embodiments, the methods for producing an antibody or antigen-binding fragment thereof as described herein further include isolating the produced antibody or antigen binding fragment from (i) the transgenic animal or (ii) recombinant cell and/or the medium in which the recombinant cell is cultured. In some embodiments, the mammalian animal is a non human primate. Accordingly, the antibodies or antigen-binding fragments produced by the methods disclosed herein are also within the scope of the disclosure.

[0404] In some embodiments, antibodies and antigen-binding fragments of the present disclosure include immunoglobulin chains having the amino acid sequences set forth herein as well as cellular modifications and in vitro post-translational modifications to the antibody and antigen-binding fragment. For example, the present disclosure includes antibodies and antigen binding fragments thereof that specifically bind to CoV-S comprising heavy and/or light chain amino acid sequences set forth herein ( e.g. , HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3) as well as antibodies and fragments wherein one or more amino acid residues is glycosylated, one or more Asn residues is deamidated, one or more residues (e.g, Met, Trp and/or His) is oxidized, the N-terminal Gin is pyroglutamate (pyroE) and/or the C-terminal Lysine is missing.

Pharmaceutical compositions

[0405] The antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures of the disclosure can be incorporated into compositions, including pharmaceutical compositions.

[0406] In another aspect, the antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures of the disclosure can be incorporated into compositions suitable for various downstream applications, for example, pharmaceutical compositions. Exemplary compositions of the disclosure include pharmaceutical compositions which generally include one or more of the antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures as described herein and a pharmaceutically acceptable excipient, e.g., carrier. In some embodiments, the composition is a sterile composition. In some embodiments, the composition is formulated as a vaccine. In some embodiments, the composition further includes an adjuvant.

[0407] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to an individual. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. The scope of the present disclosure includes desiccated, e.g. , freeze-dried, compositions comprising an anti-CoV-S antigen-binding polypeptides, e.g. , antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof that includes a pharmaceutically acceptable carrier but substantially lacks water. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. In some embodiments, the pharmaceutical composition is sterilely formulated for administration into an individual or an animal (some non-limiting examples include a human, or a mammal). In some embodiments, the individual is a human.

[0408] The terms “administration” and “administering”, as used herein, refer to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, oral, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering. [0409] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated to be suitable for the intended route of administration to an individual. For example, the pharmaceutical composition can be formulated to be suitable for parenteral, intraperitoneal, colorectal, intraperitoneal, and intratumoral administration. In some embodiments, the pharmaceutical composition can be formulated for oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intra-arterial administration. One of ordinary skilled in the art will appreciate that the formulation should suit the mode of administration.

[0410] For example, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N. J.), or phosphate buffered saline (PBS). In some embodiments, the composition should be sterile and should be fluid to the extent that easy syringability exists. It can be stabilized under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and/or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0411] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0412] In some embodiments, the pharmaceutical composition of the disclosure further includes a further therapeutic agent. Non-limiting examples of further therapeutic agents include

(i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds the serine protease TMPRSS2 of a target cell, and (iv) a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. Accordingly, in some embodiments, the pharmaceutical composition of the disclosure further includes a further therapeutic agent selected from the group consisting of: (i) an antiviral agent,

(ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds the serine protease TMPRSS2 of a target cell, and (iv) a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. In some embodiments, the further therapeutic agent is a second antibody or antigen-binding fragment described herein. In some embodiments, one, two, three, four, or more antibodies, or antigen binding fragments thereof can be used in combination.

[0413] In some embodiments, the one or more further therapeutic agents includes an antiviral drug or a vaccine. One of ordinary skill in the art will understand that the antiviral drug of the disclosure can include any anti-infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject. In some embodiments, the antiviral drug includes, but is not limited to a cationic steroid antimicrobial, leupeptin, aprotinin, ribavirin, or interferon- alpha2b (IFN-a2b). Methods for treating or preventing virus (e.g. , coronavirus) infection in a subject in need of said treatment or prevention by administering an antibody or antigen-binding fragment in association with a further therapeutic agent are part of the present disclosure.

[0414] For example, in some embodiments of the disclosure, the further therapeutic agent is a vaccine, e.g., a coronavirus vaccine. In some embodiments, a vaccine is an inactivated/killed virus vaccine, a live attenuated virus vaccine or a virus subunit vaccine.

KITS

[0415] Further provided herein are kits for the practice of a method described herein. In some embodiments, provided herein are kits for identification or characterization of antibodies and antigen-binding fragments having binding affinity for an antigen. Such kits can include: (a) a plurality of target antigens and non-target antigens, and wherein each of the target antigens and non-target antigens include a reporter oligonucleotide including (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence; and (b) instructions for performing a method of the disclosure.

[0416] In some embodiments, provided herein are kits for identification of antibodies and antigen-binding fragments having binding affinity for a CoV-S. Such kits can include: (a) a plurality of CoV-S antigens and non-CoV-S antigens, and wherein each of the antigens comprise a reporter oligonucleotide comprising (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence; and (b) instructions for performing a method of identifying an antibody or antigen-binding fragment having binding affinity for CoV-S as described herein.

[0417] Also provided, in some embodiments of the disclosure, are kits for (i) for producing an antibody or antigen-binding fragment thereof, (ii) detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, or (iii) treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject. A kit can include instructions for use thereof and one or more of the antibodies or antigen-binding fragments thereof, recombinant nucleic acids, recombinant cells, and pharmaceutical compositions as described and provided herein. For example, some embodiments of the disclosure provide kits that include one or more of the antibodies described herein and/or antigen-binding fragments thereof, and instructions for use. In some embodiments, provided herein are kits that include one or more recombinant nucleic acids, recombinant cells, and pharmaceutical compositions as described herein and instructions for use thereof.

[0418] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container. Accordingly, in some embodiments of the disclosure, the kit includes an anti-CoV-S antigen binding polypeptide, e.g ., an antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof in one container (e.g, in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g, in a sterile glass or plastic vial).

[0419] In another embodiment, the kit includes a combination of the compositions described herein, including an anti-CoV-S antigen-binding polypeptide, e.g, antibody or antigen-binding fragment thereof as described herein, or pharmaceutical composition thereof in combination with one or more further therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

[0420] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device ( e.g ., an injection device or catheter) for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above containing the anti-CoV-S antigen-binding polypeptide, e.g., antibody or antigen-binding fragment thereof of the present disclosure.

[0421] In some embodiments, a kit can further include instructions for using the components of the kit to practice a method described herein. For example, the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and intellectual property information.

[0422] The instructions for practicing the method are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g, associated with the packaging or sub packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g, via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0423] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0424] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0425] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0426] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

EXAMPLES

EXAMPLE 1 Biological samples

[0427] Sample procurement: The experiments described in the below Examples were performed with peripheral blood mononuclear cells (PBMCs) collected from convalescent human survivors of natural infection with SARS-2. Specifically, Donor 531 PBMCs were purchased from Cellero (~112m/vial product, Cat. # 1146-4785JY20) and used in these experiments.

[0428] Sample background/timeline: The donor tested positive via nasopharyngeal swab while presenting asymptomatic/presymptomatic on Day 0. Hospitalization was not required. The donor tested negative for SARS-2 on Day 23. Plasma and apheresis sample collection were performed on Day 104. The donor is also seropositive for cytomegalovirus, a ubiquitous human herpesvirus.

EXAMPLE 2

Enrichment of B cells

[0429] A vial of frozen peripheral blood mononuclear cells (PBMCs) was thawed for 1-2 min in a water bath, then transferred into 8-10 mL of 10% Fetal Bovine Serum (FBS) in PBS, and centrifuged for 5 min at 350g. The cell pellet was washed three times by resuspending in 0.04% Bovine Serum Albumin (BSA) in PBS and centrifuging at RT at 350g for 5 min each wash, with the final pellet resuspended to a concentration of ~20 million cells per mL in a total volume of 5 mL (-100 million cells total). B cells were enriched using the B Cell Isolation Kit II (human; MACS™ Miltenyi) according to manufacturer’s instructions, and approximately 50 million cells were applied to each of two LS columns designed for positive selection of cells.

The effluent was concentrated and prepared for cell labeling.

EXAMPLE 3

Antigen sourcing preparation and conjugation [0430] Biotinylated antigens were sourced from suppliers as follows:

[0431] 1) Biotinylated trimerized S (SARS-2) was sourced from ACRO Biosystems, catalog # SPN-C82E9-25 (https://www.acrobiosystems.com/P3345-Biotinylated-SARS-CoV-2- S-protein-HisAvitag™-Superstable-trimer-%28MALS-verified%29.html). This protein carries a polyhistidine tag at the C-terminus, followed by an Avi tag. Biotinylation of this product is performed using Avitag™ technology. Briefly, the single lysine residue in the Avitag is enzymatically labeled with biotin.

[0432] 2) Biotinylated trimerized S D614G (SARS-2), from ACRO Biosystems, catalog

# SPN-C82E3-25 (https://www.acrobiosystems.com/P3431-Biotinylated-SARS-CoV-2-S- protein-%28D614G%29-HisAvitag™-Super-stable-trimer-%28MALS-verified%29.html). This protein contains D614G mutation, which has become increasingly common in SARS-CoV-2 viruses from around the world. This protein also carries a polyhistidine tag at the C-terminus, followed by an Avi tag. Biotinylation of this product is performed using Avitag™ technology. Briefly, the single lysine residue in the Avitag is enzymatically labeled with biotin.

[0433] 3) Biotinylated Human Serum Albumin (HSA) HSA-H82E3, from Sapphire, catalog # (https://sapphireusa.eom/product.i sp?q=ns%3ANS0000368507Y

[0434] Biotinylated antigens were each solubilized per manufacturer’s instructions. In each case, they were thawed and dissolved in sterile deionized water for 30-60 minutes at room temperature with occasional gentle mixing for a final concentration of 100 microgram/mL (for HSA) or 200 microgram/mL for both of the trimerized S antigens.

[0435] Solubilized antigens were each conjugated with, e.g ., allowed to form a complex with (or bind to)) one of the following Total SeqC reagents, supplied by BioLegend, which each contain a unique barcoded DNA oligonucleotide (i.e., a reporter oligonucleotide) supplied by the vendor as follows:

[0436] 1) TotalSeq-C0951 PE Streptavidin was conjugated to biotinylated trimerized S glycoprotein (SARS-2).

[2] 2) TotalSeq-C0952 PE Streptavidin was conjugated to biotinylated human serum albumin ( i.e ., a non-SARS-CoV-S antigen).

[0437] 3) TotalSeq-C0956 APC Streptavidin was conjugated to biotinylated trimerized S

D614G glycoprotein (SARS-2).

[0438] 4) TotalSeq-C0957 APC Streptavidin was conjugated to biotinylated human serum albumin. Briefly, each Total Seq-C barcoded streptavidin PE or APC reagent was diluted to 0.1 mg/mL and then mixed with biotinylated antigen at a 5X molar excess of antigen to streptavidin, based on a fixed amount of 0.5 pg PE-SA. One fifth of the streptavidin-oligo PE or APC conjugate was added to the antigen every 20 minutes at 4°C. The reaction was then quenched with 5 mΐ 4mM biotin (Pierce, Thermo Fisher) for 30 minutes for a total probe volume of 20 pL. The final conjugated antigen probes (streptavidin antigen complexes) were then immediately used for cell labeling at a dilution of 1:50.

EXAMPLE 4 Cell labeling

[0439] This Example describes experiments performed to stain B cells with a number of barcoded antibodies and conjugated antigens. In these experiments, approximately 4.4 million enriched B cells were first resuspended in labeling buffer (1% BSA in PBS) and performed Fc blocking for 10 minutes on ice using Human TruStain FcX (BioLegend).

[0440] Next, cells were stained with the following cocktail of antibodies, antigens and dyes: CD19 PE-Cy7 (clone SJ25C1, BD Pharmingen) for discrimination of CD19+ cells by using fluorescence-activated cell sorting (FACS).

