WO2023156186A1

WO2023156186A1 - Markers for high-affinity sars-cov-2 spike protein antibodies

Info

Publication number: WO2023156186A1
Application number: PCT/EP2023/052289
Authority: WO
Inventors: Hedda WARDEMANN; Rajagopal MURUGAN; Ralf Bartenschlager; Heeyoung Kim
Original assignee: Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts; Universität Heidelberg
Priority date: 2022-02-16
Filing date: 2023-01-31
Publication date: 2023-08-24
Also published as: WO2023156187A1

Abstract

The present invention relates to a method for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity, the method comprising the steps of: (a) determining the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of said antibody; and (b) identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity if IGKV1-39 is present in its light chain and/or IGHV3-9 is present in its heavy chain. The present invention also relates to a method for manufacturing an antibody, a method for assessing whether a subject produces antibodies that bind the SARS-CoV-2 spike protein with high affinity, and to uses, kits, antibodies, and polynucleotides related thereto.

Description

Markers for high-affinity SARS-CoV-2 spike protein antibodies

The present invention relates to a method for identifying an antibody that binds the SARS-CoV- 2 spike protein with high affinity, the method comprising the steps of (a) determining the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of said antibody; and (b) identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity if IGKV1-39 is present in its light chain and/or IGHV3-9 is present in its heavy chain. The present invention also relates to a method for manufacturing an antibody, a method for assessing whether a subject produces antibodies that bind the SARS-CoV-2 spike protein with high affinity, and to uses, kits, antibodies, and polynucleotides related thereto.

In 2020, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the Covid- 19 disease has become a pandemic due to its high transmissibility and deadly outcome. The infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of Spike protein to host receptor, primarily human angiotensin-converting enzyme 2 (hACE2). Upon binding, fusion of viral and host membranes occurs allowing the virus to enter the host cell and start viral replication.

There have been some reports on neutralizing antibodies. Those antibodies typically bind to the receptor binding domain of the Spike protein and thereby inhibit binding to hACE2 and, thus, finally entering of the virus into the host cell (Asamow 2021, Cell 184, 3192-3204; Sharma 2021, Proteins. 2021; 1—11; WO2021/1207433). The antibodies reported so far, however, have affinities in the nano-molar range. While antibodies that block this interaction are in emergency use as early Covid- 19 therapies, precise determinants of neutralization potency remain unknown.

There is a present need to develop further anti-SARS-CoV-2 antibodies having higher affinities and increased neutralization potential in terms of strength and range in order to efficiently and reliably treat and prevent as well as diagnose SARS-CoV-2 infections. This is even more important given the fact that the global pandemic is producing constantly novel viral variants of concern. As of today, there have been reports for multiple variants, including: SARS-CoV- 2 alpha variant (B. l.1.7), SARS-CoV-2 beta variant (B.1.1351) SARS-CoV-2 delta variant (B.1.617.2) or SARS-CoV-2 omicron variant (B.1.1.529) including the currently known omicron sub-variants BA.l, BA.2 and BA.3, in a preferred embodiment further including subvariant BA.5.

Also, high-affinity antibodies are typically identified by screening and determining affinities to the antigen of interest of a large number of antibodies. This proceeding requires massive efforts with regards to resources, in particular with regards to time and equipment and any method reducing this effort is desirable.

The technical problem underlying the present invention may be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

In accordance, the present invention relates to a method for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity, the method comprising the steps of:

(a) determining the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of said antibody; and

(b) identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity if IGKV1-39 is present in its light chain and/or IGHV3-9 is present in its heavy chain.

In general, terms used herein are to be given their ordinary and customary meaning to a person of ordinary skill in the art and, unless indicated otherwise, are not to be limited to a special or customized meaning. As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Also, as is understood by the skilled person, the expressions "comprising a" and "comprising an" preferably refer to "comprising one or more", i.e. are equivalent to "comprising at least one". In accordance, expressions relating to one item of a plurality, unless otherwise indicated, preferably relate to at least one such item, more preferably a plurality thereof; thus, e.g. identifying "a cell" relates to identifying at least one cell, preferably to identifying a multitude of cells.

Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.

The methods specified herein below, preferably, are in vitro methods. The method steps may, in principle, be performed in any arbitrary sequence deemed suitable by the skilled person, but preferably are performed in the indicated sequence; also, one or more, preferably all, of said steps may be assisted or performed by automated equipment. Moreover, the methods may comprise steps in addition to those explicitly mentioned above.

As used herein, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ± 20%, more preferably ± 10%, most preferably ± 5%. Further, the term "essentially" indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ± 20%, more preferably ± 10%, most preferably ± 5%. Thus, “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like. Preferably, a composition consisting essentially of a set of components will comprise less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of non-specified component(s).

The degree of identity (e.g. expressed as "%identity") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the sequence it is compared to for optimal alignment. The percentage is calculated by determining, preferably over the whole length of the polynucleotide or polypeptide, the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. In the context of biological sequences referred to herein, the term "essentially identical" indicates a %identity value of at least 80%, preferably at least 90%, more preferably at least 98%, most preferably at least 99%. As will be understood, the term essentially identical includes 100% identity. The aforesaid applies to the term "essentially complementary" mutatis mutandis.

The term "fragment" of a biological macromolecule, preferably of a polynucleotide or polypeptide, is used herein in a wide sense relating to any sub-part, preferably subdomain, of the respective biological macromolecule comprising the indicated sequence, structure and/or function. Thus, the term includes sub-parts generated by actual fragmentation of a biological macromolecule, but also sub-parts derived from the respective biological macromolecule in an abstract manner, e.g. in silico. Thus, as used herein, an Fc or Fab fragment, but also e.g. a singlechain antibody, a bispecific antibody, and a nanobody may be referred to as fragments of an immunoglobulin.

Unless specifically indicated otherwise herein, the compounds specified, in particular the polynucleotides and polypeptides, may be comprised in larger structures, e.g. may be covalently or non-covalently linked to further sequences, carrier molecules, retardants, and other excipients. In particular, polypeptides as specified may be comprised in fusion polypeptides comprising further peptides, which may serve e.g. as a tag for purification and/or detection, as a linker, or to extend the in vivo half-life of a compound. The term “detectable tag” refers to a stretch of amino acids which are added to or introduced into the fusion polypeptide; preferably, the tag is added C- or N- terminally to the fusion polypeptide. Said stretch of amino acids preferably allows for detection of the polypeptide by an antibody which specifically recognizes the tag; or it preferably allows for forming a functional conformation, such as a chelator; or it preferably allows for visualization, e.g. in the case of fluorescent tags. Preferred detectable tags are the Myc-tag, FLAG-tag, 6-His-tag, HA-tag, GST-tag or a fluorescent protein tag, e.g. a GFP-tag. These tags are all well known in the art. Other further peptides preferably comprised in a fusion polypeptide comprise further amino acids or other modifications which may serve as mediators of secretion, as mediators of blood-brain-barrier passage, as cell-penetrating peptides, and/or as immune stimulants. Further polypeptides or peptides to which the polypeptides may be fused are signal and/or transport sequences, e.g. an IL-2 signal sequence, and linker sequences.

The term “polypeptide”, as used herein, refers to a molecule consisting of several, typically at least 20 amino acids that are covalently linked to each other by peptide bonds. Molecules consisting of less than 20 amino acids covalently linked by peptide bonds are usually considered to be "peptides". Preferably, the polypeptide comprises of from 50 to 1000, more preferably of from 75 to 1000, still more preferably of from 100 to 500, most preferably of from 110 to 400 amino acids. Preferably, the polypeptide is comprised in a fusion polypeptide and/or a polypeptide complex. The term “antibody” as used herein refers to any polypeptide which comprises amino acid sequence stretches that are capable of forming a binding pocket that is sufficient for specific binding to the SARS-CoV-2 spike protein, more preferably the receptor binding domain (RBD) thereof, with an equilibrium dissociation constant (Kd) as referred to herein. Such an antibody may be, preferably, a monoclonal antibody, a single chain antibody, a chimeric antibody or any fragment or derivative of such antibodies being still capable of binding to the SARS-CoV-2 spike protein, preferably the receptor binding domain (RBD) thereof, specifically as referred to herein. Fragments and derivatives comprised by the term antibody as used herein encompass a bispecific antibody, a synthetic antibody, a Fab, F(ab)2 Fv or scFv fragment or a chemically modified derivative of any of these antibodies. Antibodies or fragments thereof, in general, can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by the techniques which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals and, preferably, immunized mice. Antibodies may also be produced recombinantly by techniques well known in the art. The antibody of the present invention can be, preferably, generated by using the techniques described in the accompanying Examples below. Preferably, the antibody comprises at least one light chain and at least one heavy chain, preferably as specified elsewhere herein.

The antibody of the invention shall bind to, preferably specifically bind to, the SARS-CoV-2 spike protein, more preferably the receptor binding domain (RBD) of SARS-CoV-2 spike protein. The term “receptor binding domain (RBD)” as used herein refers to a region of the SARS-CoV-2 spike protein which is involved in binding of the said spike protein of the virus to the human Angiotensin Converting Enzyme (hACE)-2 receptor on host cells. The RBD consists of amino acids 319 to 541 of the SARS-CoV-2 spike protein of WT SARS-CoV-2 (see preferably, BetaCoV/Wuhan/IVDC-HB-01/2019, accession ID: EPI ISL 402119; BetaCoV/Wuhan/IVDC-HB-04/2020, accession ID: EPI_ISL_402120;

BetaCoV/Wuhan/IVDC-HB-05/2019, accession ID: EPI ISL 402121). It will be understood that in virus variants of SARS-CoV-2, the position may differ due to the presence of one or more additional amino acids and/or deletions of one or more amino acids. Typically, such variants, however, shall also comprise a RBD which consists of amino acids corresponding to the amino acids of the RBD in WT SARS-CoV-2 at amino acid positions 319 to 541. The spike protein is a glycoprotein that forms a homotrimer at the surface of SARS-CoV-2 (Wrapp et al, Science, 367: 1260-1263). In the trimeric structure of the spike protein, the RBD may be exhibited by each monomer in either a so-called “up” or a so-called “down” configuration. The structure and amino acid composition of the SARS-CoV-2 spike protein is well known in the art, for WT SARS-CoV-2 as well as for various variants of the virus, such as SARS-CoV-2 alpha variant (B.l.1.7), SARS-CoV-2 beta variant (B.1.1351) SARS-CoV-2 delta variant (B.1.617.2) or SARS-CoV-2 omicron variant (B.1.1.529) including its sub-variants. Exemplary amino acid sequences are found in Genbank Acc Nos. [MT380725.1] (spike proteins), as well as Genbank Acc Nos. [MT380724.1] (RBDs).

The phrase “specifically binds to” as used in accordance with the present invention means that the antibody shall not cross-react significantly with components or regions other than the compounds or regions thereof indicated; thus, the antibody referred to herein preferably binds specifically to the SARS-CoV-2 spike protein, preferably the RBD of the SARS-CoV-2 spike protein. Specific binding of an antibody as referred to herein can be tested by the skilled person by various techniques including immunological technologies such as Western blotting, ELISA or RIA based assays or measuring of binding affinities using, e.g., surface plasmon resonance technology. Preferably, the affinity, e.g. measured as Kd as specified elsewhere herein, of the antibody to a non-cognate polypeptide or epitope preferably is at least a factor of 10, more preferably at least a factor of 100, even more preferably a least a factor of 10³, most preferably at least a factor of 10⁴, lower than for a spike polypeptide, preferably an RBD of a spike protein.

