Nothing Special   »   [go: up one dir, main page]

CN116813789A - Fully human bispecific antibody for broad spectrum neutralization of novel coronavirus Omicron subvariant - Google Patents

Fully human bispecific antibody for broad spectrum neutralization of novel coronavirus Omicron subvariant Download PDF

Info

Publication number
CN116813789A
CN116813789A CN202310792691.2A CN202310792691A CN116813789A CN 116813789 A CN116813789 A CN 116813789A CN 202310792691 A CN202310792691 A CN 202310792691A CN 116813789 A CN116813789 A CN 116813789A
Authority
CN
China
Prior art keywords
antibody
amino acid
monoclonal antibody
seq
chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202310792691.2A
Other languages
Chinese (zh)
Other versions
CN116813789B (en
Inventor
陈薇
于长明
迟象阳
孙韩聪
张冠英
李建民
范鹏飞
房婷
黄平
侯利华
陈郑珊
陈旖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Academy of Military Medical Sciences AMMS of PLA
Original Assignee
Academy of Military Medical Sciences AMMS of PLA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Academy of Military Medical Sciences AMMS of PLA filed Critical Academy of Military Medical Sciences AMMS of PLA
Priority to CN202310792691.2A priority Critical patent/CN116813789B/en
Priority claimed from CN202310792691.2A external-priority patent/CN116813789B/en
Publication of CN116813789A publication Critical patent/CN116813789A/en
Application granted granted Critical
Publication of CN116813789B publication Critical patent/CN116813789B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a fully human bispecific neutralizing antibody capable of resisting a new coronavirus, which is formed by modifying two strains of fully human neutralizing antibodies of the new coronavirus. The antibodies are capable of effectively neutralizing currently popular Omicron sub-variants bf.7, ba.4, xbb.1.5 and xbb.1.16 with strong escape capacity. Compared with the parent monoclonal antibody, the bispecific antibody disclosed by the invention has stronger antiviral activity and the capability of inhibiting escape mutation, is a feasible and effective strategy for treating and preventing severe new crowns, and has potential to become a candidate medicament for intervening in the new crowns.