[0441] Barcoded Antibodies for lOx Single Cell Immune profiling, which included the following TotalSeq-C oligo barcoded antibodies:

[0442] - TotalSeq-C0389 anti-human CD38.

[0443] - Total Seq-CO 154 anti-human CD27.

[0444] - Total Seq-CO 189 anti-human CD24.

[0445] - TotalSeq-C0384 anti-human IgD.

[0446] - TotalSeq-COlOO anti-human CD20.

[0447] - TotalSeq-C0050 anti-human CD19 (clone HIB19, to distinguish it from the flow clone). [0448] - TotalSeq-C0049 anti-human CD3E.

[0449] - TotalSeq-C0045 anti-human CD4.

[0450] - TotalSeq-C0046 anti-human CD8A.

[0451] - TotalSeq-C0051 anti-human CD14.

[0452] - TotalSeq-C0083 anti -human CD 16.

[0453] - TotalSeq-C0090 mouse IgGl K isotype control.

[0454] - TotalSeq-C0091 mouse IgG2a K isotype control.

[0455] - TotalSeq-C0092 mouse IgG2b K isotype control.

[0456] Final conjugated antigens:

[0457] - Total Seq-C0951 PE trimerized S (SARS-2).

[0458] - TotalSeq-C0952 PE Human Serum Albumin.

[0459] - TotalSeq-C0956 APC trimerized S D614G (SARS-2).

[0460] - TotalSeq-C0957 APC Human Serum Albumin.

[0461] - 7AAD for live/dead cell discrimination.

[0462] Cells were stained in labeling buffer (1% BSA in PBS) in the dark for 30 minutes on ice, then cells were washed 3 times with 2 mis of cold labeling buffer at 350g for 5 minutes at 4°C, resuspended in cold labeling buffer and a 1:200 addition of live/dead cell discriminating agent 7AAD for 10 minutes on ice in the dark, then washed one more time with labeling buffer at 350*g for 5 minutes at 4°C, then resuspended in labeling buffer and loaded into a Sony MA900 Cell Sorter using a 70 microM sorting chip.

EXAMPLE 5

Antigen-specific enrichment via FACS

[0463] Cells were initially gated on being single, live (7AAD^negative) and PE-Cy7-CD19+ and then sorted on their PE and/or APC status directly into master mixed and water based on one of four criteria:

[0464] 1) PE+, representing trimerized S (SARS-2) antigen+ and/or HSA+ control antigen cells (gate Q1 in FIG. 12; 2,430 cells);

[0465] 2) APC+, representing trimerized S D614G (SARS-2) antigen and/or HSA control antigen cells (gate Q3 in FIG. 12; 728 cells);

[0466] 3) Dual PE+ and APC+, representing a combination of trimerized S (SARS-2) antigen+, trimerized S D614G (SARS-2) antigen+ and/or HSA control antigen-positive cells (gate Q2 in FIG. 12; 828 cells);

[0467] 4) PE and APC negative cells, representing cells not binding either SARS-2 antigen or control HSA antigen (gate Q4 in FIG. 12; 5,000 cells).

[0468] In FIG. 12, the Y axis represents PE (representing trimerized S (SARS-2) antigen+ and/or HSA+ control antigen cells) signal. The X axis represents APC trimerized S (SARS-2) D614G antigen+ and/or HSA+ control antigen cells. The numbers adjacent to each gate name represent the fraction of events of the parent population (single, live, CD 19+ cells) for that gate. FACS data were analyzed with FlowJo.

[0469] The resulting volume was adjusted with additional water to match the recommended volume and target for loading with the lOx 5’ V2 Single Cell Immune Profiling kit. FACS data were analyzed using FlowJo. Standard gene expression, V(D)J, and barcoded antigen libraries were constructed using the lOx 5Ύ2 Single Cell Immune Profiling kit per manufacturer's instructions. Additional information in this regard can be found at “support.10xgenomics.com/permalink/getting-started-immune-profiling-feature-barcoding.”

EXAMPLE 6 Sequencing analysis

[0470] The libraries resulting from the experiments described in Example 5 above were sequenced on a NovaSeq 3 using a NovaSeq S4200 cycles 2020 vl .5 kit, targeting using read 28, 10, 10, and 90 cycles targeting 20,000, 30,000, or 6000 reads per cell for gene expression, barcoded antigen, or Ig libraries, respectively. Sequence analysis (described further herein, see, e.g., Example 7) identified a total of 239 antibodies. The binding affinity of 159 antibodies to a trimerized wild-type SARS-CoV-2 spike protein (SEQ ID NO: 3045) and a SARS-CoV-2 spike protein variant with D614G substitution (SEQ ID NO: 3046) is summarized in Tables 1 A and IB below. In these experiments, the binding affinity of an antigen-binding molecule (e.g, antibody) to a target antigen (wild-type S protein or a variant thereof) was determined based on quantity/numbers of unique molecular identifiers (UMIs) associated with each of the antigen binding molecules bound to the target antigen. Generally, the higher target antigen UMI counts were used as a predictor of higher binding affinity. When an antibody was found “fluorophore reactive” or “biotin-reactive” then it was categorized as a non-specific antibody, even if it had non-zero target antigen UMI counts. As shown in Tables 1A-1B below, all of the identified antibodies displayed high target antigen counts and low non-target antigen counts. As such, they were predicted to have specific binding affinity for the target antigen and were distinguishable from non-specific binders.

TABLE lA: Binding affinity of 159 exemplary antibodies. The integer values displayed in the table below represent antigen UMI counts for each of the individual on-target (Wild-type S or D416G mutant (Mutant S) and off-target (human serum albumin control/HSA 1, human serum albumin control/HSA 2) antigens.

TABLE IB: Binding affinity of 80 exemplary antibodies. The integer values displayed in the table below represent antigen UMI counts for each of the individual on-target (Wild-type S or D416G mutant (Mutant S) and off-target (human serum albumin control/HSA 1, human serum albumin control/HSA 2) antigens.

[0471] It was observed that the antibodies identified in these experiments were diverse in their VH, VL, and isotype/subclass (see Table 1C below).

[0472] As demonstrated, BEAM-Ab directly captures full length antibody sequences, enabling rapid expression of the native antibody, including somatic hypermutation. Furthermore, BEAM-Ab is highly reproducible: 220 of 240 screened binders were re-identified in separate samples from the same blood draw.

EXAMPLE 7 Statistical analysis

[0473] Binding antibodies with a maximum spike antigen count greater than 40 UMIs (as summarized in Tables 1 A and IB above) were selected for further analysis using lOx Genomics “Enclone” (available at https://bit.ly/enclone), which is a computational tool developed for clonal grouping to study the adaptive immune system. In this computational tool, the lOx Genomics Chromium Single Cell V(D)J data containing B cell receptor (BCR) and T cell receptor (TCR) RNA sequences are provided as input data to Enclone. Based on the input, Enclone finds and organizes cells arising from the same progenitors into groups ( e.g ., clonotype families) and compactly displays each clonotype along with its salient features, including mutated amino acids. Antibodies in the dataset were classified into 3 categories, as listed below, via a process termed “barcode-enabled antigen mapping by sequencing” (BEAM-seq).

[0474] Category 1 : Antibodies are classified into this category if the mix of antigens includes target and non-target antigens linked to different fluorophores, and counts are detected for target and non-target antigen linked to fluorophore 1 but not fluorophore 2, which indicates that the antibodies bind to the fluorophore and not the target antigen. In this particular Example, antibodies were classified into this category if counts were detected for only one spike protein and the corresponding albumin labeled with the same fluorophore.

[0475] Category 2: Antibodies are classified into this category if the mix of antigens includes target and non-target antigens linked to different fluorophores, and counts are detected for target and non-target antigen linked to both fluorophores, which indicates that the antibodies does not bind the antigen but instead binds to a core component of the reagent (e.g., streptavidin, biotin) or is polyreactive (e.g., sticky and non-specific). In this particular Example, antibodies were classified into this category (e.g., classified as biotin-reactive, streptavidin-reactive, or polyreactive, if counts were detected for both spike proteins (trimerized wild-type S and trimerized S D614G) and both albumins (PE-HSA and APC-HSA).

[0476] Category 3: Antibodies are classified into this category if counts are detected for target antigen but absent or at lower levels for non-target antigen, which indicates that the antibodies specifically binds the target antigen and has affinity for the target antigen. In this particular Example, antibodies were classified into this category (e.g., classified as candidate SARS-2-reactive antibodies, if counts were considerably higher for one or both spike proteins relative to the albumins; most antibodies bound the wild-type spike protein and the common population variant D614G.

[0477] In a dataset where there is considerable enrichment for genuine antigen-binding cells, the binding affinity of an antigen-binding molecule ( e.g ., antibody or antigen-binding fragment) to a target antigen (such as S protein) were determined based on a quantity/number of unique molecular identifiers (UMIs) associated with the antigen bound to each cell. BEAM scores are approximately normally distributed, increase exponentially as target antigen-binding relative to expressed antibody and control antigen increases, are correlated with generation probability of the HCDR3 junction, e.g., following the known general relationship of somatic hypermutation (SHM) and increasing affinity, and also reveal that class switching increases predicted relative affinity in concordance with the literature (FIGS. 13 and 14). BEAM scores are also generally higher within sublineages that contain more daughter antibodies than narrow sublineages (representative example shown in FIG. 15).

EXAMPLE 8

Antibody synthesis cloning expression and purification [0478] Variable heavy chain and light chain domains of anti-target antigen antibodies are reformatted to IgGl and synthesized and cloned into mammalian expression vector pTwist CMV BG WPRE Neo utilizing the Twist Bioscience eCommerce portal. Light chain variable domains are reformatted into kappa and lambda frameworks accordingly. Clonal genes are delivered as purified plasmid DNA ready for transient transfection in human embryonic kidney (HEK) Expi293 cells (Thermo Scientific). Cultures in a volume of 1.2 ml are grown to four days, harvested and purified using Protein A resin (PhyNexus) on the Hamilton Microlab STAR platform into 43 mM Citrate 148 mM HEPES, pH 6.

EXAMPLE 9

Further characterization of binding affinity

[0479] This Example describes the results of experiments performed to further characterize binding affinity of select antibodies described herein.

[0480] Generally, antibodies predicted to have binding affinity based on antigen EIMI count profiles were selected for further screening and analysis.

[0481] Subsequently, surface plasmon resonance (SPR) analyses were performed on the selected antibodies by using a Carterra LSA SPR biosensor equipped with a HC30M chip at 25°C in HBS-TE (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20). In these experiments, antibodies were diluted to 5 pg/ml in sodium acetate buffer, pH 4.5, and amine-coupled to the sensor chip by EDC/NHS activation, followed by ethanolamine HC1 quenching. Increasing concentrations of ligand were flowed over the sensor chip in HBSTE (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20) with 0.5 mg/ml BSA with 5 minute association and 15 minute dissociation. Following each injection cycle the surface was regenerated with 2X 30 second injections of IgG elution buffer (Thermo). The following antigens and catalog #s from Aero Biosystems were used for serial analysis at the specified concentration ranges:

[0482] (1) SARS-CoV-2 S protein, His Tag, Super stable trimer (MALS & NS-EM verified), SPN-C52H9; 0 - 100 nM.

[0483] (2) SARS-CoV-2 S protein (D614G), His Tag, Super stable trimer (MALS verified), SPN-C52H3; 0 - 100 nM.

[0484] (3) SARS-CoV-2 (COVID-19) S protein RBD (triple mutant K417N, E484K,

N501Y), His Tag (MALS verified), SPD-C52Hp; 0 - 500 nM.

[0485] (4) SARS-CoV-2 (COVID-19) SI protein NTD, His Tag, S1D-C52H6; 0 - 500 nM.

[0486] (5) SARS-CoV-2 (COVID-19) S2 protein, His Tag, S2N-C52H5; 0 - 500 nM.

[0487] (6) MERS SI protein, His Tag, S1N-M52H5; 0 - 500 nM.