The term “equilibrium dissociation constant (Kd)” as used herein indicates the propensity for the antib ody/antigen (e.g. RBD) complex to dissociate into its free components, i.e. free antibody and free antigen. The equilibrium dissociation constant (Kd) for a chemical association reaction A + B <-> AB can be expressed as follows:

Kd = [A] * [B] / [AB] wherein [A], [B], and [AB] are the concentrations of A, B and AB at the equilibrium, respectively. Thus, the smaller the equilibrium dissociation constant, the more tightly bound the ligand is, or the higher the affinity between ligand and protein. For example, an antibody with a picomolar equilibrium dissociation constant (Kd in the range of 10'¹² M) binds more tightly to a particular antigen than an antibody with a nanomolar equilibrium dissociation constant (Kd in the range of 10'⁹ M). The binding of the antibody as specified herein and the RBD shall be with an equilibrium dissociation constant (Kd) of less than 5 xlO'⁹ M, preferably, between 10'⁹ and IO'¹⁰ M or, preferably, even less than IO'¹⁰ M. The equilibrium dissociation constant referred herein can be determined by techniques well known in the art, preferably, it is to be determined using surface plasmon resonance described in the accompanying Examples, below.

Antibodies exhibiting binding to the receptor binding domain (RBD) of SARS-CoV-2 spike protein with an equilibrium dissociation constant (Kd) of less than 5 xlO'⁹ M, preferably, have CDRs as listed in Tables 1 and 2, below, or CDRs having an amino acid sequence which differs by at least one amino acid exchange, deletion and/or addition from any sequence as shown in any one of the SEQ ID NOs mentioned in Table 1 and 2, while retaining binding to spike protein, preferably, RBD with Kd of less than 5 xlO'⁹ M. Preferably, such antibodies comprise at most two amino acid exchanges, deletions and/or additions compared to any of the sequences as shown in any one of the SEQ ID NOs mentioned in Table 1 or 2, while retaining binding to RBD with Kd of less than 5 xlO'⁹ M. Preferably, such antibodies are useful for the diagnostic and/or therapeutic purposes referred to herein.

Depending on the antibody type envisaged, the antibody may further comprise amino acids or amino acid sequence from the framework regions, preferably as specified elsewhere herein. The term "framework regions" (FRs), which may also be referred to as framework (FW) sequences, refers to amino acid sequences interposed between CDRs, i.e. refers to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. From N-terminal to C-terminal, light chain variable region and heavy chain variable region both typically have the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

Numbering systems have been established for assigning numbers to amino acids that occupy positions in each of above domains. Complementarity determining regions and framework regions of a given antibody can be identified using the Kabat system. However, the CDRs can also be redefined according to an alternative nomenclature scheme based on IMGT definition (Lefranc 2003). Typically, CDR and FWR sequences are given herein according to the IMGT system in the IgBlast version 1.13.0.

The antibody preferably comprises an Ig kappa variable light chain 1-39 (IGKV1-39) amino acid sequence in the light chain and/or an Ig variable heavy chain 3-9 (IGHV3-9) amino acid sequence in the heavy chain. Corresponding sequences are known in the art, e.g. from Lefranc et al. (2020) biomedicines 8:319. The antibody, preferably, further comprises six complementary determining regions. The term “complementary determining region (CDR)” as used herein refers to regions in the variable domains of the heavy and light chain of an antibody that define the binding affinity and specificity of the antibody. There are three CDRs for the heavy chain, CDR1-H, CDR2-H and CDR3-H, and three CDRs for the light chain, CDR1-L, CDR2-L, and CDR3-L.

Preferably, the antibody light chain comprises at least the 1-39 framework region sequences FR1, FR2, and FR3, preferably comprising amino acid sequences as shown in SEQ ID NOs:l, 4 or 5, and 7 or 8, respectively. Also preferably, the antibody light chain further comprises at least one, preferably both, of the CDR1-L and CDR2-L sequences as shown in SEQ ID NOs:2 or 3 and 6, respectively. More preferably, the antibody further comprises a CDR3-L amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs:9 to 13. Preferably, the antibody comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO: 15 or 16 or a sequence at least 90%, preferably at least 95%, more preferably at least 98%, most preferably at least 99% identical thereto. Even more preferably, the antibody comprises a light chain comprising an amino acid sequence selected from the amino acid sequences as shown in SEQ ID NOs: 17 to 21 or a sequence at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% identical thereto. Most preferably, the antibody comprises a light chain comprising an amino acid sequence selected from the amino acid sequences as shown in SEQ ID NOs: 17 to 21. Preferably, the aforesaid antibody light chains further comprise a 1-39 FR4 sequence, preferably comprising the sequence of SEQ ID NO: 14.

Table 1 : Preferred light chain 1-39 amino acid sequences; FR: framework region.

Preferably, the antibody heavy chain comprises at least the variable heavy chain 3-9 framework region sequences FR1, FR2, and FR3, preferably comprising amino acid sequences as shown in SEQ ID NOs:22, 24 or 25, and 27 or 28, respectively. Also preferably, the antibody heavy chain further comprises at least one, preferably both, of the CDR1-H and CDR2-H sequences as shown in SEQ ID NOs:23 and 26, respectively. More preferably, the antibody further comprises a CDR3-H amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs:29 and 30. Preferably, the antibody comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO:32 or 33 or a sequence at least 90%, preferably at least 95%, more preferably at least 98%, most preferably at least 99% identical thereto. Even more preferably, the antibody comprises a heavy chain comprising an amino acid sequence selected from the amino acid sequences as shown in SEQ ID NOs:34 and 35 or a sequence at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% identical thereto. Most preferably, the antibody comprises a light chain comprising an amino acid sequence selected from the amino acid sequences as shown in SEQ ID NOs:34 and 35. Preferably, the aforesaid antibody heavy chains further comprise a 3-9 FR4 sequence, preferably comprising the sequence of SEQ ID NO:31.

Table 2: Preferred heavy chain 3-9 amino acid sequences; FR: framework region.

More preferably, the antibody comprises a variable light chain 1-39 amino acid sequence, preferably as specified herein above; and a heavy chain 3-9 amino acid sequence, preferably also as specified herein above. Thus, preferably, the antibody comprises the amino acid sequences as shown in SEQ ID NOs: 15 and 32, 15 and 33, 16 and 32, 16 and 33, more preferably as shown in SEQ ID NOs: 17 and 34, 18 and 34, 19 and 34, 20 and 34, 21 and 34, 17 and 35, 18 and 35, 19 and 35, 20 and 35, or 21 and 35.

The method for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity comprises step (a) determining the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of said antibody. Presence of IGKV1-39 and/or IGHV3-9 amino acid sequences can be determined by the skilled person without further ado by methods known in the art, e.g. by amino acid sequencing of the antibody polypeptide chain(s) or fragments thereof, mass spectrometry of the antibody polypeptide chain(s) or fragments thereof, by immunological methods, and/or by sequencing a polynucleotide encoding a light and/or heavy chain of an antibody, e.g. from a hybridoma cell. As the skilled person will understand, for step (a) of the method it will typically not be required to sequence a whole variable chain or polynucleotide encoding the same in order to establish the determination; thus, preferably, a sequence of at least 4 amino acids, preferably at least 5 amino acids, still more preferably at least 6 amino acids, is determined, or the corresponding number of nucleotides in an encoding polynucleotide, i.e. at least 12, 15, or 18 bases of the polynucleotide. As will be understood by the skilled person, presence of IGKV1-39 and/or IGHV3-9 amino acid sequences may also be determined by other methods as deemed appropriate by the skilled person; e.g. in an experimental animal comprising IGKV1-39 and/or IGHV3-9 amino acid sequences, said sequences may be labeled by one or more tags, which may enable detection by antibody binding or which may in themselves provide detectable signals, such as fluorescence provided by one of the fluorescent group of proteins, such as GFP, YFP, and the like. Furthermore, sequencespecific antibodies may be generated, which may facilitate IGKV1-39 and/or IGHV3-9 amino acid sequence detection e.g. in immunoblots and/or on the surface of antibody producer cells. The method for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity further comprises step (b) identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity if IGKV1-39 is present in its light chain and/or IGHV3-9 is present in its heavy chain. In accordance with the above, an antibody that binds the SARS-CoV-2 spike protein with high affinity, preferably, is identified if the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain is determined in step (a). Preferably, an antibody that binds the SARS-CoV-2 spike protein with high affinity is identified if it is determined in step (a) that a sequence of at least 4 amino acids, preferably at least 5 amino acids, still more preferably at least 6 amino acids, from an amino acid sequence selected from SEQ ID NOs: l to 8, or 14, preferably from SEQ ID NO: 1, 4, 5, 7, 8, or 14, is present in the light chain, and/or from an amino acid sequence selected from SEQ ID NOs:22 to 28 or 31, preferably from SEQ ID NO:22, 24, 25, 27, 28, or 31, is present in the heavy chain of said antibody. Thus, determination of the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody is preferably based on determining at least one sequence or subsequence from a framework region.

As will be understood by the skilled person, the method may comprise further, optional steps. E.g., the method may further comprise determining binding of said antibody to the SARS-CoV- 2 spike protein by means known in the art. Also, the method may comprise verifying that a subject from which an antibody may be derived from was contacted with SARS-CoV-2 spike protein before the antibody was obtained. Also, the method may comprise generation of hybridoma cells producing candidate antibodies, e.g. from a subject immunized against SARS- CoV-2 spike protein, according to standard methods, and, optionally, culturing such hybridoma cells. However, also methods of culturing antibody producing B cells are known and may be applied. Such antibody producing may be cultured polyclonally, preferably are cultured clonally. Preferably, polynucleotides encoding the light and/or the heavy chain are prepared and at least partially sequenced from such cultured cells.

Preferably, the antibody identified and, in particular, an antibody useful for therapeutic purposes as mentioned before, neutralizes SARS-CoV-2 in vitro with an IC50 of at most 1.0 pg/ml, at most 0.1 pg/ml or at most 0.01 pg/ml. Also preferably, the antibody identified binds the SARS-CoV-2 spike protein, preferably the RBD, with a Kd of less than 5 xlO'⁹ M; said antibody does not necessarily have to be a neutralizing antibody as specified herein below. Due to its high affinity, the antibody, even if being non-neutralizing, still is may be particularly useful in detection of the SARS-CoV-2 spike protein, in particular in diagnostic applications.

Neutralization of SARS-CoV-2 in vitro as referred to herein can be tested in vitro by applying antibodies to be tested for neutralizing potential to SARS-CoV-2 virus preparations, adding this antibody- virus mixture to a culture of hACE2 expressing cells and determining infection of the hACE2 expressing cells. If said cells are infected, SARS-CoV-2 will replicate in these cells, which can be measured by quantifying the amount of viral RNA or protein produced in these cells or by determining the degree of cell damage caused by virus replication in the inoculated cell culture, which is known in the field as plaque assay. If the antibody to be tested has neutralizing potential, the hACE2 expressing cells will be protected from infection and thus, do not produce viral RNA or protein or be protected from damage and cell death. Preferably, neutralization and neutralizing potential of an antibody can be tested as described in the accompanying Examples, below. Also preferably, neutralizing potential of an antibody may also be determined by using a surrogate neutralization assay. To this end, an enzyme-conjugated or labeled SARS-CoV-2 spike protein or RBD thereof may be applied to immobilized hACE2 in the presence of antibodies to be tested. If no binding to hACE2 or significantly reduced binding - as measured by the enzymatic activity or presence of the label - occurs in the presence of the antibodies, this will be an indicator for the neutralizing potential of said antibodies. Using the aforementioned examples of in vitro tests for the neutralizing potential of an antibody by testing the antibody of the invention, IC50 values as referred to above may be determined for a given antibody.