Description

Fully human bispecific antibody for broad spectrum neutralization of novel coronavirus Omicron subvariant
Technical Field
The invention discloses a bispecific antibody, and belongs to the technical field of polypeptides.
Background
The novel coronavirus is a single-stranded positive sense RNA virus with a capsule, the genome of which can be mutated by various mechanisms during replication, and the evolution rate is extremely high. From 2019 outbreak to date, new variant strains are continuously appeared, so that epidemic situation is repeated, and the number of infected people is continuously increased. The Omicron variant strain which is spread on a large scale in the world at present has the characteristics of strong infectivity, high spreading speed, strong immune escape capability and the like, and the variant strain continuously evolves to evolve various sub-variants such as BA.5, BF.7, XBB, XBB.1.5, XBB.1.16 and the like, and the spreading of the variant strain greatly increases the risks of reinfection and breakthrough infection, thus bringing great challenges to epidemic prevention work in the world.
In the epidemic prevention and control process, therapeutic antibody medicaments make an important contribution, and monoclonal antibody medicaments or monoclonal antibody combined cocktail therapies developed by large-scale pharmaceutical enterprises such as Gelanin, gift, gelanin Smith, and the like are all used for prevention and clinical treatment by the FDA emergency use authorization in the United states. However, the continued adaptive evolution of viruses has impaired the efficacy of existing vaccines and escaped almost all of the developed neutralizing monoclonal antibodies. There is thus a need to develop new generation neutralizing antibody drugs that provide broad spectrum protection.
Bispecific antibodies are a novel antibody drug model, integrating two specific antibodies into one molecule, exhibiting unique properties that differ from a single antibody. Compared with strategies such as single monoclonal antibody, antibody cocktail and the like, the bispecific antibody has the following advantages: (1) The cocktail therapy needs to produce two antibodies separately, while the bispecific antibody only needs to produce one, so that the production cost is low, and the pre-clinical and clinical development are more convenient; (2) IgG- (scFv) 2 The form of the diabody has tetravalent antigen binding capacity, has improved neutralizing activity on viruses, and particularly integrates two strains of neutralizing antibodies with synergistic effect, wherein the neutralizing activity has 1+1>2', a synergistic effect; (3) The different binding epitopes of the two monoclonal antibodies are combined, so that more virus antigen site mutations can be tolerated, and the probability of occurrence of virus escape mutations in the treatment process can be reduced, thus the method has important significance for clinical treatment. Thus, development of an economical and efficient bispecific antibody drug is urgent.
Bispecific antibodies are not present in nature and can only be prepared manually. Through many years of research and technological development, bispecific antibodies have emerged in many different design strategies in structure. The full-length diabodies containing the Fc region and the fragment diabodies not containing the Fc region are largely classified into 2 classes according to whether they contain the Fc functional region. Full length diabodies are characterized by the retention of the Fc region, which provides longer half-life and stability in vivo, and antibodies with the Fc region are more convenient to purify. In addition, the Fc region may mediate some immune-related functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). Full length bispecific antibodies containing Fc regions are Knobs-into-holes, crossmab, DVD-Ig, igG- (scFv) 2 Etc. Compared with the traditional antibody, the fragment double antibodyDue to lack of Fc, the size is smaller, generally only 50 kDa, the permeability of tissues is better, and the method has certain advantages in the aspect of solid tumor treatment. Currently, such bispecific antibodies are mainly bi-Nanobody, biTE, DART, tandAbs, etc.
The invention aims to construct a novel bispecific antibody for resisting a novel coronavirus variant strain so as to cope with persistent viral immune escape, and provides candidate medicines for resisting the novel coronavirus variant strain which is popular at present and has strong escape capacity and possibly appears in the future.
Disclosure of Invention