[0488] (7) HCoV-HKUl (isolate N5) SI protein, His Tag, SIN-V52H6; 0 - 500 nM.

[0489] Traces were analyzed and fit using Carterra's Kinetics Tool software, fit to a 1:1 receptor-ligand binding model.

[0490] Table 2 below provides a summary of the binding affinity of the exemplary antibodies to the following antigens: (1) a trimerized wild-type SARS-CoV-2 S protein (SEQ ID NO: 3045), (2) a SARS-CoV-2 S protein variant with D614G substitution (SEQ ID NO: 3046). For comparative analysis, also included in this study were the four following FDA-approved therapeutic antibodies previously reported to bind SARS-CoV-2 S protein: (1) imdevimab (REGN-COV2), (2) bamlanivimab (Eli Lilly / AbCellera), (3) etesevimab (Eli Lilly / AbCellera), and sotrovimab (Vir / GlaxoSmithKline). It was observed that the majority of antibodies tested in this study could bind to both wild-type S protein and D614G mutant in picomolar and nanomolar range. Remarkably, several antibodies described herein were found to have binding affinities as good as or superior to FDA-approved antibodies or antibodies in late clinical development. See also, FIGS. 16A-16B and 19.

TABLE 2: Binding affinity of exemplary antibodies. CTRL-0005: Imdevimab. CTRL-0006: Bamlanivimab. CTRL-0007: Etesevimab. CTRL-0008: Sotrovimab. ND: not determined.

[0491] Furthermore, as shown in Table 3 below, it was observed that several antibodies described herein demonstrated high binding affinity to a RBD fragment of SARS-CoV-2 S protein with triple amino acid substitutions K417N, E484K, and N501 Y. For comparative analysis, also included in this study were two therapeutic antibodies previously reported to bind RBD of SARS-CoV-2 S protein (i.e., etesevimab, and sotrovimab). It was observed that 21 antibodies tested in this study could bind to the triple mutant RBD in low to mid nanomolar range. In addition, several antibodies were found to bind the triple escape variant of RBD with binding affinities as good as or superior to FDA-approved antibodies or antibodies in late clinical development. See also, FIGS. 17A-17B. TABLE 3 : Binding affinity of exemplary antibodies to a RBD variant of SARS-CoV-2 S protein. Triple mutant RBD tested in this study contained a combination of three amino acid substitutions K417N, E484K, and N501Y. CTRL-0007: Etesevimab. CTRL-0008: Sotrovimab.

[0492] Remarkably, antibody TXG-0091 was found to be a pan-coronavirus antibody that recognizes a conserved epitope in the S 1 subunit and bind with high affinity to the S 1 subunit of a new human coronavirus strain HCoV-HKU 1 (KD = 543 pM) (see, e.g, Table 4). Accordingly, without being bound to any particular theory, this antibody could be particularly useful in therapeutic combination against SARS-CoV-2 and other coronaviruses and in combination with RBD-binding, NTD-binding, or non-Sl binding therapeutic antibodies.

[0493] As shown in Table 4, antibody TXG-0063 was found to bind N-terminal domain of SARS-CoV-2 S protein with high affinity (KD = 102 nM). Accordingly, this antibody could be particularly useful in therapeutic combination against SARS-2 with RBD-binding and non-Sl binding therapeutic antibodies. TABLE 4: Binding affinity of antibodies TXG-0091 and TXG-0063 to the N-terminal domain of SARS-CoV-2 S protein or the SI subunit of HCoV-HKUl.

EXAMPLE 10

Identification of antibodies with desired kinetic profiles

[0494] Further analyses are performed using SPR binding curves to identify antibodies with particularly low K_0ff constants.

[0495] As shown in Table 5 below, it was observed that antibodies could be stratified into two major classes: optimal binding kinetics and less optimal binding kinetics. For example, it was found that one could identify antibodies with visibly longer half-lives have a K_0ff lower than 4e-4, with an additional subclass of antibodies that have exceptionally long half lives and a K_off lower than le-4 (see, e.g ., Table 5 and FIGS. 18A-18D). Antibody half-life values and mean-life values reported in Table 5 were calculated by using formula ln(2)/K_0ff and formula 1/Koff, respectively.

[0496] A smaller number of antibodies have less optimal binding kinetics due to their higher K_0ff constants, which produce a less ideal KD even given their acceptable K_on constants (see, e.g. , Table 5 and FIGS. 18A-18D). For comparative analysis, etesevimab and sotrovimab were also included in this study.

TABLE 5: Half-lives of exemplary antibodies. CTRL-0007: Etesevimab. CTRL-0008:

Sotrovimab.

EXAMPLE 1 1

Functional characterization of antibodies

[0497] To further characterize the antibodies and antigen-binding fragments described in Examples 1-10 above, ligand-blocking assays are performed using GFP+ reporter cells expressing ACE2 and dose competition of pre-fusion D614G spike protein in dual-fluorescent format, where the antigens being competed are in tetrameric format. In these experiments, a relative KD value for each mAh can be generated.

[0498] In addition, to determine whether the antibodies and antigen-binding fragments of the disclosure have a neutralizing activity ( e.g ., antagonistic activity) against SARS-CoV-2, e.g. , able to bind to and neutralize the activity of SARS-CoV-S, additional live virus or pseudovirus neutralization assays are performed using these mAbs in a dose-dependent manner to generate an IC50 of neutralization activity. In some experiments, a neutralization activity IC50 value for each antibody can be determined in a quantitative focus reduction neutralization test (FRNT) described previously by Zost et al. (Nature, 584:443-449, 2020). In some experiments, neutralization assays are used to determine infectivity of SARS-CoV-2 S protein-containing virus-like particles. In these experiments, a neutralizing or antagonistic CoV-S antibody or antigen-binding fragment can be identified based on its ability to inhibit an activity of CoV-S to any detectable degree, e.g., inhibits or reduces the ability of CoV-S protein to bind to a receptor such as ACE2, to be cleaved by a protease such as TMPRSS2, or to mediate viral entry into a host cell or mediate viral reproduction in a host cell.

EXAMPLE 12

Additional surface plasmon resonance (SPR) analysis [0499] This Example describes the results of an additional round of experiments performed to further characterize binding affinity of select antibodies described herein.

[0500] A new lot of all 239 antibodies identified in Example 6 were synthesized, cloned, expressed, and purified according to the methods described in Example 8.

[0501] A second SPR experiment was performed under the same experimental settings (flow times, antigen concentration, coupling method, buffer, etc.) with one set of measurements per antibody (in comparison to the triplicate measurements completed as part of the first SPR experiment). Trimeric forms of the SARS-CoV-2 Wuhan entry strain (WT), beta, gamma, and kappa pre-fusion spike, SARS-CoV-2 NTD, HcoV-HKUl spike trimer, and human serum albumin were used as antigens to assess the affinity and reactivity of each antibody. The antigens used in these experiments were purchased from ACROBiosystems (His-tagged wild-type SARS- CoV-2: Cat# SPN-C52H9; His-tagged SARS-CoV-2 gamma variant: Cat# SPN-C52Hg; His- tagged SARS-CoV-2 kappa variant: Cat# SPN-C52Hr; His-tagged SARS-CoV-2 beta variant: Cat# SPN-C52Hk; His-tagged SARS-CoV-2 NTD: Cat# SPN-C52H6; and His-tagged HcoV- HKU1 (isolate N5) spike trimer: Cat# SPN-C52H5). In addition, His-tagged human serum albumin (HSA) was also purchased from ACROBiosystems (Cat# HSA-H5220). Mutations identified in the beta, gamma, and kappa variants are as follows.

[0502] Beta variant: L18F, D80A, D215G, 242-244del, R246I, K417N, E484K, N501Y, D614G, A701V.

[0503] Gamma variant: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F.

[0504] Kappa variant: T95I, G142D, E154K, L452R, E484Q, D614G, P681R, and Q1071H.

[0505] It should be noted that the delta and kappa variants share two mutations E484Q and L452R. They were identified in India’s second COVID-19 wave, and have been reported to share significant similarity, which is presumably due to the fact that they are from the same lineage.

[0506] As shown in Tables 6A and 6B below, it was observed that several antibodies described herein demonstrated high binding affinity to various spike variants of interrest (VoC), e.g ., beta, gamma, and kappa variants. For comparative analysis, also included in this study were several control antibodies (denoted as CTRL) that had been previously described as having binding affinity for SARS-CoV-2 S protein. A total of 51 control antibodies (CTRL) were included in these experiments, including: CTRL-0004: Casirivimab; CTRL-0005: Imdevimab; CTRL-0006: Bamlanivimab; CTRL-0007: Etesevimab; CTRL-0008: Sotrovimab; and CTRL-0009: Tixagevimab; (-): not determined. Of these control antibodies, CTRL-0019, CTRL-0020, CTRL- 0021, CTRL-0024, CTRL-0025, CTRL-0026, CTRL-0027, CTRL-0028, CTRL-0029, CTRL- 0030, CTRL-0031, CTRL-0032, and CTRL-0036 were discovered in a human phage display campaign, and CTRL-0022 and CTRL-0023 were discovered in a mouse phage display campaign. CTRL-0017 was discovered in a mouse hybridoma campaign. CTRL-0010, CTRL-0011, CTRL- 0012, CTRL-0013, CTRL-0014, CTRL-0015, and CTRL-0016 were discovered in a humanized mouse hybridoma campaign. CTRL-0005, CTRL-0006, CTRL-0007, CTRL-0008, CTRL-0009, CTRL-0018, CTRL-0033, CTRL-0034, CTRL-0035, CTRL-0037, CTRL-0038, CTRL-0039, CTRL-0040, CTRL-0041, CTRL-0042, CTRL-0043, CTRL-0044, CTRL-0045, CTRL-0046, CTRL-0047, CTRL-0048, CTRL-0049, CTRL-0050, CTRL-0051, CTRL-0052, and CTRL-0053 where discovered in human samples. It was observed that several antibodies tested in this experiment could bind to one or more spike variants in low to mid nanomolar range. In addition, several antibodies were found to bind beta, gamma, and/or kappa variants with binding affinities as good as or superior to FDA-approved antibodies or antibodies in late clinical development.

TABLES 6 A and 6B: Binding affinity of exemplary antibodies to beta, gamma, and kappa spike variants. A total of 51 control antibodies (CTRL) were included in these experiments, including: CTRL-0004: Casirivimab; CTRL-0005: Imdevimab; CTRL-0006: Bamlanivimab; CTRL-0007: Etesevimab; CTRL-0008: Sotrovimab; and CTRL-0009: Tixagevimab; (-): not determined.

[0507] An UpSet plot was generated (see, e.g, FIG. 25) wherein antibodies are binned into antigen bins based on two rounds of SPR binding affinity data. For an antibody to be placed into a bin a detectable kinetic fit at all concentrations of antigen was required from at least one of the SPR experiments described in Examples 9 and 12, or orthogonal neutralization data. The results described in FIG. 25 illustrate that the BEAM-seq process described in the present disclosure allows for rapid identification of many antibodies with broad and robust binding affinity against several coronavirus S antigens, including several variants of concern (VoC), e.g. , , beta, gamma, and kappa, as well as HKU 1 (which is a different coronavirus).

[0508] Remarkably, several antibodies were found to be a pan-coronavirus antibodies that recognizes a conserved epitope in the SI subunit and bind with high affinity to N-terminal domain of SARS-CoV-2 S protein (NTD) and/or to the SI subunit of a new human coronavirus strain HCoV-HKUl (see, e.g , TXG-0053, TXG-0054, TXG-0078, TXG-0091, TXG-0114, TXG-0187, TXG-0203, and TXG-0206 of Table 9). Accordingly, without being bound to any particular theory, these antibodies could be particularly useful in therapeutic combination against SARS-CoV-2 and other coronaviruses and in combination with RBD-binding or non-Sl binding therapeutic antibodies.