In addition to the aforementioned strength of neutralization, the antibody of the present invention, typically, neutralizes a significant range of SARS-CoV-2 variants. More preferably, the antibody neutralizes at least two, at least three, at least four or at least five of the SARS- CoV-2 variants. Preferably, said antibody neutralizes at least two, at least three, at least four or at least five variants that are selected from the currently known SARS-CoV-2 variants, i.e. wildtype (WT) SARS-CoV-2, SARS-CoV-2 alpha variant (B.1.1.7), SARS-CoV-2 beta variant (B.1.1351), SARS-CoV-2 delta variant (B.1.617.2) or SARS-CoV-2 omicron variant (B.1.1.529) including its sub-variants. The term “SARS-CoV-2” as used herein refers except as specified otherwise to the wildtype (WT) SARS-CoV-2 as well as to all variants thereof. Variants of SARS-CoV-2 include all SARS-CoV-2 virus mutants that are derived from SARS-CoV-2 WT or any variant thereof by natural mutagenesis or which are artificially designed based on said SARS-CoV-2 WT or any mutant thereof. In particular encompassed are the variants of concern and, more preferably, SARS-CoV-2 is selected from the group consisting of wildtype (WT) SARS-CoV-2, SARS- CoV-2 alpha variant (B. l.1.7), SARA-CoV-2 beta variant (B.1.1351) SARS-CoV-2 delta variant (B.1.617.2) or SARS-CoV-2 omicron variant (B.1.1.529) including its sub-variants.

Preferably, the antibody of the invention can block at least one antibody belonging to Classi, Class2 or Class3 from binding to the RBD of WT SARS-CoV-2.

Existing antibodies that bind to RBD have been classified based on their putative epitope region on RBD into different classes (Barnes 2020, Nature 588, 682-687). The antibody of the present invention shall, preferably, block binding of at least one antibody that has been classified either as a Classi, Class2 or Class3 antibody (i.e. belonging into the group of antibodies of Classi, Class2 and Class3). It will be understood that there are antibodies that may block binding of more than one antibody of either one class or of different classes. Moreover, the antibody of the invention my also block binding of an antibody allocated to different classes. Blocking binding of an antibody belonging to Classi, Class2 and Class3 from binding to the RBD of WT SARS-CoV-2 can be determined by well-known techniques including those described in the Examples, below.

Advantageously, it has been found in accordance with the studies underlying the present invention that anti-SARS-CoV-2 antibodies can be generated which are capable of specifically binding to the RBD of the SARS-CoV-2 spike protein with high affinity. These antibodies are particularly useful for treating, preventing and/or diagnosing SARS-CoV-2 infection. Those antibodies that display a particularly high affinity and neutralizing capacity in terms of strength and range are particularly useful for the treatment and/or prevention of SARS-CoV-2 infection, and/or they may be used for diagnostic purposes. Antibodies that exhibit a particular high affinity but less neutralizing capacity are particularly useful as diagnostic antibodies, although they may be useful for therapeutic and/or prophylactic purposes as well. Moreover, it was surprisingly found that a significantly increased portion of high-affinity antibodies comprise amino acid sequences derived from the IGKV1-39 and/or IGHV3-9 genes, so pre-selecting candidate antibodies for sequences from the aforesaid gene or genes can significantly reduce the effort required for identifying high-affinity antibodies. Moreover, identifying the aforesaid gene(s) in vaccinated or post-infection subjects can be used as a surrogate marker for successful vaccination or immune clearance, respectively, and for that high-affinity antibodies were generated by the subject.

The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

The present invention also relates to a method for manufacturing an antibody comprising the steps of

(a) identifying an antibody that binds its antigen with high affinity by the method for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity as specified herein; and

(b) manufacturing the antibody identified in step (a).

The method for manufacturing an antibody, preferably, is an in vitro method. Optionally, one or more steps of the method may be performed in vivo, e.g. by culturing cells producing the antibody identified in step (a) in an experimental or farming animal in step (b); preferably, the experimental or farming animal is sacrificed after the manufacturing in such case. More preferably, however, all steps of the method are performed in vitro. Also, one or more of said steps may be assisted or performed by automated equipment. Methods for manufacturing antibodies are well known in the art and are in particular described herein above.

The present invention also relates to a method for assessing whether a subject produces antibodies that bind the SARS-CoV-2 spike protein with high affinity comprising the steps of: (a) determining the presence or absence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample of said subject; and (b) identifying a subject that produces antibodies that bind the SARS-CoV-2 spike protein with high affinity based on the presence of IGKV1-39 in the light chain and IGHV3-9 in the heavy chain of an antibody produced by said B-cell in said B-cell containing sample.

The method for assessing, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to providing a sample for step a), and/or determining binding of antibodies from a sample of the subject to the SARS-CoV-2 spike protein before, concomitant to, or after step b). Moreover, one or more of said steps may be assisted or performed by automated equipment.

As will be understood in view of the description herein above, a subject producing antibodies that bind the SARS-CoV-2 spike protein with high affinity is preferably identified in case it is identified in step (a) that IGKV1-39 is present in the light chain and/or IGHV3-9 is present in the heavy chain of antibodies of the subject. As the skilled person will understand, in case the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain is determined from a mixture of antibodies or polynucleotides encoding the same, which may be the case e.g. in case the determination is made from a mixture of antibody producing cells, e.g. in a blood sample, it shall preferably be sufficient that presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain above the detection limit is determined. In accordance, the method for assessing preferably is to be used for assessing the immune status of said subject, in particular for assessing whether a vaccination and/or SARS-CoV infection caused production of high-affinity antibodies against SARS-CoV-2 in said subject. Said vaccination preferably comprises administering a vaccine comprising the SARS-CoV-2 spike protein, preferably, the RBD thereof.

The present invention also relates to a use of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody ex vivo as a biomarker for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity.

Also, the present invention relates to IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample for use as a biomarker for whether a subject produces antibodies that bind the SARS-CoV-2 spike protein with high affinity. In such case, said biomarker is to be preferably used for assessing the immune status of said subject; and/or for determining whether a vaccination against SARS-CoV-2 in said subject was successful. Also in the aforesaid cases, the vaccination preferably comprises administering a vaccine comprising the SARS-CoV-2 spike protein, preferably, the RBD thereof.

The present invention also relates to an antibody which binds to the SARS-CoV-2 spike protein, wherein said antibody comprises an IGKV1-39 light chain comprising the amino acid sequence of SEQ ID NO: 17, 18, 19, 20, or 21; and/or comprises an IGHV3-9 heavy chain comprising the amino acid sequence of SEQ ID NO:34 or 35.

The antibody of the instant invention has been described herein above. The antibody comprises the amino acid sequence of SEQ ID NO: 17, 18, 19, 20, or 21 in the light chain, preferably in the variable domain of the light chain and/or comprises the amino acid sequence of SEQ ID NO:34 or 35 in the heavy chain, preferably in the variable domain of the heavy chain. Thus, the light chain is an IGKV1-39 light chain and/or the heavy chain is an IGHV3-9 heavy chain.

It will be understood that a variant amino acid sequence which differs by at least one amino acid exchange, deletion and/or addition from any of the aforementioned amino acid sequences shall still be capable of exhibiting essentially the same immunological properties as the concrete amino acid sequence identified by a SEQ ID NO.

More preferably, such a variant amino acid sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the concrete amino acid sequence identified by a SEQ ID NO. Sequence identity between two amino acid sequences as referred to herein, preferably is determined as specified herein above. Preferably, in the CDR amino acid sequences comprised in said SEQ ID NOs, the amino acid sequence of the CDR differs by at most three amino acid exchanges, deletion and/or addition from the specific sequence shown in any one of the CDR SEQ ID NOs in Table 1 and/or 2. Preferably at most 3, at most 2 or at most 1 amino acid(s) may be deleted, exchanged, or added.

An antibody as referred to herein may be a full-length antibody (i.e. antibodies comprising two heavy chains and two light chains). In such a case, the light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). It will be understood that there may be modifications such as mutations reducing FcR binding that may be introduced into the antibody of the invention to increase halflife and/or to reduce or improve effector functions. The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions. The light chains of human antibodies generally are classified as kappa and lambda light chains, and each of these contains one variable region and one constant domain. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon chains, and these define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Human IgG has several subtypes, including, but not limited to, IgGl, lgG2, lgG3, and lgG4. Human IgA subtypes include IgAl and lgA2. In humans, the IgA isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains ten or twelve heavy chains and ten or twelve light chains. Antibodies according to the invention may be IgG, IgE, IgD, IgA, or IgM immunoglobulins or fragments thereof.

The antibody may also be a humanized antibody, the term "humanized antibody" relating to immunoglobulins or fragments thereof (such as Fab, Fab', F(ab)2, Fv, or other antigen binding sub-sequences of antibodies), which contain minimal sequence (but typically, still at least a portion) derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (the recipient antibody) in which CDR residues of the recipient antibody are replaced by CDR residues from a non-human species immunoglobulin (the donor antibody) such as a mouse, rat or rabbit having the desired specificity, affinity and capacity. As such, at least a portion of the framework sequence of said antibody or fragment thereof may be a human consensus framework sequence. In some instances, Fv framework residues of the human immunoglobulin need to be replaced by the corresponding non-human residues to increase specificity or affinity. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically at least two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, which (e.g. human) immunoglobulin constant region may be modified (e.g. by mutations or glycol-engineering) to optimize one or more properties of such region and/or to improve the function of the (e.g. therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.

The antibody may also be a chimeric antibody, the term "chimeric antibody" as referred to herein relating to an antibody whose light and/or heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant regions which are identical to, or homologous to, corresponding sequences of different species, such as mouse and human. Alternatively, variable region genes derive from a particular antibody class or subclass while the remainder of the chain derives from another antibody class or subclass of the same or a different species. It covers also fragments of such antibodies. For example, a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species may be used.

The present invention further relates to a polynucleotide encoding the antibody of the invention.

The term “polynucleotide” as used in accordance with the present invention refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double- stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). The term as used herein includes any and all molecules which encompass or encode a sequence specified herein, optionally further comprising the complementary or reverse-complementary sequence thereof. Preferably, the polynucleotide is RNA or DNA. The term also encompasses DNAs or RNAs with backbones modified for stability or for other reasons. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are also encompassed as polynucleotides. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. Every nucleic acid sequence herein that encodes a certain polypeptide of the invention may due to the degeneracy of the genetic code have silent variations. The degeneracy of the genetic code yields a large number of functionally identical polynucleotides that encode the same polypeptide. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are silent variations.

The polynucleotide of the invention shall encode the antibody of the invention, i.e. it shall comprise a nucleic acid sequences which encodes said antibody of the invention. In addition, the polynucleotide of the present invention may comprise additional nucleic acid sequences. Preferably, the polynucleotide of the present invention may comprise in addition to an open reading frame further untranslated sequence at the 3’ and at the 5’ terminus of the coding gene region: at least 500, preferably 200, more preferably 100 nucleotides of the sequence upstream of the 5’ terminus of the coding region and at least 100, preferably 50, more preferably 20 nucleotides of the sequence downstream of the 3’ terminus of the coding gene region.

The polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. purified or at least isolated from its natural context such as its natural gene locus) or in genetically modified or exogenously (i.e. artificially) manipulated form. An isolated polynucleotide can, for example, comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. The polynucleotide, preferably, is provided in the form of double or single stranded molecule. It will be understood that the present invention by referring to any of the aforementioned polynucleotides of the invention also refers to complementary or reverse complementary strands of the specific sequences or variants there-of referred to before. The polynucleotide encompasses DNA, including cDNA and genomic DNA, or RNA polynucleotides.

Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificial modified ones such as biotinylated polynucleotides.

The present invention contemplates a vector or expression construct comprising the polynucleotide of the invention.

The term “vector”, preferably, encompasses phage, plasmid, cosmids, viral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes (YAC). The vector encompassing the polynucleotide of the present invention, preferably, further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art. If introduced into a host cell, the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences which allow for homologous recombination or heterologous insertion. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection”, conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of prior-art processes for introducing foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, f-mating, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for the transformation or transfection of host cells, including plant cells, can be found in text books such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Alternatively, a plasmid vector may be introduced by heat shock or electroporation techniques. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells. Preferably, the vector of the present invention is an expression vector. In such an expression vector, i.e. a vector which comprises the polynucleotide of the invention having the nucleic acid sequence operatively linked to an expression control sequence (also called “expression cassette”) allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof. Suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDM8, pRc/CMV, pcDNAl, pcDNA3 (Invitrogene) or pSPORTl (GIBCO BRL). Further examples of typical fusion expression vectors are pGEX, pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), where glutathione S transferase (GST), maltose E-binding protein and protein A, respectively, are fused with the recombinant target protein. Examples of suitable inducible non-fusion E. coli expression vectors are, inter alia, pTrc and pET l id. The tar-get gene expression of the pTrc vector is based on the transcription from a hybrid trp-lac fusion promoter by host RNA polymerase. The target gene expression from the pET l id vector is based on the transcription of a T7-gnl0-lac fusion promoter, which is mediated by a co-expressed viral RNA polymerase (T7 gnl). This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident lambda-prophage which harbors a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. The skilled worker is familiar with other vectors which are suitable in prokaryotic organisms; these vectors are, for example, in E. coli, pLG338, pACYC184, the pBR series such as pBR322, the pUC series such as pUC18 or pUC19, the Ml Bmp series, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-IIIl 13-B1, lambdagtl l or pBdCl, in Streptomyces plJlOl, plJ364, plJ702 or plJ361, in Bacillus pUBUO, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667. Examples of vectors for expression in the yeast S. cerevisiae comprise pYep Seel, pMFa, pJRY88 and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and processes for the construction of vectors which are suitable for use in other fungi, such as the filamentous fungi, comprise those which are described in detail in text books such as van den Hondel, C.A.M.J.J., & Punt, P.J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi (J.W. Bennett & L.L. Lasure, Ed., pp. 396-428: Academic Press: San Diego). Further suitable yeast vectors are, for example, pAG-1, YEp6, YEpl3 or pEMBLYe23. As an alternative, the polynucleotides of the present invention can be also expressed in insect cells using baculovirus expression vectors. Baculovirus vectors which are available for the expression of proteins in cultured insect cells, e.g., Sf9 cells, comprise the pAc series and the pVL series.

Yet the vector may be an integration vector. An integration vector refers to a DNA molecule, linear or circular, that can be incorporated, e.g., into a microorganism's genome, such as a bacteria’s genome, and provides for stable inheritance of a gene encoding a polypeptide of interest, such as the alcohol acyl transferase of the invention. The integration vector generally comprises one or more segments comprising a gene sequence encoding a polypeptide of interest under the control of additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and one or more segments that drive the incorporation of the gene of interest into the genome of the target cell, usually by the process of homologous recombination. Typically, the integration vector will be one which can be transferred into the target cell, but which has a replicon which is non— functional in that organism. Integration of the segment comprising the gene of interest may be selected if an appropriate marker is included within that segment. One or more nucleic acid sequences encoding appropriate signal peptides that are not naturally associated with a polypeptide to be expressed in a host cell of the invention can be incorporated into (expression) vectors. For example, a DNA sequence for a signal peptide leader can be fused in-frame to a nucleic acid of the invention so that the alcohol acyl transferase of the invention is initially translated as a fusion protein comprising the signal peptide. Depending on the nature of the signal peptide, the expressed polypeptide will be targeted differently. A secretory signal peptide that is functional in the intended host cells, for instance, enhances extracellular secretion of the expressed polypeptide. Other signal peptides direct the expressed polypeptide to certain organelles, like the chloroplasts, mitochondria and peroxisomes. The signal peptide can be cleaved from the polypeptide upon transportation to the intended organelle or from the cell. It is possible to provide a fusion of an additional peptide sequence at the amino or carboxyl terminal end of the polypeptide.

The term “gene construct” as used herein refers to polynucleotides comprising the polynucleotide of the invention and additional functional nucleic acid sequences. A gene construct according to the present invention is, preferably, a linear DNA molecule. Typically, a gene construct in accordance with the present invention may be a targeting construct which allows for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below. In both cases, the construct must be, preferably, impeccable, with structures to control gene expression, such as a promoter, a site of transcription initiation, a site of polyadenylation, and a site of transcription termination. Moreover, it will be understood that a gene construct in accordance with the present invention may also be generated by using genomic modification techniques such as genome editing using the CRISPR/Cas technology.

Yet, the present invention provides a host cell comprising the polynucleotide of the invention or the vector or expression construct of the invention.

The host cell of the invention is capable of expressing the polypeptide of the invention comprised in the vector or gene construct of the invention. The host cell is, typically transformed or transduced with said vector or gene construct such that the polypeptide of the invention can be expressed from the vector or gene construct. The transformed vector or gene construct may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome as specified elsewhere herein in more detail. A host cell according to the invention may be produced based on standard genetic and molecular biology techniques that are generally known in the art, e.g., as described in standard text books such as Sambrook, J., and Russell, D.W. "Molecular Cloning: A Laboratory Manual" 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001); and F.M. Ausubel et al, eds., "Current protocols in molecular biology", John Wiley and Sons, Inc., New York (1987), and later supplements thereto.

Preferably, said host cell is a bacterial cell, a fungal cell, an animal cell or a plant cell.

Bacterial cells may be gram-positive or gram-negative bacterial cells. Preferred bacterial cells may be selected from the genera Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Amycolatopsis, Rhodobacter, Pseudomonas, Paracoccus, Lactococcus or Pantoea. More preferably, useful gram positive bacterial host cells may be Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus Jautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptomyces spheroides, Streptomyces thermoviolaceus, Streptomyces lividans, Streptomyces murinus, Streptoverticillum verticillium ssp. verticillium. Rhodobacter sphaeroides, Rhodomonas palustri, or Streptococcus lactis. Also more preferably, useful gram negative bacterial host cells may be Escherichia coli, Pseudomonas sp., preferably, Pseudomonas purrocinia, Pseudomonas fluorescens, Rhodobacter capsulatus, Rhodobacter sphaeroides, Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens or Pantoea ananatis.

Preferred fungal host cells may be Aspergillus, Fusarium, Trichoderma, Yeast, Pichia, or Saccharomyces host cells. Yeast as used herein includes ascosporogenous yeast, basidiosporogenous yeast, and yeast belonging to the Blastomycetes.

Preferred animal host cells may comprise mammalian host cells, avian host cells, reptilian host cells or insect host cells. Preferred animal host cells are HeLa cells, HEK293T, F or E cells, U2OS cells, A549 cells, HT1080 cells, CAD cells, P19 cells, NIH3T3 cells, L929 cells, N2a cells, CHO cells, MCF-7 cells, Y79 cells, SO-Rb50 cells, HepG2 cells, DUKX-X11 cells, J558L cells or BHK cells.

Preferred plant host cells comprise tobacco, rice, wheat, pea or tomato cells.

The present invention relates to a non-human transgenic organism comprising the polynucleotide of the invention or the vector or expression construct of the invention.

The term “non-human transgenic organism” as used herein refers to an organism which has been genetically modified in order to comprise the polynucleotide, vector or gene construct of the present invention. Said genetic modification may be the result of any kind of homologous or heterologous recombination event, mutagenesis or gene editing process. Accordingly, the transgenic non-human organism shall differ from its non-transgenic counterpart in that it comprises the non-naturally occurring (i.e. heterologous) polynucleotide, vector or gene construct in its genome. Non-human organisms envisaged as transgenic non-human organisms in accordance with the present invention are, preferably, multi-cellular organisms, such as an animal, plant, multi-cellular fungi or algae. Preferably, said non-human organism is an animal or a plant. Preferred animals are mammals, in particular, laboratory animals such as rodents, e.g., mice, rats, rabbits or the like, or farming animals such as sheep, goat, cows, horses or the like. Preferred plants are crop plants or vegetables, in particular, selected from the group consisting of tobacco, rice, wheat, pea and tomato. Methods for the production of transgenic non-human organisms are well known in the art; see, standard text books, e.g. Lee-Yoon Low et al., Transgenic Plants: Gene constructs, vector and transformation method. 2018. DOI.10.5772/intechopen.79369; Pinkert, C. A. (ed.) 1994. Transgenic animal technology: A laboratory handbook. Academic Press, Inc., San Diedo, Calif.; Monastersky G. M. and Robl, J. M. (ed.) (1995) Strategies in Transgenic Animal Science. ASM Press. Washington D.C); Sambrook, loc.cit, Ausubel, loc.cit).

The present invention also provides a method for producing the antibody of the invention comprising (i) expressing the polynucleotide or the vector of the invention in a host cell and (ii) obtaining the said antibody from said host cell.

The term “producing” as used herein refers to the process of recombinant production of the antibody in a host cell. The manufacture may also comprise further steps such as purifying the produced antibody or formulating the antibody or purified antibody as a pharmaceutical composition. Accordingly, the aforementioned method of the present invention may consist of the aforementioned steps or may comprise further additional steps.

Expressing the polynucleotide or the vector of the invention in a host cell may, for example, also include the step of generating the polynucleotide or vector of the invention as well as the step of introducing said polynucleotide or vector into the host cell.

Generating the polynucleotide of the invention or the vector comprising it may, e.g., also comprise the step of generating a polynucleotide sequence encoding the antibody of the invention on the basis of sequences for antibodies obtained from B-cells. Preferably, said B- cells are obtained from patients which have successfully survived an infection by an infectious agent sharing at least one B-cell epitope with SARS-CoV-2 or subjects that have been successfully vaccinated against said infectious agent. Infectious agents sharing at least one B- cell epitope with SARS-CoV-2 can be identifed by the skilled person without further ado, in particular infectious agents for which partial or full genome sequences are avaiable in databases. Tools for predicting and identifying B-cell epitopes are available as well. Preferably, the epitope shared between the infectious agent and SARS-CoV-2 is an epitope of an SARS-CoV-2 spike polypeptide, more preferably of the the RBD domain thereof. Preferably said infectious agent is a virus, more preferably a member of the family coronaviridae, more preferably a SARS- CoV or a MERS-CoV. Thus, preferably, the B-cells are obtained from patients which have successfully survived an infection with SARS-CoV-2, or subjects that have been successfully vaccinated against SARS-CoV-2. More specifically, generating the polynucleotide sequence may comprise the following steps: (a) Single cell sorting, e.g. performed on bait+ memory B cells; (b) Ig gene amplification and sequencing (Murugan et al., 2015 Eur J Immunol. 45(9):2698-700); (c) Obtaining Ig gene features of the antibody sequences (Imkeller et al., 2016 BMC Bioinformatics 17, 67); (d) Analyzing Ig gene features and cloning antibodies exhibiting the following features: all isotypes with preference for IgG, germline and somatic hypermutations, diverse Ig segments in heavy and light chain, from all time points of B-cell sample collection; (e) Cloning and expression of the antibodies (Tiller et al., 2008, J Immunol Methods. 329(1-2): 112-124). Functional assessment of antibodies may be carried out as described in the accompanying Examples, below. It will be understood that the polynucleotide of the invention may also be obtained by using other techniques known in the art.