Based on the above purpose, the invention is to construct a fully human bispecific antibody for resisting novel coronavirus variants by taking the monoclonal antibody H4D12 (Chinese patent application CN 202211732654.4) with strong neutralizing effect on wild type and Delta, BA.2, BA.2.75, BA.2.76 and other variants of the novel coronavirus and the monoclonal antibody ZW2G10 (Chinese patent application CN 202210023619.9) with strong neutralizing effect on Alpha, beta, gamma, delta, BA.1 and other variants as parent monoclonal antibodies and utilizing the characteristics of different epitopes of H4D12 and ZW2G10 antibodies.
The invention firstly provides a fully human bispecific antibody of an anti-novel coronavirus variant, which consists of two polypeptide chains and two monoclonal antibody light chains, wherein the polypeptide chains are defined as being formed by connecting a monoclonal antibody heavy chain and a single chain antibody in series, the monoclonal antibody heavy chain and the monoclonal antibody light chain are from the same female parent monoclonal antibody, and in the bispecific antibody of the invention, the monoclonal antibody heavy chain and the monoclonal antibody light chain from the same female parent monoclonal antibody form a first antigen recognition region. The single chain antibody refers to a monovalent antibody form formed by connecting an antibody heavy chain variable region and a light chain variable region in series; in the bispecific antibody of the present invention, the single chain antibody heavy chain variable region and the single chain antibody light chain variable region constitute a second antigen recognition region; in the present invention, a complete bispecific antibody comprises two first antigen recognition regions and two second antigen recognition regions.
According to the technical scheme provided by the invention, the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody are respectively shown as the 26 th to 33 th, 51 th to 58 th and 97 th to 114 th amino acid sequences of SEQ ID NO.1, the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the light chain of the monoclonal antibody are respectively shown as the 27 th to 32 th, 50 th to 52 th and 89 th to 96 th amino acid sequences of SEQ ID NO.3, the amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain of the single chain antibody are respectively shown as the 495 th to 502 th, 520 th to 527 th and 566 th amino acid sequences of SEQ ID NO.1, and the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the light chain of the single chain antibody are respectively shown as the 637 th to 645 th, 663-665 th and 702 th amino acid sequences of the light chain of the single chain antibody.
In the design of the present invention, the heavy chain of the monoclonal antibody and the light chain of the monoclonal antibody are derived from the parent monoclonal antibody H4D12 and form the first antigen recognition region of the bispecific antibody of the present invention, so that in the present invention, the heavy chain of the monoclonal antibody refers to the heavy chain of the parent monoclonal antibody H4D12, and the light chain of the monoclonal antibody refers to the light chain of the parent monoclonal antibody H4D 12. The heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are derived from the parent monoclonal antibody ZW2G10 and form the second antigen recognition region of the bispecific antibody, so that the heavy chain of the single-chain antibody refers to the heavy chain of the parent monoclonal antibody ZW2G10 and the light chain of the single-chain antibody refers to the light chain of the parent monoclonal antibody ZW2G 10. The results of previous antibody competition experiments showed that H4D12 and ZW2G10 recognized different epitopes of the RBD region of the new coronavirus. As taught by the general knowledge in the art, the variable regions of the heavy and light chains of antibodies are responsible for binding to antigen, wherein the hypervariable regions of the variable regions (also called complementarity determining regions, CDRs) constitute the antigen binding sites of the antibody molecules, in the solution of the present invention the bispecific antibody is composed of the 4 sets of CDR regions described above, namely, the monoclonal antibody heavy chain variable regions CDR1, CDR2, CDR3 and monoclonal antibody light chain variable regions CDR1, CDR2, CDR3 from H4D12 constitute two first antigen recognition regions that recognize the RBD region of the novel coronavirus; the single chain antibody heavy chain variable region CDR1, CDR2, CDR3 and single chain antibody light chain variable region CDR1, CDR2 and CDR3 from ZW2G10 constitute two second antigen recognition regions that recognize the new coronavirus RBD region.
In a preferred embodiment, the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is shown in amino acid sequences 1-125 of SEQ ID NO.1, the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown in amino acid sequences 1-107 of SEQ ID NO.3, the amino acid sequence of the variable region of the heavy chain of the single chain antibody is shown in amino acid sequences 470-597 of SEQ ID NO.1, and the amino acid sequence of the variable region of the light chain of the single chain antibody is shown in amino acid sequences 612-722 of SEQ ID NO. 1.