[0509] As shown in Table 7A, several antibodies (e.g., T TXG-0058, TXG-0060, TXG- 0063, TXG-0064, TXG-0065, TXG-0066, TXG-0071, TXG-0076, TXG-0078, TXG-0079, TXG- 0099, TXG-0104, TXG-0116, TXG-0119, TXG-0131, TXG-0132, TXG-0146, TXG-0162, TXG- 0163, TXG-0164, TXG-0168, TXG-0170, TXG-0175, TXG-0184, TXG-0225, and TXG-0232) were found to bind N-terminal domain of SARS-CoV-2 S protein with high affinity. Accordingly, these antibodies could be particularly useful in therapeutic combination against SARS-2 with RBD-binding and non-Sl binding therapeutic antibodies.

TABLE 7A: Binding affinity of exemplary antibodies to the N-terminal domain (NTD) of SARS-CoV-2 S protein or the SI subunit of HCoV-HKUl. (ND: not determined).

[0510] In addition, several antibodies were found to be pan-coronavirus antibodies that recognizes a conserved epitope in the S 1 subunit and bind with high affinity to the S 1 subunit of a new human coronavirus strain HCoV-HKUl (see, e.g. , TXG-0085, TXG-0112, TXG-0136, TXG- 0150, TXG-0192, TXG-0227, TXG-0228, TXG-0229, and TXG-0230 in Table 7B). It was observed that several antibodies tested in this experiment could bind to human coronavirus strain HCoV-HKUl in low to mid nanomolar range. Accordingly, without being bound to any particular theory, these antibodies could be particularly useful in therapeutic combination against SARS- CoV-2 and other coronaviruses and in combination with RBD-binding, NTD-binding, or non-Sl binding therapeutic antibodies

[0511] Furthermore, as shown in Table 7B, several antibodies (e.g., TXG-0072, TXG-0137, TXG-0173, TXG-0174, and TXG-0230) were found to also bind N-terminal domain of SARS- CoV-2 S protein with high affinity. Accordingly, these antibodies could be particularly useful in therapeutic combination against SARS-2 with RBD-binding and non-Sl binding therapeutic antibodies.

TABLE 7B: Binding affinity of thirteen (13) exemplary antibodies to the N-terminal domain of SARS-CoV-2 S protein or the SI subunit of HCoV-HKUl. ND: Not determined.

EXAMPLE 13

Neutralization assays

[0512] All 239 antibodies identified in Example 6 above were screened in addition to 49 control antibodies of known SARS-2 and other viral binding. Assays were performed with a clinical isolate of the SARS-CoV-2 B.l lineage (MEX-BC2/2020). This virus carries the D614G mutation in the spike protein (full sequence available at the GISAID/EpiCoV database ID:

EPI ISL 747242). The screen was performed with a microneutralization assay that utilizes prevention of the virus-induced cytopathic effect (CPE) in Vero E6 cells. All antibodies (i.e., test-items) were provided at varying concentrations (0.06 to 0.23mg/mL), and they were stored at 4°C until use. The screen was performed in ten different experiments performed in ten days, each one assessing the activity of approximately 30 Abs in parallel. All plates included a positive control — plasma from a convalescent patient who had also received the first dose of the Pfizer/BioNTech mRNA vaccine (BNT162b2). Plasma was collected 21 days after vaccination.

[0513] For this screen, Vero E6 cells were used to evaluate the neutralization activity of the antibody test-items against a replication competent SARS-CoV-2 virus. Antibodies were pre incubated first with the virus for 1 hour at 37°C before addition to cells. Following pre incubation of Ab/virus samples, Vero E6 cells were challenged with the mixture. After addition to cells, antibodies were present in the cell culture for the duration of the infection (96 hours), at which time a “ Neutral Red” uptake assay was performed to determine the extent of the virus- induced CPE. Prevention of the CPE was used as a surrogate marker to determine the neutralization activity of the test-items against SARS-CoV-2.

[0514] Eight dilutions of the antibodies were tested in duplicates for the neutralization assay using a five- fold dilution scheme starting at l,000ng/mL. Representative raw data from neutralization assay is shown in FIGS. 21 and 22. When possible, IC50 values of the antibodies displaying neutralizing activity were determined using GraphPad Prism software. Plasma control was assessed on each plate using singlet data-points (8 two-fold dilutions throughout 1:20480). Representative neutralization curves (IC50) for control antibodies and antibody test-items are shown in FIGS. 23 and 24.

Data analysis of CPE-based neutralization assay

[0515] The average absorbance at 540nm (A540) observed in infected cells in the presence of vehicle alone was calculated first, and then subtracted from all samples to determine the inhibition of the virus induced CPE. Data points were then normalized to the average A540 signal observed in uninfected cells (“mock”) after subtraction of the absorbance signal observed in infected cells.

[0516] In the neutral red CPE-based neutralization assay, uninfected cells remained viable and uptake the dye at higher levels than non-viable cells. In the absence of antibodies, the virus-induced CPE leads to cell death in infected cells and lowers the A540 signal (this value equals 0% neutralization). By contrast, incubation with neutralizing antibodies prevents the virus induced CPE and leads to absorbance levels similar to those observed in uninfected cells. Full recovery of cell viability in infected cells represents 100% neutralization of the virus. Each plate assessed 3 antibodies in triplicates (rows A-C and F-H) or duplicates (rows D and E). However, data analysis avoided samples located in rows A and H to minimize “edge effects.” Therefore, all antibodies were evaluated in duplicates. Uninfected cells and infected cells in the absence of antibodies were analyzed using six replica data-points of each. Control neutralizing plasma was run in singlet data-points (1 : 160 or 1 :320 to 1 :20480). [0517] Every plate was analyzed during a QC step before data was selected for analysis. QC included signal to background values greater than 2.5, and percentage CV in uninfected lower than 20 (CV<20%). All plates passed QC and there was no need to perform repeats. In some instances, data-points identified as outliers may have been removed, or they were exchanged by an additional data-point of the extra row not used (the latter only for antibodies in A-C or F-H). However, these actions were rarely needed, and overall variation of the screen was excellent and within the ranges typically seen in the neutralization studies described herein.

Control inhibitors and quality controls in live SARS-CoV-2 assay

[0518] Quality controls for the infectivity assays were performed on every plate to determine: i) signal to background (S/B) values; ii) inhibition by plasma with neutralizing activity against SARS-CoV-2, and; iii) variation of the assay, as measured by the coefficient of variation (C. V.) of all data points. All controls worked as anticipated for the assay, and variation was within typical ranges seen in vendor laboratories.

[0519] The average of all C.V. (of all duplicate data-points) in each plate was below 10% (average CV 7.1% for the 10 plates, whereas the variation of uninfected controls (“mock”), which were repeated six times on each plate, was below 5% (average 3.2%). The ratio of signal - to-background (S/B) for the neutralization assays, estimated by dividing the average signal in uninfected cells (A540nm) by the average signal in infected cells (vehicle alone), was 4.1 -fold for the ten representative plates. When comparing the signal in uninfected cells to the signal in “no-cells” background wells, the S/B ratio of the assay was greater than 10 (data not shown). S/B values and variation for each of the plates are available in the accompanying excel file summary.

[0520] To evaluate the neutralization activity of 288 Abs against SARS-CoV-2, the clinical isolate (MEX- BC2/2020) carrying a D614G mutation in the viral spike protein was used. Full sequence of this isolate is available at the GISAID/EpiCoV database with the identifier EPI ISL 747242.

[0521] A CPE-based neutralization assay was performed by infecting Vero E6 cells in the presence or absence of antibodies. Infection of cells leads to significant cytopathic effect and cell death after 4 days of infection. In this screen, reduction of the virus CPE in the presence of antibodies was used as a surrogate marker to determine the neutralization activity of the tested items.

[0522] Vero E6 cells were maintained in DMEM with 10% fetal bovine serum (FBS), referred herein as DMEM10. Twenty-four hours after cell seeding, test samples were submitted to serial dilutions with DMEM with 2% FBS (DMEM2) in a different plate. Then, virus diluted in DMEM2 or DMEM2 alone was pre-incubated with antibody test-items for 1 hour at 37°C in a humidified incubator. Following incubation, media was removed from cells, and then cells were challenged with the SARS-CoV-2 / antibody pre-incubated mix. The amount of viral inoculum was previously titrated to result in a linear response inhibited by antibodies with known neutralizing activity against SARS-CoV-2. Cell culture media with the virus inoculum was not removed after virus adsorption, and antibodies and virus were maintained in the media for the duration of the assay (96 h). After this period, the extent of cell viability was monitored with the neutral red (NR) uptake assay.

[0523] The virus-induced CPE was routinely monitored under the microscope after 3 days of infection, and after 4 days, cells were stained with neutral red to monitor cell viability. Viable cells incorporate neutral red in their lysosomes. The uptake of neutral red relies on the ability of live cells to maintain the pH inside the lysosomes lower than in the cytoplasm, a process that requires ATP. Inside the lysosome, the dye becomes charged and is retained. After a 3-h incubation with neutral red (0.017%), the extra dye is washed away, and the neutral red is extracted from lysosomes by incubating cells for 15 minutes with a solution containing 50% ethanol and 1% acetic acid. The amount of neutral red is estimated by measuring absorbance at 540nm in a plate reader. The general procedure followed to determine the anti-SARS-CoV-2 activity of antibody test-items is summarized in FIG. 20.

[0524] In these experiments, antibodies were evaluated in duplicates using five-fold serial dilutions starting at 1 pg/mL. Controls included uninfected cells (“mock-infected”), and infected cells treated with vehicle alone. Some cells were treated with a positive control plasma derived from a convalescent patient who was also administered the first dose of Pfizer / BioNTech mRNA vaccine (BNT162b2). Plasma was collected 21 days after the vaccine injection.

Results

[0525] Of the antibodies tested against SARS-CoV-2 (lineage B.1, carrying the D614G mutation), approximately 40% of all antibodies displayed measurable neutralization activity. Exemplary antibodies that displayed measurable neutralization activity include TXG-0001, TXG-0002, TXG-0003, TXG-0004, TXG-0005, TXG-0006, TXG-0007, TXG-0008, TXG-0009, TXG-0017, TXG-0046, TXG-0047, TXG-0048, TXG-0050, TXG-0052, TXG-0055, TXG-0057, TXG-0060, TXG-0063, TXG-0066, TXG-0069, TXG-0070, TXG-0071, TXG-0073, TXG-0074, TXG-0076, TXG-0077, TXG-0078, TXG-0079, TXG-0080, TXG-0081, TXG-0086, TXG-0088, TXG-0091, TXG-0093, TXG-0094, TXG-0099, TXG-0100, TXG-0101, TXG-0102, TXG-0104, TXG-0107, TXG-0109, TXG-0116, TXG-0119, TXG-0120, TXG-0126, TXG-0127, TXG-0128, TXG-0129, TXG-0131, TXG-0132, TXG-0133, TXG-0139, TXG-0141, TXG-0142, TXG-0144, TXG-0151, TXG-0157, TXG-0158, TXG-0161, TXG-0162, TXG-0163, TXG-0164, TXG-0166, TXG-0168, TXG-0170, TXG-0175, TXG-0176, TXG-0178, TXG-0180, TXG-0181, TXG-0183, TXG-0184, TXG-0188, TXG-0189, TXG-0197, TXG-0198, TXG-0200, TXG-0201, TXG-0202, TXG-0204, TXG-0207, TXG-0209, TXG-0210, TXG-0222, TXG-0233, TXG-0049, TXG-0051, TXG-0068, TXG-0072, TXG-0098, TXG-0108, TXG-0115, TXG-0136, TXG-0137, TXG-0140, TXG-0147, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0208, TXG-0213, and TXG-0226.

[0526] Table 8 below provides a summary of neutralization activity of 55 exemplary potently neutralizing antibodies as determined by IC₅₀ in live SARS-CoV-2 assays.

TABLE 8: Neutralization activity of exemplary potently neutralizing TXG antibodies as determined in testing against SARS-CoV-2 (lineage B.l, carrying the D614G mutation).