Introducing the polynucleotoide or vector generated as described before into a host cell for expression can be done by all techniques available in the art, including salt-based transfection, lipofection, electroporation, injection, viral transfection techniques and the like. The polynucleotide or vector may be stably integrated into the genome of the host cell or may be transiently present.

Obtaining the antibody from the host cell can be achieved by purifying or partially purifying the antibody from the host cells or host cell culture. For protein purification, various techniques may be used including precipitation, filtration, ultra-filtration, extraction, chromatography techniques such as ion-exchange-, affinity- and/or size exclusion chromatography, HPLC or electrophoresis. The skilled person is well aware of how an antibody may be purified in order to provide it in isolated form. Preferred techniques are those described in the accompanying Examples below.

Yet, the invention relates to the use of the host cell of the invention for producing the antibody of the invention. The host cell of the present invention may, typically, be cultured under suitable conditions and for time sufficient for expression of the polynucleotide or vector of the invention such that the antibody will be produced. The antibody may be obtained from a host cell culture as described elsewhere herein.

The present invention relates to an antibody, a polynucleotide or a vector as defined herein above in accordance with the invention for use in diagnosing, treating and/or preventing a disease or condition. Preferably, the disease or condition referred to herein is associated with SARS-CoV-2 infection or infection with an infectious agent sharing at least one B-cell epitope with SARS-CoV-2 in a subject.

The antibody, polynucleotide or vector according to the present invention may be formulated as a medicament for use in in treating and/or preventing a disease or condition. Such a medicament is, preferably, for topical or systemic administration. Conventionally a medicament will be administered intra-muscularly or subcutaneously. However, depending on the nature and the desired therapeutic effect and the mode of action, the medicament may be administered by other routes as well. In particular, in accordance with the present invention, aerosol formulations or sprays applying medicament in the respiratory systems such as the nasal tract or the lung are also conceivable. The medicament is, preferably, administered in conventional dosage forms prepared by combining the ingredients with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing or dissolving the ingredients as appropriate to the desired preparation. Preferably, a solution is envisaged for the medicament. It will be appreciated that the form and character of the pharmaceutical acceptable carrier is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. A carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may include a solid, a gel, or a liquid. Examples for solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil, water, emulsions, various types of wetting agents, are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution, and the like. Similarly, the carrier may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. For polynucleotides or vectors, liposomal carriers or genetically engineered viruses may be considered as well. In particular, if a long-term application of the antibody is envisaged, a genetically engineered virus may be administered that produces the antibody of the invention over a long period within an organism to be treated. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. In addition, the medicament may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. It is to be understood that the formulation of a medicament takes place under GMP standardized conditions or the like in order to ensure quality, pharmaceutical security, and effectiveness of the medicament.

A therapeutically effective dosage of the antibody or polynucleotide of the invention refers to an amount to be used in medicament. A therapeutically effective dosage is an amount of the antibody or polynucleotide that prevents, ameliorates or treats the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity of the compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. The medicament referred to herein is administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said medicament may be administered more than one time.

The term "treating" as used herein refers to any improvement, cure or amelioration of the disease or condition as referred to herein. It will be understood that treatment may not occur in 100% of the subjects to which the antibody has been administered. The term, however, requires that the treatment occurs in a statistically significant portion of subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well-known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney-U test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.05, 0.01, 0.005, 0.001, or 0.0001.

The term “preventing” as used herein refers to significantly reducing the likelihood with which the disease or condition develops in a subject within a defined window (prevention window) starting from the administration of the antibody onwards. Typically, the prevention window is within 1 to 5 days, within 1 to 3 weeks, within 1 to 3 months or within 3 to 6 months or 3 to 12 months. However, it will be understood that the preventive window may, dependent on the kind of medicament, also be several years up to the entire life time. The prevention window depends on the amount of antibody, polynucleotide or vector which is administered and the applied dosage regimen. Typically, suitable prevention windows can be determined by the clinician based on the amount of antibody or polynucleotide to be administered and the dosage regimen to be applied without further ado. It will be understood that prevention may not occur in 100% of the subjects to which the antibody has been administered. The term, however, requires that the prevention occurs in a statistically significant portion of subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well-known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney-U test etc. Details are described elsewhere herein.

The term “disease or condition associated with SARS-CoV-2 infection or infection with an an infectious agent sharing at least one B-cell epitope with SARS-CoV-2 in a subject” refers to any disease or condition resulting directly or indirectly from an infection by SARS-CoV-2 or with an infectious agent sharing at least one B-cell epitope with SARS-CoV-2. Preferred infectious agents sharing at least one B-cell epitope with SARS-CoV-2 are known in the art and are described herein above, as are disease and conditions associated with or caused by them. E.g., in case the infectious agent is MERS-CoV, the disease is Middle East respiratory syndrome (MERS), with typical symptoms including fever, cough, diarrhea, and shortness of breath; in case the infectious agent is SARS-CoV-1 or -2, the disease is severe acute respiratory syndrome (SARS). Preferably, the disease referred to herein is Covid 19, caused by SARS- CoV-2. Moreover, the term also encompasses any symptom associated with SARS-CoV-2 infection. Typically, such symptoms may be fever, cough, headache, fatigue, breathing difficulties, loss of smell, and loss of taste, respiratory failure, or multi-organ dysfunction.

The present invention also relates to a composition comprising (i) an antibody as defined herein above and can block at least one of the antibodies belonging to Classi, Class2 and Class3 and hACE2 from binding to WT SARS-CoV-2 RBD and (ii) an antibody as defined herein above and can block at least one of the antibodies belonging to Classi, Class2 and Class3 but not hACE2 from binding to the RBD of WT SARS-CoV-2 RBD for use in treating and/or preventing a disease or condition.

The present invention also relates to an antibody as defined herein above in accordance with the present invention for use in diagnosing SARS-CoV-2 infection.

The term “diagnosing” as used herein means assessing whether a subject has been infected by SARS-CoV-2, or not. Alternatively, the term also encompasses determining the virus load of an infected subject, i.e. the amount of virus present in said subject or a sample thereof. As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be diagnosed. The term, however, requires that the assessment is correct for a statistically significant portion of the subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc.

The present invention also relates to the use of the antibody of the invention for determining the presence of SARS-CoV-2 and/or an infectious agent sharing at least one B-cell epitope with a SARS-CoV-2, in a non-diagnostic sample.

The term “non-diagnostic sample” as used herein refers to any sample from the environment that is not used for the diagnosis of an infection, e.g. SARS-CoV-2 infection. Such samples may be samples of gaseous samples such as air or liquid samples. For example, gaseous samples may be investigated for the presence or absence of SARS-CoV-2 when monitoring and evaluating the indoor quality of air, the efficacy of air filtration systems, the quality of gaseous products and the like. Liquid samples may be investigated for the presence or absence of SARS- CoV-2 in order to monitor the distribution of the virus, e.g., by assessing its presence or absence in waste waters, the quality of liquids, the quality of water in the environment or drinking water and the like.

The present invention also relates to a method for diagnosing SARS-CoV-2 infection in a subject suspected to be infected by said SARS-CoV-2 comprising the steps of: a) contacting the antibody of the invention with a sample of said subject; and b) determining binding of SARS-CoV-2 to said antibody, whereby the SARS-CoV-2 infection is to be diagnosed.

The term “subject” as used herein relates to animals, preferably mammals, and, more preferably, humans. The subject according to the present invention shall be a subject suffering from or suspected to suffer from SARS-CoV-2 infection. Typically, such a subject shows already symptoms associated with SARS-CoV-2 infection or has been in contact with one or more other subjects known to suffer from SARS-CoV-2 infection and, thus, is at risk of being infected as well.

The term “sample” refers to a sample of a body fluid, to a sample of separated cells or to a sample from a tissue or an organ. Samples of body fluids can be obtained by well-known techniques and include, preferably, samples of blood, plasma, serum, urine, saliva or exhausted air more preferably, samples of blood, plasma or serum. Tissue or organ samples may be obtained from any potentially infected tissue or organ by, e.g., biopsy or surface scratching.

The term “contacting” as referred to herein means that the sample of the subject is brought into physical proximity to the antibody of the invention such that SARS-CoV-2 viruses comprised in the sample may be bound by said antibody. Contacting is typically carried out under conditions and for a time sufficient to allow for specific binding of the antibody to a SARS- CoV-2 virus. The skilled person is well aware of how to choose suitable conditions and how long those conditions shall be applied. Preferred conditions are also described in the accompanying Examples, below.

Upon binding of the antibody to the virus, said binding shall be determined. For determining binding of SARS-CoV-2 to said antibody, various well-known techniques may be used. For example, the formation of a complex between the antibody of the invention and the virus may be determined by a secondary antibody capable of generating a detectable signal upon binding. Alternatively, the virus upon binding to the antibody may release a component from said antibody which upon release generated a detectable signal. The aforementioned principles may be realized by assays that are carried out in solution using beads comprising the antibody or the invention or can be carried out on test stripes that comprise the antibody of the invention in immobilized form. Moreover, the formation of the complex between the antibody and the virus may be determined by measuring differences in physical or chemical properties of the antibody with and without virus bound thereto. Various formats for electrochemical assays are known to the person skilled in the art.

The aforementioned method may be used qualitatively, i.e. an infection will be diagnosed, or quantitatively, i.e. the virus load during an infection will be diagnosed. For the latter case, it will be understood that the determined amount of viruses may be compared to a reference in order to determine the virus load.

The present invention also relates to a method for determining SARS-CoV-2 and/or an infectious agent sharing at least one B-cell epitope with SARS-CoV-2, in a non-diagnostic sample comprising the steps of: a) contacting the antibody of the invention with said sample; and b) determining binding of SARS-CoV-2 and/or said infectious agent to said antibody, whereby the presence of SARS-CoV-2 and/or said infectious agent is to be determined.

The term “determining” as used herein refers to determining the presence, absence or amount of SARS-CoV-2 and/or an infectious agent sharing at least one B-cell epitope with SARS-CoV- 2 in the non-diagnostic sample. Thus, the term typically encompasses qualitative determination as well as quantitative determination. The skilled artisan is well aware of how quantification of SARS-CoV-2 and/or said infectious agent can be made in the context of a quantitative determination and how suitable calibration measures can be provided.

Yet, the invention relates to a kit for diagnosing SARS-CoV-2 infection in a subject comprising the antibody of the invention and detection reagents for determining binding of SARS-CoV-2 to said antibody. The present invention also relates to a kit for identifying an antibody that binds the SARS-CoV- 2 spike protein with high affinity, said kit comprising detection agents for detecting the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody.

The term “kit” as used herein refers to a collection of components as referred to before required for diagnosing SARS-CoV-2 infection in a subject. Typically, the components of the kit are provided in separate containers or within a single container. The kit shall preferably in addition to the antibody of the invention also comprise detection reagents for binding of SARS-CoV-2 to said antibody. These reagents may include components that may generate a detectable signal upon binding of the antibody of the invention to the SARS-CoV-2. Moreover, such reagents may encompass any washing or buffer solutions required for determining binding. The container also typically comprises instructions for diagnosing SARS-CoV-2 infection in a subject. These instructions may be in the form of a manual or may be provided by a computer program code.

The kit for identifying an antibody may in particular be used for assessing the immune status of a subject or assessing whether a vaccination against SARS-CoV-2 in a subject was successful or whether a SARS-CoV-2 infection caused production of high-affinity antibodies.

In view of the above, the following embodiments are particularly envisaged:

Embodiment 1 : A method for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity, the method comprising the steps of:

Embodiment 2: The method of embodiment 1, wherein said antibody binds to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.