In a more preferred embodiment, the constant region amino acid sequence of the heavy chain of the monoclonal antibody is shown in amino acid sequences 126-455 of SEQ ID NO.1, and the constant region amino acid sequence of the light chain of the monoclonal antibody is shown in amino acid sequences 108-213 of SEQ ID NO. 3.
In a particularly preferred embodiment, the monoclonal antibody heavy chain and the single chain antibody, and/or the single chain antibody heavy chain variable region and the single chain antibody light chain variable region between flexible connecting peptides. The flexible connecting peptides of the invention generally comprise small amino acids, non-polar such as Gly, polar such as Ser. These smaller size amino acids provide flexibility in linking peptides, allowing some mobility of the two proteins being linked. And, the addition of Ser can make the connecting peptide and water molecule form hydrogen bond, and endow the connecting peptide with stability in aqueous solution, so as to reduce the interaction between the connecting peptide and the front and back proteins. The most predominant flexible connecting peptide today consists of Gly and Ser residues ("GS" linker). The most widely used flexible connecting peptide has the sequence (Gly-Gly-Gly-Gly-Ser) n. By adjusting the value of n (the number of repeats), the length of the GS-linked peptide can be altered so that the two linked proteins can be optimally separated or made to interact.
In a specific embodiment of the invention, the amino acid sequence of the flexible connecting peptide is as shown in amino acid sequence 456-469, i.e., GGGGSGGGGSGGGG.
In a specific embodiment of the invention, the amino acid sequence of the polypeptide chain is shown as SEQ ID NO.1, and the amino acid sequence of the monoclonal antibody light chain is shown as SEQ ID NO. 3. The bispecific antibody is defined herein as "4D12-H-2G10". The invention constructs an IgG- (scFv) 2 The form of bispecific antibody, i.e., a single chain antibody (scFv) is linked to the C-terminus of the Fc functional region of a normal IgG antibody molecule to achieve bifunctional binding to a target molecule via CDR regions at both ends of the molecule.
In a further embodiment of the present invention, the polynucleotide encoding the bispecific antibody is shown in SEQ ID NO.2, and the polynucleotide encoding the light chain of the monoclonal antibody is shown in SEQ ID NO. 4. It is reasonable to expect by those skilled in the art that the coding sequence of the bispecific antibody can be appropriately optimized without changing the amino acid sequence to facilitate the expression and purification of the protein, and thus other coding sequences of the bispecific antibody can also implement the bispecific antibodies defined in the present invention.
Third, the present invention provides an expression vector containing the above polynucleotide. In a specific embodiment of the invention, the expression vector is pcDNA3.4. It is reasonable to expect by the skilled person that other eukaryotic or prokaryotic expression vectors of the prior art may also be used for the expression of the bispecific antibodies of the invention.
Fourth, the present invention provides a host cell containing the above expression vector. Expression of the bispecific antibody is performed using an Expi293 expression system in one particular embodiment of the invention. It is reasonable to expect by the skilled person that other engineering cells of the prior art, such as CHO cells etc. can also be used for the expression of the bispecific antibodies of the invention.
Finally, the invention provides the application of the bispecific antibody in preparing novel coronavirus therapeutic drugs.
The cross-binding activity identification of the invention shows that the bispecific antibody 4D12-H-2G10 can cross-bind to the S-ECD proteins of BA.1, BA.2, BA.2.75, BA.2.76, BA.3, BA.4, BA.4.6, BF.7, XBB, XBB.1.5, and the bispecific antibody 4D12-H-2G10 has a broader binding activity compared to the parent monoclonal antibody.
IC of specific antibody 4D12-H-2G10 for neutralizing BA.1 pseudovirus 50 1 ng/mL, and neutralizing BA.2 pseudovirus IC 50 14 ng/mL, neutralizing BA.2.75 pseudovirus IC 50 IC for neutralizing BA.2.76 pseudovirus at 2 ng/mL 50 1 ng/mL, and neutralizing BA.3 pseudovirus IC 50 54 ng/mL, and neutralizing BA.4 pseudovirus IC 50 IC for neutralizing BA.4.6 pseudovirus at 5 ng/mL 50 11 ng/mL, neutralizing BF.7 pseudovirus IC 50 Is 5 ng/mL, and the IC for neutralizing XBB.1.5 pseudovirus 50 IC of 2 ng/mL for neutralizing XBB.1.16 pseudovirus 50 Is 2 ng/mL. The results show that compared with the parent monoclonal antibody, the bispecific antibody 4D12-H-2G10 has more broad-spectrum efficient neutralization activity on different sub-variants of Omicron pseudoviruses, so that the development of the Omicron pseudoviruses into a powerful antibody drug for resisting current and future epidemic new crown variant strains is possible.