[0527] Excluding the 29 control antibodies, at least twenty-two (24) of the hits (9.8% of all antibodies, excluding controls) displayed IC50 values below 100 ng/mL, including TXG-0001 (44 ng/mL), TXG-0004 (90 ng/mL), TXG-0006 (11 ng/mL), TXG-0009 (97 ng/mL), TXG-0063 (89 ng/mL), TXG-0069 (9 ng/mL), TXG-0077 (21 ng/mL), TXG-0080 (42 ng/mL), TXG-0088 (65 ng/mL), TXG-0093 (54 ng/mL), TXG-0109 (32 ng/mL), TXG-0120 (39 ng/mL), TXG-0126 (35 ng/mL), TXG-0129 (15 ng/mL), TXG-0178 (20 ng/mL), TXG-0189 (53 ng/mL), TXG-0197 (79 ng/mL), TXG-0200 (42 ng/mL), TXG-0202 (45 ng/mL), TXG-0204 (47 ng/mL), TXG-0209 (50 ng/mL), TXG-0210 (41 ng/mL), TXG-140 (40 ng/mL), and TXG-154 (22 ng/mL).

[0528] A summary of the antibodies’ neutralization potency is also shown in FIGS. 28A and 28B. As indicated in Table 8, the most potent neutralizing antibodies (excluding those identifying as controls) were TXG-0006, TXG-0069, TXG-0077, and TXG-0129.

[0529] In these experiments, positive plasma controls (CS478 pi_vac_pfl, plasma of Pfizer vaccine) were run on every plate to determine the intra-plate and inter-day variation. The average NT50 value and standard deviation of all plates was 2,183 ± 551(CV=25.2%), with lower variation when plates in the same day were evaluated. Of note, NT50 values for the plasma control were generated with dose-response curves using singlet data-points (as compared to duplicates), and that may have increased the variation observed in these controls.

[0530] Additionally, an UpSet plot of antibodies identified as having neutralization activity against live SARS-COV-2 was generated (see, e.g. , FIG. 26), wherein the antibodies are binned into antigen bins as described in FIG. 25. The data described in this figure illustrates that the BEAM-seq process described in the present disclosure allows for rapid identification of many antibodies with broad and robust neutralizing activity against several SARS-CoV-2 S variants of concern (VoC), e.g., beta, gamma, and kappa, as well as HKU1 (which is a different coronavirus).

[0531] In addition, two UpSet plots of the potently (IC50 <= 1000 ng / ml) neutralizing antibodies retrieved from the BEAM-seq workflow described herein were generated (see, e.g, FIGS. 27A and 27B). FIG. 27A is an Upset plot of the potently neutralizing antibodies selected from 239 antibodies identified in Example 6. FIG. 27B is an Upset plot of the potently neutralizing antibodies selected from the antibodies of Table 3. In these UpSet plots, rows represent the binding of these neutralizing antibodies to pre-fusion spike trimers from major SARS-CoV-2 variants of concern, the endemic HKU1 coronavirus spike protein and the SARS- CoV-2 N terminal domain. The data described in these figures illustrate that the BEAM-seq process described in the present disclosure allows for rapid identification of many antibodies with potent and broad neutralizing activity against several SARS-CoV-2 S variants of concern (VoC), and that are diverse in their VH/VL usage. These potently neutralizing antibodies were found to use 18 diverse VH genes and 46 unique VH :VL pairings.

[0532] As illustrated in FIGS. 29-31, analysis of the IC50 values associated with external control antibodies previously characterized as having binding affinity for SARS-CoV-2 spike protein, including FDA authorized and/or approved external controls, as well as several control antibodies discovered via traditional means (e.g, originating from phage display, hybridoma, humanized mouse), shows that the BEAM-seq workflows disclosed herein yield greater numbers of antibodies with superior properties as compared to traditional antibody discovery workflows. For example, the data presented in FIG. 29 demonstrated that the ON rate of antibodies isolated with BEAM is correlated with the detected antigen UMIs. Accordingly, the BEAM-seq workflows disclosed herein identified higher numbers of antibody hits that neutralize live SARS- CoV-2 at greater potency, and within a much shorter timeframe than traditional discovery approaches. The antibody hits identified via BEAM-seq workflows are likely to have lower developability burden than those identified using display methodologies. Collectively, the figures and data disclosed here demonstrate that the affinity and functional profiles of BEAM-seq- derived antibodies are typically superior or non-inferior to those of antibodies derived using slower and lower-throughput approaches.

EXAMPLE 14 Epitope binning assays

[0533] This Example describes the results of experiments performed to assess binding characteristics of select antibodies described herein.

Methodology

[0534] 1) Antigens:

[0535] a) Pre-fusion trimerized spike protein from SARS-CoV-2 USA-WA 1 /2020 isolate, ACRO Biosystems.

[0536] b) Pre-fusion trimerized spike protein from SARS-CoV-2 delta variant,

ACRO Biosystems.

[0537] c) SARS-CoV-2 NTD, ACRO Biosystems.

[0538] 2) Surface Plasmon Resonance fSPR) techniques:

[0539] a) Competition/sandwich.

[0540] b) Bidirectional/premix.

[0541] Epitope binning experiments were performed in a premix format using a Carterra LSA SPR biosensor equipped with a HC30M chip at 25°C in HBS-TE (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20). First, antibodies were amine-coupled to the sensor chip by EDC/NHS activation, followed by ethanolamine HC1 quenching. Antibodies and SARS-CoV-2 prefusion stabilized S trimers were combined and incubated for 1 hour in HBS-TE with 0.5 mg/ml BSA at 120 nM and 7.5 nM, respectively. This constituted a 5-molar excess relative to the S trimer antigens, accounting for three (3) binding sites for each molecule. Each premix sample was injected over the immobilized antibodies to determine blocking, partial blocking, or non -blocking activity. The sensor chip was regenerated between injections with Pierce IgG Elution Buffer (Thermo Fisher Scientific).

[0542] Data were analyzed using Carterra’s Epitope Tool software. Briefly, blocking assignments were determined relative to the binding responses for S turner alone (normalized to 1); premixes giving binding responses less than 0.5 were determined to be blocking, 0.5-0.7 were intermediate blocking, and above 0.7 were not blocking. Heat maps representing the competition results were generated where red, yellow, and green cells represent blocked, intermediate, and not blocked analyte/ligand pairs, respectively. A summary of binding characteristics of exemplary antibodies described herein is presented in FIGS. 32-33 and Table 9 below.

TABLE 9: Epitope binning of exemplary antibodies as determined in testing against a pre fusion trimerized spike protein from SARS-CoV-2 USA-WA 112020 isolate (WA1) and/or SARS-CoV-2 delta variant (Delta) by using SPR competition assay and bidirectional assay. A number of control antibodies (CTRL) were included in these experiments, including: CTRL- 0004: Casirivimab; CTRL-0005 (Imdevimab), CTRL-0006: Bamlanivimab; CTRL-0007: (Etesevimab), CTRL-0008: Sotrovimab; and CTRL-0009: Tixagevimab. NA: not applicable. Other: antibodies capable of binding to an epitope different that the antibodies in any other of the bins identified in the same column of Table 9.

Data interpretation

[0543] In these experiments, antibodies that share an epitope bin are antibodies which compete for binding to the same epitopes in a dose-dependent manner.

[0544] As shown in the table above and further described below, the discovered antibodies group into five prominent epitope bins. Furthermore, a number of the discovered antibodies group into unique bins outside of the five prominent bins.

A. NTD targeting antibodies

[0545] As shown in Table 11, twenty-four (24) antibodies tested in these epitope binning experiments grouped into WA1 trimer bins 1 or 2. Sixteen (16) of these antibodies grouped into WA1 trimer bin 1 and 8 antibodies grouped into WA1 trimer bin 2. These likely represent two groups of antibodies targeting at least two distinct NTD epitopes of the spike protein from SARS-CoV-2 USA-WA1/2020 isolate (WA1), based on SPR data indicating that antibodies that group into bins 1 and 2 exhibit high (nM) affinity for the N-terminal domain (NTD) of SARS- CoV-2 S protein and the observation that they do not compete for binding with the antibodies in WA1 trimer bins 3, 4, or 3/4 (which include the FDA-authorized antibodies which have been shown to target RBD epitopes). Of these antibodies, as least 20 antibodies were found to display measurable neutralization activity as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies that target NTD of the WA1 isolate and potently neutralize live SARS-CoV-2 include TXG-0066, TXG-0071, TXG-0078, TXG-0104, TXG-0116, TXG-0170, TXG-0173, and TXG-0174.

[0546] As shown in Table 11, eighteen (18) antibodies tested in these epitope binning experiments grouped into delta trimer bins 1, 2, or 1/2. Eight (8) of these antibodies grouped into delta trimer bin 1, nine (9) antibodies grouped into delta trimer bin 2, and one antibody was grouped into delta trimer bin 1/2. These likely represent two groups of antibodies targeting at least two distinct NTD epitopes of the spike protein from SARS-CoV-2 delta variant, based on SPR data indicating that antibodies that group into these bins exhibit high (nM) affinity for the N-terminal domain (NTD) of SARS-CoV-2 S protein and the observation that they do not compete for binding with the antibodies in delta trimer bins 3, 4, or 3/4 (which include the FDA- authorized antibodies which have been shown to target RBD epitopes). Of these 18 antibodies, as least fifteen (15) antibodies were found to display measurable neutralization activity as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies that target NTD of the WA1 isolate and potently neutralize live SARS-CoV-2 include TXG-0071, TXG-0078, TXG- 0091, TXG-0099, TXG-0116, TXG-0170, and TXG-0174.

[0547] B. RBD targeting antibodies

[0548] As shown in Table 11, 46 antibodies tested in these epitope binning experiments grouped into WA1 trimer bins 3, 4, or 3/4. These likely represent three groups of antibodies targeting at least three partially distinctive RBD of the spike protein from SARS-CoV-2 USA- WAl/2020 isolate (WA1), based on the observation that they compete for binding with FDA- authorized antibodies which have been shown to target RBD epitopes. Ten (10) of these antibodies grouped into WA1 trimer bin 3, 16 antibodies grouped into WA1 trimer bin 4, and 20 antibodies grouped into WA1 trimer bin 3/4. All of these 46 antibodies were found to display measurable neutralization activity as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies that target primarily RBD of the WA1 isolate and potently neutralize live SARS- CoV-2 include TXG-0001, TXG-0002, TXG-0004, TXG-0005, TXG-0006, TXG-0008, TXG- 0009, TXG-0057, TXG-0063, TXG-0069, TXG-0077, TXG-0080, TXG-0081, TXG-0086, TXG-0088, TXG-0091, TXG-0093, TXG-0094, TXG-0100, TXG-0109, TXG-0115, TXG-0120, TXG-0126, TXG-0128, TXG-0129, TXG-0132, TXG-0140, TXG-0141, TXG-0144, TXG-0153, TXG-0154, TXG-0178, TXG-0180, TXG-0181, TXG-0183, TXG-0189, TXG-0197, TXG-0198, TXG-0200, TXG-0201, TXG-0202, TXG-0204, TXG-0207, TXG-0209, and TXG-0210.

[0549] As shown in Table 11, ten (10) antibodies tested in these epitope binning experiments grouped into WA1 trimer bin 3, along with CTRL-0008 (sotrovimab). These likely represent antibodies that target a WA1 RBD epitope that is at least partially distinctive from those targeted by bins 3/4 or 4, e.g. , a distinctive RBD epitope from those targeted by the tested FDA-approved antibodies save for sotrovimab. All of these 10 antibodies were found to display measurable neutralization activity as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies in WA1 trimer bin 3 and which potently neutralize live SARS-CoV-2 include TXG-0057, TXG-0063, TXG-0091, TXG-0093, TXG-0094, TXG-0120, TXG-0153, TXG-0181, and TXG-0183.