Embodiment 3: The method of embodiment 1 or 2, wherein said antibody that binds the

SARS-CoV-2 spike protein, preferably the RBD, with high affinity characterized by an equilibrium dissociation constant (Kd) of less than 5 x 10'⁹ M . Embodiment 4: The method of any one of embodiments 1 to 3, wherein said method further comprises culturing producer cells producing at least one candidate antibody.

Embodiment 5: The method of any one of embodiments 1 to 4, wherein in step (a) the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of said antibody is identified by sequencing a polynucleotide encoding said light chain and/or sequencing a polynucleotide encoding said heavy chain.

Embodiment 6: A method for manufacturing an antibody comprising the steps of

(a) identifying an antibody that binds its antigen with high affinity by the method of any one of embodiments 1 to 3; and

(b) manufacturing the antibody identified in step (a).

Embodiment 7: The method of embodiment 6, wherein said antibody referred to in step

(a) is obtained from a B-cell or a hybridoma cell.

Embodiment 8: A method for assessing whether a subject produces antibodies that bind the SARS-CoV-2 spike protein with high affinity comprising the steps of

(a) determining the presence or absence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample of said subject; and

(b) identifying a subject that produces antibodies that bind the SARS-CoV-2 spike protein with high affinity based on the presence of IGKV1-39 in the light chain and IGHV3-9 in the heavy chain of an antibody produced by said B-cell in said B-cell containing sample.

Embodiment 9: The method of embodiment 8, wherein said antibodies bind to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.

Embodiment 10: The method of embodiment 8 or 9, wherein said method is to be used for assessing the immune status of said subject.

Embodiment 11 : The method of any one of embodiments 8 to 10, wherein said method is to be used for assessing whether a vaccination and/or SARS-CoV infection caused production of high-affinity antibodies against SARS-CoV-2 in said subject.

Embodiment 12: The method of embodiment 1, wherein said vaccination comprises administering a vaccine comprising the SARS-CoV-2 spike protein, preferably, the RBD thereof.

Embodiment 13: The method of any one of embodiments 8 to 12, wherein said antibody binds the SARS-CoV-2 spike protein with high affinity characterized by an equilibrium dissociation constant (Kd) of less than 5 xlO'⁹ M. Embodiment 14: Use of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody ex vivo as a biomarker for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity.

Embodiment 15: The use of embodiment 14, wherein said antibody that binds its antigen with high affinity is binding to the antigen with an equilibrium dissociation constant (Kd) of less than 5 xlO'⁹ M.

Embodiment 16: IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample for use as a biomarker for determining whether a subject produces antibodies that bind the SARS-CoV-2 spike protein with high affinity.

Embodiment 17: The IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample for use of embodiment 16, wherein said biomarker is to be used for assessing the immune status of said subject.

Embodiment 18: The IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample for use of embodiment 16 or 17, wherein said biomarker is to be used for whether a vaccination against SARS-CoV-2 in said subject was successful.

Embodiment 19: The IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample for use of embodiment 18, wherein said vaccination comprises administering a vaccine comprising the SARS-CoV-2 spike protein, preferably, the RBD thereof.

Embodiment 20: The IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample for use of any one of embodiments 14 to 17, wherein said antibody that binds its antigen with high affinity is binding to the antigen with an equilibrium dissociation constant (Kd) of less than 5 x 10'⁹ M.

Embodiment 21 : A kit for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity comprising detection agents for detecting the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody.

Embodiment 22: The kit of embodiment 21, wherein said antibody that binds the SARS-

CoV-2 spike protein with high affinity characterized by an equilibrium dissociation constant (Kd) of less than 5 xlO'⁹ M.

Embodiment 23 : The kit of embodiment 21 or 22, wherein said kit is to be used for assessing the immune status of a subject or assessing whether a vaccination against SARS- CoV-2 in a subject was successful or whether a SARS-CoV-2 infection caused production of high-affinity antibodies.

Embodiment 24: An antibody which binds to the SARS-CoV-2 spike protein, wherein said antibody comprises an IGKV1-39 light chain comprising the amino acid sequence of SEQ ID NO: 17, 18, 19, 20, or 21; and/or comprises an IGHV3-9 heavy chain comprising the amino acid sequence of SEQ ID NO:34 or 35.

Embodiment 25: The antibody of embodiment 25, wherein said antibody binds to the

SARS-CoV-2 spike protein with an equilibrium dissociation constant (Kd) of less than 5 xlO'⁹ M.

Embodiment 26: The antibody of embodiment 24 or 25, wherein said antibody binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein.

Embodiment 27 : The antibody of any one of embodiments 24 to 26, wherein said antibody specifically binds to the SARS-CoV-2 spike protein, preferably to the RBD of SARS-CoV-2 spike protein.

Embodiment 28: The antibody of any one of embodiments 24 to 27, wherein said antibody neutralizes SARS-CoV-2 in vitro with IC50 of at most 1.0 pg/ml, at most 0.1 pg/ml or at most 0.01 pg/ml.

Embodiment 29: The antibody of any one of embodiments 24 to 28, wherein the said antibody can block at least one antibody belonging to Classi, Class2 or Class3 from binding to the RBD of WT SARS-CoV-2.

Embodiment 30: A polynucleotide encoding the antibody of any one of embodiments 24 to 29.

Embodiment 31 : The polynucleotide of embodiment 30, wherein said polynucleotide is

RNA or DNA.

Embodiment 32: A vector or expression construct comprising the polynucleotide of embodiment 30 or 31.

Embodiment 33 : A host cell comprising the polynucleotide of embodiment 30 or 31 or the vector or expression construct of embodiment 32.

Embodiment 34: The host cell of embodiment 33, wherein said host cell is a bacterial cell, a fungal cell, an animal cell or a plant cell.

Embodiment 35: A non-human transgenic organism comprising the polynucleotide of embodiment 30 or 31 or the vector or expression construct of embodiment 32. Embodiment 36: The non-human transgenic organism of embodiment 35, wherein said organism is an animal or a plant.

Embodiment 37: A method for producing the antibody of any one of embodiments 24 to

29 comprising (i) expressing the polynucleotide of embodiment 30 or 31 or the vector or expression construct of embodiment 32 in a host cell and (ii) obtaining the said antibody from said host cell.

Embodiment 38: Use of the host cell of embodiment 33 or 34 for producing the antibody of any one of embodiments 24 to 29.

Embodiment 39: An antibody as defined in any one of embodiments 24 to 29, a polynucleotide as defined in embodiment 30 or 31 or a vector or expression construct as defined in embodiment 32 for use in diagnosing, treating and/or preventing a disease or condition.

Embodiment 40: The antibody, polynucleotide or vector of embodiment 39, wherein said disease or condition is associated with SARS-CoV-2 infection and/or infection with an infectious agent sharing at least one B-cell epitope with SARS-CoV-2 in a subject.

Embodiment 41 : A composition comprising (i) an antibody as defined in any one of embodiments 1 to 8 and can block at least one antibody belonging to Classi, Class2 or Class3 and hACE2 from binding to WT SARS-CoV-2 RBD and (ii) an antibody as defined in any one of embodiments 1 to 8 and can block at least one antibody belonging to Classi, Class2 or Class3 but not hACE2 from binding to the RBD of WT SARS-CoV-2 RBD for use in diagnosing, treating and/or preventing a disease or condition.

Embodiment 42: The antibody, polynucleotide or vector of embodiment 20, wherein said disease or condition is associated with SARS-CoV-2 infection and/or an infection with an infectious agent sharing at least one B-cell epitope with SARS-CoV-2 in a subject.

Embodiment 43 : An antibody as defined in any one of embodiments 24 to 29 for use in diagnosing SARS-CoV-2 infection.

Embodiment 44: Use of an antibody as defined in any one of embodiments 24 to 29 for determining SARS-CoV-2 and/or an infectious agent sharing at least one B-cell epitope with SARS-CoV-2 in a non-diagnostic sample.

Embodiment 45: A method for diagnosing SARS-CoV-2 infection in a subject suspected to be infected by said SARS-CoV-2 comprising the steps of:

(i) contacting the antibody of any one of embodiments 24 to 29 with a sample of said subject; and (ii) determining binding of SARS-CoV-2 to said antibody, whereby the SARS-CoV-2 infection is to be diagnosed.

Embodiment 46: A method for determining SARS-CoV-2 and/or an infectious agent sharing at least one B-cell epitope with SARS-CoV-2 in a non-diagnostic sample comprising the steps of:

(i) contacting the antibody of any one of embodiments 24 to 29 with said sample; and

(ii) determining binding of SARS-CoV-2 and/or an infectious agent sharing at least one B-cell epitope with SARS-CoV-2 to said antibody, whereby the SARS-CoV-2 is to be determined.

Embodiment 47: A kit for diagnosing SARS-CoV-2 infection in a subject comprising the antibody of any one of embodiments 24 to 29 and detection reagents for determining binding of SARS-CoV-2 to said antibody.

Embodiment 48: A kit for determining SARS-CoV-2 in a non-diagnostic sample comprising the antibody of any one of embodiments 24 to 29 and detection reagents for determining binding of SARS-CoV-2 to said antibody.

Embodiment 49: The subject matter of any of the preceding embodiments, wherein said

SARS-CoV-2 is selected from the group consisting of: wildtype (WT) SARS-CoV-2, SARS- CoV-2 alpha variant (B.1.1.7), SARA-CoV-2 beta variant (B.1.1351) SARS-CoV-2 delta variant (B.1.617.2), and SARS-CoV-2 omicron variant (B.1.1.529) including its sub-variants.

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

Figure Legends

Figure 1: Antibody binding to spike protein and RBD detected in ELISA. Binding of reactive antibodies to spike and RBD is comparable to positive controls. Area under the binding curve (AUC) was calculated for each antibody. Out of 263 mAbs, 113 showed binding to spike protein and/or RBD based on our cut-off AUC. A) Spike, B) RBD, C) AUC Spike, D) AUC RBD.

Figure 2: Majority of spike binding antibodies (58%) target RBD while a considerable proportion (29%) of antibodies target epitopes in Spike other than RBD.13% of antibodies with weak binding to RBD may recognize hidden (inaccessible) epitopes of the trimeric spike protein.

Figure 3: Antibodies with high affinity to RBD can be identified. RBD-reactive antibodies show a range of affinities (10‘⁵ M to IO'¹⁰ M). Affinity measurement using the same method allowed for a direct comparison to the published antibodies including antibodies in clinical use. Antibodies with comparable affinity (10‘⁷ - 10'⁹ M) to or even higher affinity (< 10'⁹ M) than the published antibodies were identified.

Figure 4: RBD-binding antibodies show efficient in vitro SARS-CoV-2 neutralization. mAbs that bind to the epitopes in spike protein other than RBD do not show virus neutralization. The majority of RBD-binding antibodies show, however, no neutralizing capacity. 19% of RBD- binding antibodies show various degree of virus neutralization (IC50 < 1.0 pg/ml).

Figure 5: High antibody affinity critical but not sufficient for virus neutralization. Virusneutralizing antibodies show affinities of 10'⁷ M or lower (IC50 < 1.0 ug/ml). 27% and 23% of antibodies with affinity less 10'⁷ M and 10'⁹ M, respectively, show virus neutralization. The majority of high affinity anti-RBD antibodies do not neutralize the virus and therefore target non-neutralizing epitopes. The potency of the antibodies depends on their affinity (the higher the better) and epitope specificity. The data demonstrate that high affinity alone is not enough for neutralization.

Figure 6: Distribution of the antibodies found in the present studies to different classes. High affine antibodies are often enriched in Classl/2, S309 or none (not yet defined). High affinity is often associated with low kinetic off rate - sign of efficient selection in the germinal center driven B cell response. Rare class antibody with high affinity such as Class2/3 has high kinetic on rate and good off rate.