Drawings
FIG. 1 is a schematic diagram of the structure of a bispecific antibody of the present invention;
FIG. 2 is a SDS-PAGE profile of bispecific antibody purification;
FIG. 3 ELISA assay for cross-binding activity of 4D12-H-2G 10;
FIG. 4 neutralizing activity of antibodies against BA.1 pseudoviruses;
FIG. 5 neutralizing activity of antibodies against BA.2 pseudoviruses;
FIG. 6 neutralizing activity of antibodies against BA.2.75 pseudoviruses;
FIG. 7 neutralizing activity of antibodies against BA.2.76 pseudoviruses;
FIG. 8 neutralizing activity of antibodies against BA.3 pseudoviruses;
FIG. 9 neutralizing activity of antibodies against BA.4 pseudoviruses;
FIG. 10 neutralizing activity of antibodies against BA.4.6 pseudoviruses;
FIG. 11 neutralizing activity of antibodies against BF.7 pseudoviruses;
FIG. 12 neutralizing activity of antibodies against XBB.1.5 pseudoviruses;
FIG. 13 neutralizing activity of antibodies against XBB.1.16 pseudoviruses.
Description of the embodiments
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are only exemplary and do not limit the scope of the invention in any way, which is defined by the claims.
EXAMPLE 1 construction of bispecific antibodies
In the early stage, fully humanized monoclonal antibodies H4D12 and ZW2G10 with excellent broad-spectrum neutralization activity are obtained from memory B cells of recombinant novel coronavirus vaccinators through a flow sorting-single cell PCR technology, different epitopes of S protein RBD structural domains of SARS-CoV-2 are respectively taken as targets, wherein H4D12 has strong neutralization effect on wild type novel coronavirus and variant strains such as Delta, BA.2, BA.2.75, BA.2.76 and the like 50 Is lower than 10 ng/mL (see Chinese patent application CN 202211732654.4), ZW2G10 has strong neutralization effect on Alpha, beta, gamma, delta, BA.1 and other variants, and IC 50 Are lower than 100 ng/mL (Chinese patent ZL 202210023619.9).
VH and VL of ZW2G10 were linked using GGGGSGGGGSGGGG linker according to literature to construct scFv of ZW2G10, after which scFv of ZW2G10 was linked to the C-terminus of H4D12 heavy chain with the same linker to construct a key map as follows:
Xbai cleavage site-GCCGCCACC-Signal peptide-H4D 12 VH+CH-linker-ZW 2G10 VH-linker-ZW 2G10 VL-stop codon-BamH I cleavage site, synthesizing the sequence of interest and byXbaI andBamh I the construction of the recombinant expression plasmid for 4D12-H-2G10 heavy chain and gene synthesis was completed by general biology (Anhui) company, and then the plasmid was transfected together with the light chain expression plasmid for H4D12 into an Expi293F cell to express the 4D12-H-2G10 bispecific antibody. FIG. 1 shows a schematic diagram of the structure of a bispecific antibody of the present invention, which is an IgG- (scFv) 2 Form of bispecific antibody, i.e. a single chain antibody (scFv) derived from parent monoclonal antibody ZW2G10 linked to the C-terminal end of the Fc functional region of the parent monoclonal antibody molecule H4D12 derived from the normal structure, is achieved by binding the CDR regions at both ends of the molecule to the target moleculeBispecific recognition.
EXAMPLE 2 transient expression and affinity chromatography purification of bispecific antibodies
Using an Expi293 expression System, 100 mL of Expi293F cells were transfected after 50. Mu.g of heavy chain and 50. Mu.g of light chain were mixed, the culture broth was harvested after 5-6 days according to instructions (ThermoFisher Scientific, A14635), supernatant after centrifugation was about 30 mL, pre-loaded Protein G affinity chromatography column with volume of 5 mL was used, equilibrated with 20 mM PBS before loading, after conductivity to baseline, after loading was completed, the column was washed with 20 mM PBS to baseline plateau, the Protein of interest was eluted with 0.1M glycine buffer pH 2.7, after OD 280 After near baseline, collection was stopped.
Results: SDS-PAGE detects affinity chromatography purified bispecific antibody, see in particular FIG. 2, lanes 1, 2 and 3 are respectively the pre-column, post-column and eluent bands of 4D12-H-2G10, and protein samples can be reduced by mercaptoethanol into two fragments with the sizes of 75 kDa and 25 kDa, which correspond to the theoretical molecular weights of the heavy chain and the light chain of the antibody respectively, and are in line with expectations. Lane M is a molecular weight marker.