[0550] As shown in Table 11, 16 antibodies tested in these epitope binning experiments grouped into WA1 trimer bin 4, along with CTRL-0004 (Casirivimab), CTRL-0007 (Etesevimab), and CTRL-0009 (Tixagevimab). These likely represent antibodies that target a WA1 RBD epitope that is at least partially distinctive from those targeted by bins 3 or 3/4. All of these 16 antibodies were found to display potent neutralization activity as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies in WA1 trimer bin 4 and which potently neutralize live SARS-CoV-2 include TXG-0077, TXG-0109, TXG-0128, TXG-0132, TXG- 0141, TXG-0144, TXG-0180, TXG-0197, TXG-0198, TXG-0200, TXG-0201, TXG-0202, TXG-0204, TXG-0207, TXG-0209, and TXG-0210.

[0551] As shown in Table 11, 32 antibodies tested in these epitope binning experiments grouped into delta trimer bins 3, 4, or 3/4. These likely represent antibodies targeting primarily RBD of the spike protein from SARS-CoV-2 delta variant, based on the observation that they compete for binding with FDA-authorized antibodies which have been shown to target RBD epitopes. Nearly all of these antibodies also grouped into WA1 trimer bins 3, 4, or 3/4. Of these 32 antibodies, as least 30 antibodies were also found to display measurable neutralization activity as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies that target primarily RBD of the SARS-CoV-2 delta variant and potently neutralize live SARS-CoV-2 include TXG- 0001, TXG-0002, TXG-0004, TXG-0005, TXG-0006, TXG-0008, TXG-0009, TXG-0063, TXG-0077, TXG-0086, TXG-0093, TXG-0094, TXG-0100, TXG-0120, TXG-0126, TXG-0128, TXG-0129, TXG-0140, TXG-0141, TXG-0154, TXG-0180, TXG-0181, TXG-0197, TXG-0198, TXG-0200, TXG-0201, TXG-0202, TXG-0204, TXG-0209, and TXG-0210.

[0552] As shown in Table 11, five (5) antibodies tested in these epitope binning experiments grouped into delta trimer bin 3, along with CTRL-0008 (sotrovimab). These likely represent antibodies that target a delta RBD epitope that is at least partially distinctive from those targeted by bins 3/4 or 4, e.g ., a distinctive delta RBD epitope from those targeted by the tested FDA-approved antibodies save for sotrovimab. These 5 antibodies also grouped into WA1 trimer bin 3. All of these 5 antibodies were found to display potent neutralization activity as determined by IC50 in live SAR.S-CoV-2 assays. Antibodies that distinctively target RBD of the SAR.S-C0V- 2 delta variant and potently neutralize live SAR.S-CoV-2 include TXG-0063, TXG-0093, TXG- 0094, TXG-0120, and TXG-0181.

[0553] Ten (10) antibodies tested in these epitope binning experiments grouped into delta trimer bin 4, along with CTRL-0004 (Casirivimab), CTRL-0007 (Etesevimab) and CTRL-0009 (Tixagevimab). These likely represent antibodies that target a delta RBD epitope that is at least partially distinctive from those targeted by bins 3 or 3/4. All of these 10 antibodies were found to display potent neutralization activity as determined by IC50 in live SAR.S-CoV-2 assays. Examples of antibodies in delta trimer bin 4 and which potently neutralize live SAR.S-CoV-2 include TXG-0077, TXG-0128, TXG-0180, TXG-0197, TXG-0198, TXG-0200, TXG-0201, TXG-0202, TXG-0204, and TXG-0209.

[0554] Twenty (20) antibodies tested in these epitope binning experiments grouped into WA1 trimer bin 3/4, along with CTRL-0005 (Imdevimab) and CTRL-0006 (Bamlanivimab). These likely represent antibodies that target a WA1 RBD epitope that is at least partially distinctive from those targeted by bin 3 or bin 4, in that they partially compete with antibodies such bins. All of these antibodies displayed potent neutralization activity as determined by IC50 in live SAR.S-CoV-2 assays. These antibodies include: TXG-0001, TXG-0002, TXG-0004, TXG-0005, TXG-0006, TXG-0008, TXG-0009, TXG-0069, TXG-0080, TXG-0081, TXG-0086, TXG-0088, TXG-0100, TXG-0115, TXG-0126, TXG-0129, TXG-0140, TXG-0154, TXG-0178, and TXG-0189.

[0555] Seventeen (17) antibodies tested in these epitope binning experiments grouped into delta trimer bin 3/4, along with CTRL-0005 (Imdevimab). These likely represent antibodies that target a delta RBD epitope that is at least partially distinctive from those targeted by bin 3 or bin 4, in that they partially compete with antibodies such bins. Of these, 15 antibodies displayed potent neutralization activity as determined by live SARS-CoV-2 assays. Examples of antibodies in delta trimer bin 3/4 and which potently neutralize live SARS-CoV-2 include: TXG-0001, TXG-0002, TXG-0004, TXG-0005, TXG-0006, TXG-0008, TXG-0009, TXG-0086, TXG-0100, TXG-0126, TXG-0129, TXG-0140, TXG-0141, TXG-0154, and TXG-0210.

[0556] C. Other epitopes

[0557] Eighteen (18) antibodies tested in these epitope binning experiments grouped into WA1 trimer bins “Other” or “NA”. These likely represent antibodies that target epitopes that are distinctive from the epitopes targeted by any other of the WA1 trimer bins. Exemplary antibodies in these categories include TXG-0010, TXG-0053, TXG-0054, TXG-0064, TXG-0076, TXG- 0085, TXG-0099, TXG-0112, TXG-0114, TXG-0146, TXG-0187, TXG-0192, TXG-0203, TXG-0206, TXG-0227, TXG-0228, TXG-0229, and TXG-0230. Of these 18 antibodies, at least two are potent neutralizers. Example antibodies that belong in the WA1 trimer “Other” or “NA” bin and potently neutralize live SARS-CoV-2 include TXG-0076 and TXG-0099. Of these 18 antibodies, at least 14 exhibit high (nM) affinity for WT spike and the gamma, kappa, and beta variants by SPR. Example antibodies that belong in the WA1 trimer “Other” or “NA” bin and exhibit high (nM) affinity for WT spike and the gamma, kappa, and beta variants include TXG- 0053, TXG-0054, TXG-0085, TXG-0099, TXG-0112, TXG-0114, TXG-0146, TXG-0187, TXG-0192, TXG-0203, TXG-0227, TXG-0228, TXG-0229, and TXG-0230. Of these 18 antibodies, at least five exhibit nanomolar affinity for NTD. Example antibodies that belong in the WA1 trimer “Other” or “NA” bin and exhibit high (nM) affinity for NTD include TXG-0064, TXG-0076, TXG-0099, TXG-0146, and TXG-0230. Of these 18 antibodies, at least 12 exhibit nanomolar affinity for HKU. Example antibodies that belong in the WA1 trimer “Other” or “NA” bin and exhibit nanomolar affinity for HKU include TXG-0053, TXG-0054, TXG-0085, TXG-0112, TXG-0114, TXG-0187, TXG-0192, TXG-0203, TXG-0227, TXG-0228, TXG-0229, and TXG-0230.

[0558] Thirteen (13) antibodies tested in these epitope binning experiments grouped into delta trimer bin 5. These likely represent antibodies that target a distinct delta variant epitope from other binned groups, e.g ., distinct from any of the tested FDA approved antibodies. Exemplary antibodies in this category include: TXG-0053, TXG-0080, TXG-0115, TXG-0132, TXG-0136, TXG-0162, TXG-0175, TXG-0178, TXG-0192, TXG-0206, TXG-0207, TXG-0230, and TXG-0232. Of these, five displayed potent neutralization activity as determined by live SARS-CoV-2 assays. Examples of antibodies in delta trimer bin 5 and which potently neutralize live SARS-CoV-2 include: TXG-0080, TXG-0115, TXG-0132, TXG-0178, and TXG-0207.

[0559] 23 antibodies tested in these epitope binning experiments grouped into delta trimer bins “Other”. These likely represent antibodies that target epitopes that are distinctive from the epitopes targeted by any other of the delta trimer bins. Exemplary antibodies in these categories include TXG-0054, TXG-0057, TXG-0069, TXG-0076, TXG-0081, TXG-0085, TXG-0088, TXG-0104, TXG-0109, TXG-0112, TXG-0114, TXG-0144, TXG-0146, TXG-0164, TXG-0168, TXG-0173, TXG-0183, TXG-0187, TXG-0189, TXG-0203, TXG-0227, TXG-0228, and TXG-0229. Of these 23 antibodies, 13 exhibit measurable neutralization activity and 11 are potent neutralizers. Examples of antibodies in delta trimer bin “Other” and which neutralize live SARS-CoV 2 include: TXG-0057, TXG-0069, TXG-0076, TXG-0081, TXG-0088, TXG-0104, TXG-0109, TXG-0144, TXG-0164, TXG-0168, TXG-0173, TXG-0183, and TXG-0189. Examples of antibodies in delta trimer bin “Other” and which potently neutralize live SARS- CoV 2 include: TXG-0057, TXG-0069, TXG-0076, TXG-0081, TXG-0088, TXG-0104, TXG- 0109, TXG-0144, TXG-0173, TXG-0183, and TXG-0189. Of these 23 antibodies, at least 19 exhibit high (nM) affinity for WT spike and the kappa and gamma variants by SPR, and at least 14 exhibit high (nM ) affinity for WT spike and the beta, gamma, and kappa variants by SPR. Exemplary antibodies that belong in the delta trimer “Other” bin and exhibit high (nM) affinity for WT spike and the gamma and kappa variants include TXG-0054, TXG-0057, TXG-0076, TXG-0085, TXG-0104, TXG-0109, TXG-0112, TXG-0114, TXG-0144, TXG-0146, TXG-0164, TXG-0168, TXG-0173, TXG-0183, TXG-0187, TXG-0203, TXG-0227, TXG-0228, and TXG- 0229. Exemplary antibodies that belong in the delta trimer “Other “ bin and exhibit high (nM) affinity for WT spike and the beta, gamma, and kappa variants include TXG-0054, TXG-0057, TXG-0085, TXG-0109, TXG-0112, TXG-0114, TXG-0144, TXG-0146, TXG-0183, TXG-0187, TXG-0203, TXG-0227, TXG-0228, and TXG-0229.

[0560] These results illustrate that the BEAM-seq process described in the present disclosure, in addition to the advantages described elsewhere herein, can beneficially and rapidly identify a large set of specific antibodies that bind to a highly diverse range of epitopes for a target antigen of interest.

[0561] Antibodies that group into different epitope bins can advantageously be used in a therapeutic antibody cocktail or combination therapy regimen. For example, a neutralizing antibody from bin A can thus be combined with a neutralizing antibody from bin B effectively as the two antibodies do not bind in the same location. Examples of such complementary bins that may be advantageously used in a combination therapy or antibody cocktail include:

[0562] NTD and RBD targeting combination: a. Antibody 1 : an antibody from bin 1 or bin 2, both of which represent NTD- binding antibodies b. Antibody 2: an antibody from bin 3, 4, or 3/4, which represent antibodies targeting primarily RBD

[0563] RBD distinctive targeting combination: a. Antibody 1 : an antibody from bin 3 (sotrovimab-like antibodies) b. Antibody 2: an antibody from bin 4

[0564] RBD partially distinctive targeting combination: a. Antibody 1 : an antibody from bin 3 or 4

[0565] Antibody 2: an antibody from bin 3/4 which partially competes with an antibody from bin 3 or 4.