Figure 7: Some of Classl/2 and Class2/3 antibodies have non-overlapping epitopes in the RBD. Three Classl/2 antibodies and one Class2/3 antibody were tested against each other to block binding to the RBD. Two of three Classl/2 antibodies did not block Class2/3 antibody from binding to the RBD. Classl/2/3 mAb (2939) blocks Class2/3 (3279) but not Classl/2 (1255) mAbs from binding to the RBD. Figure 8: High affine antibodies show broad VoC virus neutralization. High affine antibodies belonging to Classl/2 and Class2/3 show high neutralization capacity against three Variants of Concern (VoCs) tested. High affine antibodies with S309 profile show specific neutralization against alpha variant but not against WT or other variants.

Figure 9: General trend in loss of antibody affinity to Omicron variant RBD. Only three control mAbs (S309, REGN10933 and CR3022) have measurable binding to Omicron RBD, while the rest lost binding completely. mAbs from this study show increased, similar, decreased or complete loss in affinity to Omicron RBD compared to WT RBD.

Figure 10: Two high affine mAbs bind to Omicron RBD without blocking each other. High affine antibodies (1255 and 2939) can both bind to WT and Omicron RBD without blocking each other - suggesting they bind non-overlapping epitopes.

Figure 11: Two high affine mAbs differ in blocking hACE2 binding to Omicron RBD. 1255 blocks whereas 2939 does not block hACE2 interaction with both WT and Omicron RBD, indicating the difference in their binding site overlap with hACE2 interaction site.

Figure 12: 2939 and 1255 show high Omicron variant neutralization capacity. mAb 2939 shows better Omicron neutralization than therapeutically used mAb S309 - VIR Biotechnology (Sotrovimab). Given antibodies were mixed with defined amounts of SARS-CoV-2 virus particles and after 30 minutes, the mixture was added to VeroE6 cells. After 24 hours, SARS- CoV-2 replication in the inoculated cells was measured by fixing the cells and immunostaining of the viral nucleocapsid protein. By testing 10 serial dilutions of the antibody solution, dosedependent neutralization capacity, expressed as IC50 value, was calculated by non-linear regression sigmoidal dose response analysis using the GraphPad Prism 7 software package.

Figure 13: Virus neutralizing capacity may differ between variants. mAbs that do not neutralize WT virus show weak neutralizing capacity of the Omicron variant. Half maximal inhibition concentration values of these mAbs (IC50 values) are still lower than those of antibodies 1255 and 2939. Figure 14: Alignments of antibody variable sequences comprising IGKV1-39 (A) or IGHV3- 9 (B); FR: framework region; CDR: complementarity determining region; *: identical amino acid.

The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLES

The invention will be illustrated by the following Examples. The Examples shall not be construed as limiting the scope of the invention.

Example 1: Isolation of anti-RBD antibodies

Plasmids encoding His-tagged versions of full-length spike protein and RBD were kindly provided by Florian Krammer (Amanat et al., 2020 Nat Med 26, 1033-1036). Spike and RBD proteins were recombinantly expressed in HEK293F cells (Thermo Fischer Scientific) and purified using affinity column chromatography. Using commercially available kits, the RBD domain was biotinylated and the spike protein was labelled with A647. Both were used as baits to detect antigen-reactive memory B cells by FACS. PBMCs from hospitalized patients and convalescent donors were incubated with 0.125 pg/ml of biotinylated RBD and 0.5 pg/ml of spike_A647 along with the following mouse anti-human antibodies at the noted dilutions: CD19-Brilliant Violet 786 (BV786) (SJ25C1) at 1: 10, CD27-phycoerythrin (M-T271) at 1 :5, IgG-BV510(G18-145) at 1 :20, CD138-BV421 (MI15) at 1 :20, IgD-allophycocyanin (APC)-H7 (IA6-2) at 1 :20 and CD38-BV605 (HB7) at 1 :20. Upon 30 min incubation on ice, the cells were washed and stained with streptavidin- fluorescein isothiocyanate (FITC) at 1: 1000 and 7- Aminoactinomycin D (7AAD) (Invitrogen) at 1 :400. Memory B cells (7AAD-CD19+CD27+ B cells) positive for binding to at least one of the baits were single-cell index sorted using a FACSAria III (BD Biosciences) with FACSDiva software.

Ig genes from single B cells were amplified by RT-PCR as previously described (Murugan et al., 2015 Eur J Immunol. 45(9):2698-700). In brief, reverse transcription was performed using random hexamers and the resulting complementary DNA was used as template to amplify IGH, IGK and IGL genes with barcoded primers in the second PCR. Pooled and purified amplicons were ligated with adaptors and sequenced using Illumina MiSeq 2x300 bp paired end sequencing. The sequence reads were analyzed using sciReptor to obtain and integrate Ig gene information of heavy and light genes with flow cytometry index data (Imkeller et al., 2016 BMC Bioinformatics 17:67). Data from single cells with paired and functional Ig genes on both loci were used for analysis.

Antibodies of different isotypes with preference for IgG, encoded by germline and somatically mutated Ig genes were selected for cloning and recombinant mAb expression as described previously (Tiller et al., 2008, J Immunol Methods. 329(1-2): 112-124). In brief, Ig genespecific primers tagged with restriction enzyme binding sites were used to amplify heavy and light chain genes and cloned into corresponding expression vectors (IgG, Igk and IgA). Vectors containing successfully cloned heavy and light genes were co-transfected into HEK293F cells (Thermo Fischer Scientific) and recombinant monoclonal antibodies were expressed and purified.

Example 2: Characterization of anti-RBD antibodies

Antibody binding to spike protein and recombinant RBD was investigated by ELISA. High binding 384-well plates were coated with 2 ng/ pl spike protein or 4 ng/ pl RBD in PBS. 25 pl/ well of the respective solution was added and incubated over night at 4 °C. The next day, ELISA plates were washed thrice with 0.05 % Tween in PBS (PBS-T) using a Tecan plate washer and blocked with 1 % BSA in PBS for 1 h at RT. Four 1 :4 serial dilutions of each antibody supernatant in PBS were prepared with an initial concentration of 4 pg/ ml. PBS and the monoclonal antibody mGO53 were used as negative controls while the monoclonal antibodies CR3022 and S309 were used as positive controls. After blocking, ELISA plates were washed three times with PBS-T and samples were loaded onto the plates. 15 pl/well of diluted antibodies was added to the plates and incubated for 2 h at RT. ELISA plates were washed thrice with PBS-T. HRP-conjugated anti-human IgG-Fcy (Jackson ImmunoResearch) was diluted 1 : 1000 in blocking buffer, loaded onto the ELISA plate (15 pl/ well) and incubated for

1 h at RT. After washing the plate three times with PBS-T, ELISAs were developed using 20 pl/well of 1 :1000 dilution H2O2 in ABTS solution. Absorbance was measured at 405 nm every

2 min for a total of 20 min using a Tecan MIOOOPro plate reader. Reactivity of the antibodies was assessed by calculating the area under the curve (AUC) of the absorbance measured for the four dilutions using GraphPad Prism 8.

It was found that binding of reactive antibodies to spike and RBD is comparable to positive controls. The area under the binding curve (AUC) was calculated for each investigated antibody. Out of 263 mAbs, 113 showed binding to spike protein and/or RBD (cut-off AUC>3); see Fig. 1 and 2.

Moreover, affinity to RBD was further investigated by Surface Plasmon Resonance (SPR). Briefly, SPR-based assays were performed to determine the affinity of RBD-binding antibodies using a Biacore T200 system and Biacore sensor chip CM5. Two flow cells were immobilized with anti-human Fab antibodies using human Fab capture kit following the manufacturer’s instructions. Test mAb samples (40 pg/ ml) and the negative control mAb mGO53 (40 pg/ ml) were captured in the sample and reference flow cells, respectively. Stabilization of both flow cells was performed by SPR running buffer at 10 pl/ min flow rate for 10 min. A serial dilution of RBD was set up in SPR running buffer and the following concentrations were injected into both flow cells: 0 nM, 12.4 nM, 37.0 nM, 111.1 nM, 333.3 nM and 1000 nM using a flow rate of 30 pl/ min. Dissociation and association took place at 25 °C for 60 s and 180 s, respectively. Between the injections of different sample antibodies, flow cells were regenerated using 10 mM glycine in HC1. Data was analyzed using a 1 : 1 binding model or steady-state kinetic analysis using Biacore T200 software V2.0.

Results are shown in Fig. 3. RBD-reactive antibodies showed broad range of affinities to RBD (10‘⁵ M to 10'¹⁰ M) in direct comparison to measurements of published antibodies including antibodies in clinical use that were expressed and tested under the same conditions. Antibodies with comparable affinity (10‘⁷ - 10'⁹ M) to or even higher affinity (< 10'⁹ M) than the published antibodies were identified.

Example 3: Virus neutralization by antibodies

Neutralization titers were determined in titration experiments using VeroE6 cells. Virus stocks were produced by isolation and amplification of the SARS-CoV-2 WT (isolate H2P4, Steuten 2021), the B.l.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta) and the B.1.1.529.1 (omicron) variant from nasopharyngeal and oropharyngeal swabs of PCR-confirmed SARS-CoV-2 positive patients (Benning et al., 2021 Clin J Am Soc Nephro 17 (1) 98-106; Mallm et al., 2021, medRxiv). SARS-CoV-2 WT, B. l.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variant were amplified in VeroE6 cells and virus titers of stocks were determined by plaque assay and Tissue Culture Infectious Dose (TCID) 50 assay in VeroE6 cells. To avoid rapid cell culture adaptation, stocks of the B.1.1.5291 (omicron) variant were produced in Calu-3 cells and titers were determined in VeroE6 cells using TCID 50 assay. For neutralization assays, monoclonal antibodies were diluted 1 :3 for 10 steps with a test range from 0.5 ng/ml to 10 pg/ml and were incubated with 6x10⁴ TCID 50 of SARS-CoV-2 WT, B.l.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta) and the B.1.1.529.1 (omicron) variant. After 1 h at 37 °C, the mixture was added to VeroE6 cells and cells were fixed in the plates with 5% formaldehyde 24 h later. Virus replication was determined by immunostaining for the viral nucleocapsid protein using an incell ELISA. S309 and mGO53 were used as positive and negative controls, respectively. Data were normalized to a mock-infected (0%) and a no-serum control (100%). The inhibitory concentration 50 (IC50) is defined by non-linear regression sigmoidal dose response analysis using the GraphPad Prism 7 software package

Virus neutralization was observed only among the antibodies that bound to the RBD, but not to the other epitopes in the spike protein. Antibodies with better neutralizing capacity compared to the published antibodies were identified. Indeed, the virus neutralizing capacity was observed only among antibodies with high affinity. (RBD Kd < 10-7 M). Nevertheless, only 27% and 23% of antibodies with RBD Kd < 10-7M and RBD Kd < 10-9M, respectively, neutralized the virus. This suggests that the potency of the antibodies depends on both epitope specificity and high affinity. Refer to Fig. 4 and 5.

Example 4: Antibody binding region in RBD elucidated by a blocking assay using published antibodies with known epitopes and hACE2

Based on the epitopes recognized in the RBD, antibodies are grouped into different classes. Antibodies of the same class do not necessarily target the same epitope or bind in the same mode. The class categories are not absolute, i.e. antibodies can belong to more than one class depending on the exact target epitope. In order to identify the potential epitope and class of the antibodies from this study, an assay was developed to measure their capacity in blocking previously reported antibodies with known epitopes/classes from binding to RBD.