Example 3 identification of Cross-binding Activity of bispecific antibodies
1. Coating: 96-well ELISA plates were prepared on day before the experiment, and the Omicron sub-variants BA.1S-ECD antigen (Acro, SPN-C522 a), BA.2S-ECD antigen (Acro, SPN-C522 b), BA.2.75S-ECD antigen (Acro, SPN-C522 f), BA.2.76S-ECD antigen (Acro, SPN-C522 i), BA.3S-ECD antigen (Acro, SPN-C522C), BA.4S-ECD antigen (Acro, SPN-C5229), BA.4.6S-ECD antigen (Acro, SPN-C522 m), BF.7S-ECD antigen (Acro, SPN-C522 q), XBB S-ECD antigen (Acro, SPN-C5248) and XBB.1.5 antigen (Acro, SPN-C524 i) were diluted with the coating solution to 2. Mu.g/mL, coated with the ELISA plates, and coated overnight at 100. Mu.L.
2. Closing: plate washer (BIO-TEK, 405_LS) was used for 3 washes, 100 μl of blocking solution was added to each well and incubated at 37deg.C for 1 h.
3. Sample incubation: plates were washed 3 times, except for the first well, 100 μl of diluent was added to each well, the antibodies were diluted to 1 μg/mL for the first well, 4-fold gradient dilution, 100 μl/well, three duplicate wells were set per antibody, and incubated 1 h at 37 ℃.
4. Secondary antibody incubation: plates were washed 3 times, and HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab 97225) was diluted 1:10000 with dilution, 100 μl per well was added to the ELISA plate corresponding well and incubated 1 h at 37 ℃.
5. Color development: washing the plate for 3 times, adding 100 mu L of TMB single-component color development liquid into each hole, developing for 6 min, keeping out of the sun at room temperature, and adding 50 mu L of termination liquid into each hole to terminate the reaction.
6. OD values at 450-630 nm were detected on a microplate reader, curves were drawn using Logistic four-parameter fitting, and the EC of the antibody was calculated 50 Values.
Results: the binding activity of the bispecific antibody to omacron subtype S protein is shown in FIG. 3, 4D12-H-2G10 binds strongly to the S-ECD proteins BA.1, BA.2, BA.2.75, BA.2.76, BA.3, BA.4, BA.4.6, BF.7, XBB, XBB.1.5, EC 50 23 ng/mL, 15 ng/mL, 17 ng/mL, 17 ng/mL, 13 ng/mL, 22 ng/mL, 25 ng/mL, 48 ng/mL, 37 ng/mL, 21 ng/mL, respectively, whereas the two parent monoclonal antibodies do not achieve full binding to the Omicron sub-variant S protein described above. Compared with the parent monoclonal antibody, the bispecific antibody 4D12-H-2G10 has a broader binding activity.
EXAMPLE 4 identification of pseudovirus neutralizing Activity of bispecific antibodies
1. Purified bispecific antibody was serially diluted 3-fold from the initial concentration with dmem+10% FBS medium, added to 96-well plates, 3 multiplex wells were set, and the volume was 50 μl/well; then 50. Mu.L of pseudovirus suspension of the novel coronavirus variant (diluted to appropriate titer with DMEM+10% FBS) was added to each well, thoroughly mixed, and a survival control (without virus and antibody) and a death control (with virus only) were additionally provided, and the mixture was subjected to 5% CO at 37 ℃ 2 Cell incubator incubate 1 h.
2. HEK293T cells were digested with 0.25% pancreatin and diluted to 2.5X10% with medium (DMEM+10% FBS) 5 cell/mL concentration, inoculating into 96-well cell culture plate, inoculating volume of 100 μl/well, and placing 5% CO at 37deg.C 2 The cell culture incubator was incubated overnight.
3.48 After h, 100. Mu.L of the cell culture supernatant was discarded, 100. Mu.L of chromogenic substrate was added and incubated for 2 min in the dark. Transfer 150 μl to 96-well white microwell plate and read Luciferase signal values using Tecan Spark multifunctional microwell plate detector.
4. With [1- (sample-surviving control signal)/(death control signal-surviving control signal)]Antibody neutralization was calculated at 100%, and antibody IC was calculated by fitting a curve to GraphPad Prism 8 50 Values.
Results: FIGS. 4-13 are graphs of pseudovirus neutralization activity of 4D12-H-2G10 with BA.1, BA.2, BA.2.75, BA.2.76, BA.3, BA.4, BA.4.6, BF.7, XBB.1.5 and XBB.1.16 as a function of concentration, respectively, for the IC of bispecific antibody 4D12-H-2G10 to neutralize BA.1 pseudovirus 50 1 ng/mL, and neutralizing BA.2 pseudovirus IC 50 14 ng/mL, neutralizing BA.2.75 pseudovirus IC 50 IC for neutralizing BA.2.76 pseudovirus at 2 ng/mL 50 1 ng/mL, and neutralizing BA.3 pseudovirus IC 50 54 ng/mL, and neutralizing BA.4 pseudovirus IC 50 IC for neutralizing BA.4.6 pseudovirus at 5 ng/mL 50 11 ng/mL, neutralizing BF.7 pseudovirus IC 50 Is 5 ng/mL, and the IC for neutralizing XBB.1.5 pseudovirus 50 IC of 2 ng/mL for neutralizing XBB.1.16 pseudovirus 50 Is 2 ng/mL. The results show that the bispecific antibody 4D12-H-2G10 has a more broad spectrum efficient neutralizing activity against different sub-variants of Omicron pseudoviruses than the parent monoclonal antibody.