EXAMPLE 15

Analysis of antibodies for binding one or more antigens under a variety of treatment conditions

[0566] As illustrated in FIG. 34, a set of reporter oligonucleotide-associated antigens comprises a first antigen (or epitope) coupled to a first reporter oligonucleotide comprising a first barcode sequence (BC1), and the same first antigen (or epitope) coupled to a second reporter oligonucleotide comprising a second barcode sequence (BC2). In some embodiments, the set of reporter oligonucleotide-associated antigens further includes a second antigen (or epitope) coupled to a third reporter oligonucleotide comprising a third barcode sequence (BC3) and the same second antigen (or epitope) coupled to a fourth reporter oligonucleotide comprising a fourth barcode sequence (BC4). The first antigen may be a target antigen and the second antigen may be a negative control antigen (e.g., as described herein). The BC1 -associated first antigen (and optionally the BC3-associated second antigen) is subjected to a first treatment condition (e.g., fixation) and the BC2-associated first antigen (and optionally the BC4-associated second antigen is subjected to a second treatment condition (e.g., no fixation). Thus, the reporter barcode sequences (e.g., BC1-BC4) are used to identify both the antigen (or epitope) and the treatment condition that the antigen (or epitope) are subjected to.

[0567] B cells are stained with the set of reporter oligonucleotide-associated antigens. Standard gene expression, V(D)J, and barcoded antigen libraries are prepared from the stained B cells using the lOx 5’ V2 Single Cell Immune Profiling kit per manufacturer’s instructions. Sequence analysis of the libarires is used to identify antigen-binding molecules (e.g., antibodies) that bind to antigens subjected to various treatment conditions.

EXAMPLE 16 BEAM denoising

[0568] This Example describes the results of experiments performed to test BEAM denoising approach by pre-blocking cells with TsC-STA loaded with Biotin.

[0569] Biomaterials. Donor 531 PBMCs as described in Example 1 were purchased from Cellero (~112m/vial product, Cat. # 1146-4785JY20) and used in these experiments.

[0570] Enrichment. Enrichment of B cells was performed as described in Example 2 above.

[0571] Pre-block. Cells were either pre-blocked with biotin-saturated streptavidin prior to cell labeling. 5 uL of 4 mM biotin was added to 5 uL of 1 : 10 diluted BioLegned TsC-STA-PE (0965). TsC-STA-PE (0965) was diluted in PBS and 5 uL of diluted material contained 9.45 pmol of the reagent. The biotin binding reaction was performed at room temperature in the dark for 30 min.

[0572] Cell labelins. Cell labelling was performed as described in Example 4 above, with the exception that biotin saturated SAV was included. COV-19 Donor D531 PBMCs were thawed and washed and blocked with PBS + 2% FBS for 30 min. B cell enrichment was performed using StemCell Technologies EasySep Human CD3 Positive Selection kit II (Cat.# 17851). Fc block was performed for 10 min. Cells that received biotin block were stained with TsC-STA-PE-Biotin (0965) 1 : 100 for 30 min on ice in the dark.

[0573] B cells were stained with TsC-STA-CoV2 antigens or TsC-STA-HSA at 1 : 100 dilution, and with anti-CD19, CD14, CD15, CD16 antibodies and 7AAD viability stain. Cells were washed two times with 1 mL of PBS + 2% FBS.

[0574] Antisen-specific enrichment by FACS antigen-specific enrichment was performed as described in Example 5 above. Live cells were selected using 7AAD negative gate. CD14 ,

CD 15 , CD 16 CD19⁺ cells were gated. CD19⁺ PE⁺ cells and/or CD19⁺ APC⁺ cells were gated and sorted.

[0575] Analysis and Results : sequencing was performed as described in Example 6 above. Statistical analysis was performed as described in Example 7 above. The analysis revealed that the same clones that were identified in Example 6 were also identified in this experiment.

EXAMPLE 17 Impact of Fc block

[0576] This Example describes the results of experiments performed to test impact of Fc block on data quality and further test impact of storage of antigens for 3 days vs. use of freshly prepared antigens.

[0577] Biomaterials. Donor 531 PBMCs as described in Example 1 were purchased from

Cellero (~112m/vial product, Cat. # 1146-4785JY20) and used in these experiments.

[0578] Cell enrichment. A vial of frozen PBMCs were thawed for 1-2 min in a water bath, then transferred into 8-10 mis of 10% Fetal Bovine Serum (FBS) in PBS, and centrifuged for 5 min at 350g. The cell pellet was washed three times by resuspending in 0.04% Bovine Serum Albumin (BSA) in PBS and centrifuging at RT at 350g for 5 min each wash, with the final pellet resuspended to a concentration of ~20 million cells per mL in a total volume of 5 mL (-100 million cells total). B cells were enriched using the B Cell Isolation Kit II (human; MACS Miltenyi) according to manufacturer’s instructions, and approximately 50 million cells were applied to each of two LS columns. The effluent was concentrated and prepared for cell labeling.

[0579] Antisen sourcins: Biotinylated antigens were sourced from suppliers as described in Example 3 above.

[0580] Cell labellins. Cell labelling was performed as described in Example 4 above.

[0581] Antisen-specific enrichment by FACS antigen-specific enrichment was performed as described in Example 5 above. Cells are gated on being single, live (7AAD-negative) and evaluated for CD19-PECy7+ and then sorted based on their PE and/or APC status directly into master mix and water based on one of four criteria:

[0582] 1) PE+, representing some combination of trimerized S (SARS-2) wt+Sl

NTD+RBD antigen+ and/or HSA+ control cells.

[0583] 2) APC+, representing trimerized S D614G (SARS-2) antigen and/or HSA control antigen+ cells);

[0584] 3) Dual PE+ and APC+, representing a combination of dual trimerized S

(SARS-2) antigen+, trimerized S D614G (SARS-2) antigen+, SI NTD+, SI ECD, RBD+ and/or HSA control antigen-positive cells;

[0585] 4) PE and APC negative cells, representing cells that are not binding any antigen, or at a level below the thresholding/gating/detection we set on FACS (note: a new, narrower gate, “J” was generated in the center of the quadrant 3 gate.

[0586] The total number of events/cells (and not aborted cells sorted into Master Mix and Water) is recorded for loading into the lOx Chromium system. The volume is adjusted with additional water to match the recommended volume and target concentration for loading with the lOx 5Ύ2 Single Cell Immune Profiling kit. FACS data is analyzed using FlowJo.

[0587] Analysis and Results : sequencing was performed as described in Example 6 above. Statistical analysis was performed as described in Example 7 above. As illustrated in FIG. 36, it was observed that performance between fresh and stored reagents was equivalent. In addition, it was also observed that Fc block increased the number of clonotypes identified and the number of expanded clonotypes.

EXAMPLE 18 Testing in mouse models

[0588] Animals: BALB/c mice were immunized on DO with 50 pg of SARS-CoV-2- S protein (His Tag, Super stable trimer: Aero Biosystems, Cat. #: SPN-C52H9). They received a booster immunization with 25 pg of the S protein on D14, D28, D42, and a final boost (50 pg) on D51. Samples (plasma, lymph nodes, spleen, and femur and tibia) were taken from the mice on D56.

[0589] Sample preparation:

[0590] Splenocytes : briefly, samples were filtered through a 70 pm filter, washed with cold buffer (e.g., PBS + 10% serum), centrifuged (e.g., at 300 g for 5 minutes), and lysed with ACK lysis buffer and then washed prior to cell counting. [0591] Lymphocytes lymphocytes were obtained from femur/tibia samples as follows: samples were flushed with cold PBS + 10% serum by a 23 G needle syringe. The sample was then centrifuged (e.g., at 300 g for 5 minutes), then washed with cold buffer (e.g., PBS + 10% serum, filtered through a 70 pm filter prior to cell counting.

[0592] Bone marrow briefly, bone marrow samples were filtered through a 70 pm filter, washed with cold buffer (e.g., PBS + 10% serum), centrifuged (e.g., at 300 g for 5 minutes), and lysed with ACK lysis buffer and then washed prior to cell counting.

[0593] Antigen sourcing preparation and conjugation: Biotinylated antigens were sourced from suppliers and conjugated to TotalSeqC reagents as follows.

[0594] Cell labelling: Cells were subjected to Fc block, and then stained with the above antigens and additional barcoded antibodies for lOx Single Cell immune profiling, as described in Example 4 above.

[0595] Antigen-specific enrichment via FACS: Cells were initially gated on being single, live (7AADnegative) and PE-Cy7-CD19+, then sorted based on PE status into master mix and water. Standard gene expression, V(D)J, and barcoded antigen libraries were constructed using the lOx 5’ V2 Single Cell Immune Profiling kit per manufacturer's instructions.

[0596] Sequencing and analysis: The libraries were sequenced as described herein (see Example 6). PE positive B cells were detected as being associated with the D614G reporter barcode sequence 0995. PE positive B cells were detected as being associated with the HSA reporter barcode sequence 0955. PE positive B cells were also detected as being associated with the NTD reporter barcode sequence 0961. These results indicate that the BEAM-seq workflows disclosed herein can advantageously discover target-specific antibodies using a variety of mouse samples.

EXAMPLE 19

Comparison with hybridoma-based discovery

[0597] A portion of the samples from Example 18 are saved and used for bulk fusions to produce hybridoma cells, followed by cell plating and ELISA tests for the three antigens according to traditional hybridoma discovery methods. The hybridoma methods yield results in 5+ weeks, as compared to the BEAM-seq method described above which yields results in 1 week. Furthermore, the BEAM-seq method identifies more target-specific antibodies than the hybridoma workflow.

EXAMPLE 20

[0598] B cells are stained with a barcoded antigen panel and panel of barcoded antibodies for profiling immune cells, including antibodies from a “T and B Natural Killer” (TBNK) panel with binding affinity for individual immune cell features such as CD3, CD4 and CD8 (for T-cells), CD56 (for NK cells), and CD 19 (for B-cells). See, e.g., Example 4. In these experiments, each barcoded antibody of the panel of barcoded antibodies comprises a reporter oligonucleotide that identifies the immune cell feature. In these experiments, separate sequencing handles are deployed for the TBNK panel and the antigen panel to facilitate downstream various downstream applications, including identification and/or isolation of antibodies that specifically bind a target antigen.

While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, the method comprising: a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a target antigen coupled to a first reporter oligonucleotide and (ii) a non-target antigen coupled to a second reporter oligonucleotide, and wherein the contacting provides a B cell bound to the target antigen; b) partitioning the B cell bound to the target antigen into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the B cell bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen; d) assessing the binding affinity of the antibody or antigen-binding fragment to the target antigen; and e) identifying the antibody or antigen-binding fragment as having a binding specificity for the target antigen if the antibody or antigen-binding fragment specifically binds to the target antigen.

2. A method for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, the method comprising: a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a target antigen coupled to a first reporter oligonucleotide and (ii) a non-target antigen coupled to a second reporter oligonucleotide, and wherein the contacting provides a B cell bound to the target antigen; b) partitioning the B cell bound to the target antigen into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the B cell bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen; and d) identifying the antibody or antigen-binding fragment as having a binding specificity for the target antigen if the antibody or antigen-binding fragment binds to the target antigen and does not significantly bind the non-target antigen.

3. A method for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, the method comprising: a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a target antigen coupled to a first reporter oligonucleotide and (ii) a non-target antigen coupled to a second reporter oligonucleotide, and wherein the contacting provides a B cell bound to the target antigen; b) partitioning the B cell bound to the target antigen into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the B cell bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen; and d) identifying the antibody or antigen-binding fragment as non-specific for the target antigen if the antibody or antigen-binding fragment binds to the non-target antigen.

4. The method of any one of claims 1 to 3, wherein the non-target antigen has been selected such that it is not expected to bind the antibody or antigen-binding fragment.

5. The method of any one of claims 1 to 3, wherein the non-target antigen is an antigen to which the B cell is not expected to bind.

6. The method of any one of claims 1 to 5, wherein the first and the second reporter oligonucleotide comprising (i) a first and a second reporter barcode sequence that identify the target antigen and the non-target antigen, respectively and (ii) a capture handle sequence, optionally wherein the first and/or second reporter barcode sequence identifies a treatment condition that the target antigen and/or non-target antigen are subjected to.