Blocking assay was performed using Biacore T200 system and Biacore sensor chip CM5. A flow cell was immobilized with anti-human IgG antibodies using human antibody capture kit by following manufacturer’s instructions. Sample antibody (mAbl) was captured in the flow cell at 20 pg/ml, followed by the capture mGO53 at 100 pg/ml. Stabilization of the flow cell was performed by SPR running buffer at 10 pl/ min flow rate for 5 min. To measure binding, injection of RBD at 1 pM was immediately followed by an injection of 20 pg/ml mAb2 using dual injection option. Binding values were calculated by measuring the difference in response units before and after the injection of mAb2 and subtracting the background binding as measured by performing the same steps while injecting the running buffer instead of RBD. Between the injections of different sample antibodies, the flow cell was regenerated using 3 M MgC12. Data was analyzed using using Biacore T200 software V2.0.

Results for blocking capabilities viz-a-viz known SARS-CoV-2 antibodies are shown in Fig. 6. Antibodies belonging to Classl/2, Class4, S309 and “none” classes showed high affinity due to reduced kinetic off rate, suggesting they are selected efficiently in the germinal center driven B cell response.

Blocking of binding to hACE2 was also investigated. Blocking assay was performed using Biacore T200 system and Biacore sensor chip CM5. A flow cell was immobilized with antihuman IgG antibodies using human antibody capture kit by following manufacturer’s instructions. Sample antibody (mAbl) was captured in the flow cell at 20 pg/ml, followed by stabilization of the flow cell by SPR running buffer at 10 pl/ min flow rate for 5 min. To measure binding, injection of RBD at 1 pM was immediately followed by an injection of 200 nM hACE2 using dual injection option. Binding values were calculated by measuring the difference in response units before and after the injection of hACE2 and subtracting the background binding as measured by performing the same steps while injecting the running buffer instead of RBD. Between the injections of different sample antibodies, flow cell was regenerated using 3 M MgC12. Data was analyzed using using Biacore T200 software V2.0. The majority of Classl/2, 1/2/3, 1/3, 1/3/4, 1/4 antibodies block hACE2 binding to WT RBD, suggesting their epitope overlaps with the hACE2 interaction site in RBD. Antibodies belonging to Classi, 4, S309 and none class do not block hACE2 binding to WT RBD, suggesting they do not bind to receptor binding motif (RBM) in RBD. High affine antibodies in Classl/2, Classl/2/3 and Class2/3 show efficient virus neutralization (protective epitopes). However, high affinity in antibodies with other binding profiles such as S309 and none classes does not result in virus neutralization (non-protective epitopes). Using SPR, hACE2 interaction with RBD upon binding to mAbl was measured. All Classl/2 mAbs block hACE2 interaction with RBD, while Class2/3 mAb does not.

This suggests Classl/2 and Class2/3 antibodies neutralize virus by binding to different, nonoverlapping epitopes. Three Classl/2 antibodies and one Class2/3 antibody were tested against each other to block binding to RBD. Two of three Classl/2 antibodies did not block Class2/3 antibody from binding to RBD. Classl/2/3 mAb (2939) blocks Class2/3 (3279) but not Classl/2 (1255) mAbs from binding to RBD (Fig. 7). Antibodies with non-overlapping epitopes show synergy in virus neutralization, e.g., high affinity binding to non-overlapping epitopes by mAbs 3279 and 1255 translates into more efficient virus neutralization when used in cocktail compared to on their own. Moreover, high affine antibodies belonging to Classl/2 and Class2/3 show high neutralization capacity against three Variants of Concern (VoCs) tested. High affine antibodies with S309 profile show specific neutralization against alpha variant but not against WT or other variants (Fig. 8).

Example 5: Antibody binding and activity against the SARS-CoV-2 omicron variant

Among all VoCs, Omicron RBD has a particular high number of mutations (15 aa) compared to the WT RBD. Affinity for SARS-CoV-2 omicron variant was investigated next. SPR based assay was performed to determine affinity of RBD-binding antibodies using Biacore T200 system and Biacore sensor chip CM5. Two flow cells were immobilized with anti-human Fab antibodies using human Fab capture kit by following manufacturer’s instructions. Antibody samples (40 pg/ ml) as well as the negative control mGO53 (40 pg/ ml) were captured in the sample and reference flow cells, respectively. Stabilization of both flow cells was performed by SPR running buffer at 10 pl/ min flow rate for 10 min. A serial dilution of Omicron RBD was performed in SPR running buffer and the following concentrations were injected into both the flow cells: 0 nM, 12.4 nM, 37.0 nM, 111.1 nM, 333.3 nM and 1000 nM using a flow rate of 30 pl/ min. High affine antibodies were independently tested at 0 nM, 3 nM, 9.2 nM, 27.7 nM, 83.3 nM and 250 nM of Omicron RBD. Dissociation and association took place at 25 °C for 60 s and 180 s, respectively. Between the injections of different sample antibodies, flow cells were regenerated using 10 mM glycine in HC1. Data was analyzed using a 1 : 1 binding model or steady-state kinetic analysis using Biacore T200 software V2.0.

Only three control mAbs (S309, REGN10933 and CR3022) have measurable binding to Omicron RBD, while the rest lost binding completely. mAbs from this study show increased, similar, decreased or complete loss in affinity to Omicron RBD compared to WT RBD (Fig. 9). High affinity in antibodies with binding profiles such as S309 does not result in WT virus neutralization. However, the majority of the mAbs belonging to the non-neutralizing antibody classes bind to Omicron RBD with similar or higher affinity in comparison to WT RBD. High affine antibodies in Class 1/2, Class 1/2/3 and Class2/3 show efficient WT virus neutralization. The majority of the mAbs belonging to the three classes bind to Omicron RBD with reduced affinity in comparison to WT RBD. mAbs 1255 in Classl/2 and 2939 in Classl/2/3 retain higher binding to Omicron RBD compared to other antibodies of the respective classes. High affine antibodies (1255 and 2939) can both bind to WT and Omicron RBD without blocking each other - suggesting they bind non-overlapping epitopes (Fig. 10). It was further found that 1255 blocks, whereas 2939 does not block hACE2 interaction with both WT and Omicron RBD, indicating the difference in their binding site overlap with hACE2 interaction site (Fig. 18). Antibodies 2939 and 1255 show high Omicron variant neutralization capacity. Moreover, mAb 2939 shows better Omicron neutralization than therapeutically used mAb S309 - VIR Biotechnology (Sotrovimab) (Fig. 12).

In general, mAbs that do not neutralize WT virus show weak neutralizing capacity to Omicron variant virus. Inhibition values of these mAbs are still lower than those of 1255 and 2939 - suggesting that the epitopes targeted by 1255 and 2939 are promising for protective vaccine responses (Fig. 13). Classl/2 (1255) and Class2/3 (3279) antibodies show broad neutralization and could be considered for therapeutic purposes or diagnostic purposes.

Surprisingly, it was found that out of the 41 antibodies with the highest affinities (Kd for RBD of 10'⁸ or lower), 7 had a IGKVl-39*01 variable light chains and two had IGHV3-9*01 variable heavy chains (Table 3). Notably, the antibodies with the IGHV3-9 heavy chains were associated with IGKV1-39 light chains. Alignments of exemplary sequences are shown in Fig. 14.

Example 6: Neutralization of SARS-CoV-2 variants

Capacity of antibodies to neutralize SARS-CoV-2 variant strains was tested by determining EC50 values for viral infection essentially as in Example 3 herein above. Results are summarized in Table 6 below. Table 6: EC50 values (in pg/ml) of selected antibodies for SARS-CoV-2 variants; NC: not calculated.

able 3: Properties of high-affinity antibodies; ab name: designation of antibody; donor id: identification number of donor; IGHV: heavy chain ariable gene; H isptype: heavy chain isotype; IGLV: kappa light chain variable gene; IGLV: lambda light chain variable gene; RBD KD (M): quilibrium dissociation constant Kd for RBD in M; class: RBD epitope recognition class.

Cited literature

Amanat 2020, Nat Med 26: 1033-1036

Asamow 2021, Cell 184, 3192-3204

Bames 2020, Nature 588, 682-687

Benning et al., 2021 Clin J Am Soc Nephro 17 (1) 98-106

Imkeller et al., 2016 BMC Bioinformatics 17, 67

Lefranc 2003, Dev Comp Immunol 27(1): 55-77

Lefranc et al. (2020) biomedicines 8:319

Mallm et al., 2021, medRxiv, doi: 10.1101/2021.04.27.21254849

Murugan et al., 2015 Eur J Immunol. 45(9):2698-700

Sharma 2021, Proteins. 2021; 1-11

Steuten 2021, ACS Infect. Dis. 7, 6, 1457-1468

Tiller et al., 2008, J Immunol Methods. 329(1-2): 112-124

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Claims

1. A method for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity, the method comprising the steps of:

2. The method of claim 1, wherein said antibody binds to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.

3. The method of claim 1 or 2, wherein said antibody that binds the SARS-CoV-2 spike protein, preferably the RBD, with high affinity characterized by an equilibrium dissociation constant (Kd) of less than 5 x 10'⁹ M.

4. The method of any one of claims 1 to 3, wherein in step (a) the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of said antibody is identified by sequencing a polynucleotide encoding said light chain and/or sequencing a polynucleotide encoding said heavy chain.

5. A method for manufacturing an antibody comprising the steps of

(a) identifying an antibody that binds its antigen with high affinity by the method of any one of claims 1 to 3; and

(b) manufacturing the antibody identified in step (a).

6. The method of claim 5, wherein said antibody referred to in step (a) is obtained from a B-cell or a hybridoma cell.

7. A method for assessing whether a subject produces antibodies that bind the SARS-CoV- 2 spike protein with high affinity comprising the steps of (a) determining the presence or absence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample of said subject; and

(b) identifying a subject that produces antibodies that bind the SARS-CoV-2 spike protein with high affinity based on the presence of IGKV1-39 in the light chain and IGHV3-9 in the heavy chain of an antibody produced by said B-cell in said B-cell containing sample. The method of claim 7, wherein said method is to be used for assessing the immune status of said subject, preferably for assessing whether a vaccination and/or SARS-CoV infection caused production of high-affinity antibodies against SARS-CoV-2 in said subject. Use of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody ex vivo as a biomarker for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity. IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample for use as a biomarker for determining whether a subject produces antibodies that bind the SARS-CoV-2 spike protein with high affinity. The IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody produced by a B-cell in a B-cell containing sample for use of claim 10, wherein said biomarker is to be used for assessing the immune status of said subject, preferably wherein said biomarker is to be used for determining whether a vaccination against SARS-CoV-2 in said subject was successful. A kit for identifying an antibody that binds the SARS-CoV-2 spike protein with high affinity comprising detection agents for detecting the presence of IGKV1-39 in the light chain and/or IGHV3-9 in the heavy chain of an antibody. An antibody which binds to the SARS-CoV-2 spike protein, wherein said antibody comprises an IGKV1-39 light chain comprising the amino acid sequence of SEQ ID NO: 17, 18, 19, 20, or 21; and/or comprises an IGHV3-9 heavy chain comprising the amino acid sequence of SEQ ID NO:34 or 35, wherein said antibody preferably binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A polynucleotide encoding the antibody of any one of claim 13. The subj ect matter of any of the preceding claims, wherein said SARS-CoV-2 is selected from the group consisting of: wildtype (WT) SARS-CoV-2, SARS-CoV-2 alpha variant

(B. l.1.7), SARA-CoV-2 beta variant (B.1.1351) SARS-CoV-2 delta variant (B.1.617.2), and SARS-CoV-2 omicron variant (B.1.1.529) including its sub-variants.