Claims (10)

1. The fully human bispecific antibody of the novel coronavirus variant is characterized in that the bispecific antibody consists of two polypeptide chains and two monoclonal antibody light chains, wherein each polypeptide chain is formed by connecting a monoclonal antibody heavy chain and a single chain antibody in series, the monoclonal antibody heavy chain and the monoclonal antibody light chain are from the same parent monoclonal antibody, the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the monoclonal antibody heavy chain are respectively shown as 26-33, 51-58 and 97-114 of SEQ ID NO.1, the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the monoclonal antibody light chain are respectively shown as 27-32, 50-52 and 89-96 of SEQ ID NO.3, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the single chain antibody are respectively shown as 495-502, 520-527 and 566 of the variable region of SEQ ID NO.1, and the amino acid sequences of CDR 586-66 and amino acid sequences of the variable region of the monoclonal antibody light chain are respectively shown as 495-66-52 and 89-96 of the single chain antibody light chain is shown as amino acid sequences of SEQ ID NO.1 and 586-645.
2. The specific antibody according to claim 1, wherein the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is shown in amino acid sequences 1 to 125 of SEQ ID NO.1, the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown in amino acid sequences 1 to 107 of SEQ ID NO.3, the amino acid sequence of the variable region of the heavy chain of the single chain antibody is shown in amino acid sequences 470 to 597 of SEQ ID NO.1, and the amino acid sequence of the variable region of the light chain of the single chain antibody is shown in amino acid sequences 612 to 722 of SEQ ID NO. 1.
3. The bispecific antibody of claim 1, wherein the constant region amino acid sequence of the heavy chain of the monoclonal antibody is shown in amino acid sequences 126-455 of SEQ ID No.1, and the constant region amino acid sequence of the light chain of the monoclonal antibody is shown in amino acid sequences 108-213 of SEQ ID No. 3.
4. The bispecific antibody of claim 3, wherein the variable region of the single chain antibody heavy chain and the variable region of the single chain antibody light chain are linked by a flexible linker peptide between the monoclonal antibody heavy chain and the single chain antibody.
5. The bispecific antibody of claim 5, wherein the amino acid sequence of the flexible linker peptide is as depicted in amino acid sequence 456-469.
6. The bispecific antibody of claim 5, wherein the amino acid sequence of the polypeptide chain is shown in SEQ ID No.1 and the amino acid sequence of the monoclonal antibody light chain is shown in SEQ ID No. 3.
7. A polynucleotide encoding the bispecific antibody of claim 6, wherein the polynucleotide of the polypeptide chain has the sequence shown in SEQ ID No.2 and the polynucleotide of the monoclonal antibody light chain has the sequence shown in SEQ ID No. 4.
8. An expression vector comprising the polynucleotide of claim 7.
9. A host cell comprising the expression vector of claim 8.
10. Use of a bispecific antibody according to any one of claims 1-6 for the preparation of a novel coronavirus therapeutic.
CN202310792691.2A 2023-06-30 Fully human bispecific antibody for neutralizing novel coronavirus Omicron variant Active CN116813789B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310792691.2A CN116813789B (en) 2023-06-30 Fully human bispecific antibody for neutralizing novel coronavirus Omicron variant