7. A method for identifying and/or characterizing an antibody, or antigen-binding fragment thereof, the method comprising: a) contacting a plurality of B cells obtained from a biological sample with a plurality of antigens, wherein the plurality of antigens comprises (i) a first antigen coupled to a first reporter oligonucleotide comprising a first reporter barcode sequence and (ii) the first antigen coupled to a second reporter oligonucleotide comprising a second reporter barcode sequence, wherein the first antigen coupled to the first reporter oligonucleotide is subjected to a first treatment condition and the first antigen coupled to the second reporter oligonucleotide is subjected to a second treatment condition, and wherein the contacting provides a labeled B cell bound to the first antigen coupled to the first reporter oligonucleotide and/or the first antigen coupled to the second reporter oligonucleotide; b) partitioning the labeled B cell into a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the labeled B cell and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the labeled B cell; and d) identifying the antibody or antigen-binding fragment as binding to the first antigen subjected to the first and/or second treatment condition, wherein optionally the plurality of antigens further comprises (iii) a second antigen coupled to a third reporter oligonucleotide comprising a third reporter barcode sequence and (iv) the second antigen coupled to a fourth reporter barcode sequence, wherein (iii) and (iv) are subjected to the first treatment condition and second treatment conditions, respectively, and wherein the labeled B cell is optionally bound to (iii) and/or (iv), optionally wherein the first antigen is a target antigen and the second antigen is a non-target control antigen, and/or wherein the method further comprises the prior step of subjecting (i) and (ii) to the first and second treatment conditions, respectively, prior to (a), and/or wherein the method further comprises the prior step of subjecting (iii) and (iv) to the first and second treatment conditions, respectively, prior to (a).

8. The method of any one of claims 1 to 7, wherein a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to the capture handle sequence of the first and/or second reporter oligonucleotide, and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further comprises a capture sequence configured to couple to an mRNA analyte or DNA analyte.

9. The method of claim 8, wherein the capture sequence is configured to couple to the capture handle sequence of the reporter oligonucleotide by complementarity base pairing.

10. The method of claim 8, wherein the capture sequence is configured to couple to the mRNA analyte, and wherein the capture sequence configured to couple to the mRNA analyte comprises a polyT sequence.

11. The method of claim 8, wherein the capture sequence is configured to couple to the mRNA analyte, and wherein the capture sequence configured to couple to the mRNA analyte comprises a targeted priming sequence, optionally wherein the targeted priming sequence targets an antibody or BCR region of the mRNA analyte, optionally wherein the targeted priming sequence targets a constant sequence of said antibody or BCR region of the mRNA analyte.

12. The method of any one of claims 1 to 11, wherein a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to the capture handle sequence, and wherein a second nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to non-templated nucleotides appended to a cDNA reverse transcribed from an mRNA analyte.

13. The method of claim 12, wherein the mRNA analyte is reverse transcribed to the cDNA utilizing a primer comprising a polyT sequence.

14. The method of claims 12-13, wherein the non-templated nucleotides appended to the cDNA comprise a cytosine.

15. The method of claim 14, wherein the capture sequence configured to couple to the cDNA comprise a guanine.

16. The method of claim 15, wherein the coupling of the capture sequence to the non-templated cytosine extends reverse transcription of the cDNA into the second nucleic acid barcode to generate the second barcoded nucleic molecule.

17. The method of claim 16, wherein the second nucleic acid barcode molecule further comprises a template switch oligonucleotide (TSO).

18. The method of any one of claims 1 to 17, wherein the first and second nucleic acid barcode molecules each comprise a unique molecule identifier (UMI).

19. The method of any one of claims 1 to 18, further comprising generating, in the partition, a first barcoded nucleic acid molecule or a plurality of first barcoded nucleic acid molecules comprising (i) a sequence of the first reporter oligonucleotide, or a reverse complement thereof, and (ii) the common barcode sequence or a reserve complement thereof, and optionally using the first barcoded nucleic acid molecule to identify the B cell as having bound to the target antigen coupled to the first reporter oligonucleotide.

20. The method of any one of claims 1 to 18, further comprising generating a first barcoded nucleic acid molecule or a plurality of first barcoded nucleic acid molecules comprising (i) a sequence of the first reporter oligonucleotide, or a reverse complement thereof, and (ii) the common barcode sequence or a reserve complement thereof, and optionally using the first barcoded nucleic acid molecule to identify the B cell as having bound to the target antigen coupled to the first reporter oligonucleotide.

21. The method of any one of claims 19 to 20, wherein the binding affinity is assessed based on the number of first barcoded nucleic acid molecules comprising (i) a sequence of the first reporter oligonucleotide or reverse complement thereof and (ii) the common barcode sequence or reverse complement thereof.

22. The method of any one of claims 1 to 21, further comprising, in the partition, generating a second barcoded nucleic acid molecule comprising (i) a nucleic acid sequence encoding the antibody, or antigen-binding fragment thereof, produced by the B cell, and (ii) the common barcode sequence or reverse complement thereof, and optionally using the second barcoded nucleic acid molecule to identify a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen.

23. The method of any one of claims 1 to 21, further comprising generating a second barcoded nucleic acid molecule comprising (i) a nucleic acid sequence encoding the antibody, or antigen binding fragment thereof, produced by the B cell, and (ii) the common barcode sequence or reverse complement thereof, and optionally using the second barcoded nucleic acid molecule to identify a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the target antigen.

24. The method of any one of claims 1 to 23, further comprising generating a third barcoded nucleic acid molecule or plurality of third barcoded nucleic acid molecules comprising (i) a sequence of the second reporter oligonucleotide, or reverse complement thereof, and (ii) the common barcode sequence or reverse complement thereof, and optionally using the third barcoded nucleic acid molecule or plurality of third barcoded nucleic acid molecules to identify the B cell as having bound to the non-target antigen coupled to the second reporter oligonucleotide, optionally wherein the generating of the third barcoded nucleic acid molecule or plurality thereof occurs in the partition.

25. The method of any one of claims 19 to 24, further comprising determining sequences of the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule, and optionally determining a sequence of the third barcoded nucleic acid molecule, optionally wherein the determining is performed by sequencing.

26. The method of claim 25, further comprising identifying the antibody or antigen-binding fragment thereof based on the determined sequence of the second barcoded nucleic acid molecule.

27. The method of claim 26, wherein the determined sequence comprises a nucleotide sequence.

28. The method of claim 27, wherein the determined sequence comprises an amino acid sequence encoded by the nucleotide sequence.

29. The method of any one of claims 19 to 28, wherein the binding affinity of the antibody or antigen-binding fragment to the target antigen is assessed based on the determined sequence of the first barcoded nucleic acid molecule.

30. The method of any one of claims 1 to 29, wherein the biological sample is from a vertebrate subj ect.

31. The method of claim 30, wherein the vertebrate subject is a non-mammalian subject.

32. The method of claim 31, wherein the non-mammalian subject is an avian species.

33. The method of claim 30, wherein the vertebrate subject is a mammalian subject.

34. The method of claim 33, wherein the mammalian subject is a human.

35. The method of any one of claims 1 to 34, wherein the first and/or the second reporter oligonucleotide is conjugated to a tag configured for detection or separation.

36. The method of claim 35, wherein the tag is configured for magnetic separation.

37. The method of claim 36, wherein the tag comprises a fluorescent agent.

38. The method of any one of claims 1 to 37, further comprising, prior to the (b) partitioning, isolating and/or enriching the plurality of single B cells.

39. The method of claim 38, wherein the enriching comprises sorting of the B cells bound to the target antigen and/or non-target antigen based on detection of one or more labelling agents coupled to the reporter oligonucleotides attached to the respective antigens.

40. The method of claim 38 or 39, wherein the enriching the plurality of B cells comprises sorting cells of the plurality of B cells according to their binding to the target antigen.

41. The method of any one of claims 1 to 40, wherein the single B cell is a plasmablast, a plasma cell, a memory B cell, a regulatory B cell, or a lymphoplasmacytoid cell.

42. The method of any one of claims 1 to 41, wherein the target antigen and/or the non-target antigen is coupled to a barcode moiety that identifies the target antigen and/or the non-target antigen, respectively.

43. The method of any one of claims 1 to 42, wherein (a) further comprises contacting the biological sample with a plurality of additional labelling agents, wherein the additional labeling agents are configured to bind or otherwise couple to one or more cell-surface features of an immune cell.

44. A method for identifying an antibody having binding affinity for a coronavirus spike protein (CoV-S), the method comprising: a) contacting a plurality of B cells obtained from a subject who has been exposed to a coronavirus with a plurality of antigens, wherein the plurality of antigens comprises a CoV-S antigen and a non-CoV-S antigen, and wherein each of the antigens comprise a reporter oligonucleotide, wherein the contacting provides a B cell bound to a CoV-S antigen; b) partitioning the B cell bound to the CoV-S antigen in a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the B cell bound to the CoV- S antigen and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the CoV-S antigen; and d) assessing the binding affinity of the antibody or antigen-binding fragment to a CoV-S protein; and e) identifying the antibody or antigen-binding fragment as having a binding specificity for the CoV-S protein if the antibody or antigen-binding fragment specifically binds to the CoV-S protein.

45. The method of claim 44, wherein the reporter oligonucleotide comprising (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence.

46. The method of any one of claims 44 to 45, further comprising coupling a barcode moiety to the antibody or antigen-binding fragment produced by the B cell to generate a barcoded antibody or antigen-binding fragment.

47. The method of any one of claims 44 to 46, wherein a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to the capture handle sequence of the reporter oligonucleotide and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further comprises a capture sequence configured to couple to an mRNA analyte.

48. The method of claim 47, wherein the capture sequence is configured to couple to the capture handle sequence of the reporter oligonucleotide is complementary to the capture handle sequence of the reporter oligonucleotide.

49. The method of claim 48, wherein the capture sequence configured to couple to an mRNA analyte comprises a polyT sequence.

50. The method of any one of claims 44 to 49, wherein the first and second nucleic acid barcode molecules each comprise a unique molecule identifier (UMI).

51. The method of any one of claims 44 to 50, wherein the antibody has a binding specificity to an epitope on a domain of the CoV-S protein.

52. The method of claim 51, wherein the domain of the CoV-S protein is the SI region.

53. The method of claim 51, wherein the domain of the CoV-S protein is the S2 region.

54. The method of any one of claims 44 to 53, wherein the antibody or antigen-binding fragment has binding affinity for a trimeric form of the CoV-S protein.

55. The method of any one of claims 44 to 54, wherein the CoV-S protein is a spike protein of SARS-CoV-1, SARS-CoV-2, orMERS-CoV.

56. The method of any one of claims 44 to 55, wherein the subject is suspected of being infected with a coronavirus, has been infected with a coronavirus, has been vaccinated, or has been recovered from a coronavirus infection.

57. The method of any one of claims 44 to 56, wherein the subject is a mammalian subject.

58. The method of claim 57, wherein the mammalian subject is a human.

59. The method of any one of claims 44 to 58, wherein the antigens are each coupled to a fluorescent label identifying the antigens.

60. The method of any one of claims 44-59, further comprising isolating and/or enriching the plurality of single B cells prior to (b).

61. The method of claim 60, wherein the enriching further comprises sorting of the B cells bound to the CoV-S antigen and/or non-CoV-S antigen based on detection of one or more of the fluorescent labels coupled to the antigens.

62. The method of any one of claims 44 to 61, wherein the CoV-S protein is coupled to a barcode moiety.

63. An isolated antibody identified by a method according to any one of claims 1 to 62.

64. A kit for identifying and/or characterizing an antibody or antigen-binding fragment having binding affinity for an antigen, the kit comprising:

(a) a plurality of target antigens and non-target antigens, and wherein each of the target antigens and non-target antigens comprise a reporter oligonucleotide comprising (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence; and

(b) instructions for performing the method of any one of claims 1 to 62.

65. A kit for identifying an antibody or anti gen -binding fragment having binding affinity for a coronavirus spike protein (CoV-S), the kit comprising:

(a) a plurality of CoV-S antigens and non-CoV-S antigens, and wherein each of the antigens comprise a reporter oligonucleotide comprising (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence; and

(b) instructions for performing the method of any one of claims 1 to 62.