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310792691.2A CN116813789B (en) 2023-06-30 Fully human bispecific antibody for neutralizing novel coronavirus Omicron variant

Publications (2)

Publication Number Publication Date
CN116813789A true CN116813789A (en) 2023-09-29
CN116813789B CN116813789B (en) 2024-11-12

Family

ID=

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113735969A (en) * 2021-09-20 2021-12-03 中国人民解放军军事科学院军事医学研究院 Fully human anti-new coronavirus broad-spectrum high-neutralization-activity monoclonal antibody and application thereof
CN114031685A (en) * 2022-01-10 2022-02-11 中国人民解放军军事科学院军事医学研究院 Fully human anti-new coronavirus broad-spectrum neutralizing antibody ZW2G10 and application thereof
WO2022148374A1 (en) * 2021-01-05 2022-07-14 中国科学院分子细胞科学卓越创新中心 Fully human broad-spectrum neutralizing antibody 76e1 against coronavirus, and use thereof
CN115850465A (en) * 2022-12-30 2023-03-28 中国人民解放军军事科学院军事医学研究院 Fully human-derived new coronavirus variant potent neutralizing antibody H4D12 and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022148374A1 (en) * 2021-01-05 2022-07-14 中国科学院分子细胞科学卓越创新中心 Fully human broad-spectrum neutralizing antibody 76e1 against coronavirus, and use thereof
CN113735969A (en) * 2021-09-20 2021-12-03 中国人民解放军军事科学院军事医学研究院 Fully human anti-new coronavirus broad-spectrum high-neutralization-activity monoclonal antibody and application thereof
CN114031685A (en) * 2022-01-10 2022-02-11 中国人民解放军军事科学院军事医学研究院 Fully human anti-new coronavirus broad-spectrum neutralizing antibody ZW2G10 and application thereof
CN115850465A (en) * 2022-12-30 2023-03-28 中国人民解放军军事科学院军事医学研究院 Fully human-derived new coronavirus variant potent neutralizing antibody H4D12 and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DAVID J. SULLIVAN: "Analysis of anti-SARS-CoV-2 Omicronneutralizing antibody titers in different vaccinated and unvaccinated convalescent plasma sources", 《NATURE COMMUNICATIONS》, vol. 13, 29 October 2022 (2022-10-29), pages 1 - 11, XP093117804, DOI: 10.1038/s41467-022-33864-y *
徐偲瑜: "新型冠状病毒疫苗及抗体药物研发进展", 《生物医学工程学杂志》, vol. 39, no. 5, 31 October 2022 (2022-10-31), pages 1059 - 1064 *

Similar Documents

Publication Publication Date Title
CN111690059B (en) Monoclonal antibody 1D7 for resisting SARS-CoV-2
AU2017237543B2 (en) Antibody that binds to envelope glycoprotein of severe fever with thrombocytopenia syndrome virus, and use for same
CN111732654B (en) Monoclonal antibody 1E10 for resisting SARS-CoV-2
RU2013125459A (en) ANTIBODIES AGAINST c-Met RECEPTOR PROTEIN
CN112521494B (en) Monoclonal antibody 2B11 for resisting SARS-CoV-2
US20170327570A1 (en) ANTI-TNF-alpha FULLY HUMAN MONOCLONAL ANTIBODIES WITH LOW IMMUNOGENICITY AND APPLICATION THEREOF
CN115991778A (en) anti-PD-L1 antibodies and uses thereof
CN106397598A (en) Expression and preparation methods for polyvalent multi-specific antibody and immune hybrid protein
WO2017011413A1 (en) Bispecific molecules comprising an hiv-1 envelope targeting arm
CN115093477A (en) Monoclonal antibody for resisting N terminal region of novel coronavirus nucleoprotein and application thereof
CN114751987A (en) Multispecific antibody for neutralizing coronavirus
CN116813789B (en) Fully human bispecific antibody for neutralizing novel coronavirus Omicron variant
CN112961250A (en) Antibody fusion protein and application thereof
CN116813789A (en) Fully human bispecific antibody for broad spectrum neutralization of novel coronavirus Omicron subvariant
CN116891531B (en) Fully human bispecific antibody for resisting novel coronavirus variant
CN107880117A (en) Identify the monoclonal antibody and purposes of viruses of human hepatitis B's X protein
CN113817051B (en) Monoclonal antibody 1B6 against SARS-CoV-2
WO2023071953A1 (en) Anti-cd26 antibody and use thereof
CN116891531A (en) Fully human bispecific antibody for resisting novel coronavirus variant
CN116987197A (en) Covalent multispecific antibodies
CN115160435A (en) Bispecific anti-HIV-1 antibody
CN114409766A (en) Neutralizing antibody of EB virus and application thereof
CN113817050B (en) Monoclonal antibody 1H8 against SARS-CoV-2
CN116715769B (en) Anti-HVEM antibody, preparation method and application thereof
CN113880947B (en) Small molecule antibody, coding gene thereof, preparation method and application thereof, and pharmaceutical composition

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant