WO2022105772A1

WO2022105772A1 - Bispecific antibody having neutralizing activity against coronavirus, and use thereof

Info

Publication number: WO2022105772A1
Application number: PCT/CN2021/131080
Authority: WO
Inventors: 郎国竣; 刘婵娟; 邵俊斌; 谭永聪; 孔超; 闫闰; 闫鑫甜; 胡宇豪
Original assignee: 三优生物医药(上海)有限公司; 上海之江生物科技股份有限公司
Priority date: 2020-11-18
Filing date: 2021-11-17
Publication date: 2022-05-27

Abstract

Provided are a bispecific antibody having a neutralizing activity against a coronavirus, and a use thereof. A neutralizing antibody which is against a coronavirus S protein and blocks the binding of the coronavirus S proteins to an ACE 2 receptor is provided to prevent and treat coronaviruses. Specifically provided is an antibody or polypeptide complex specifically binding to a coronavirus S protein, comprising: (a) a first epitope binding moiety comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH and VL form an antigen binding domain specifically binding to a first epitope of an S protein; and (b) a second epitope binding moiety comprising a single-domain antibody or a VHH fragment thereof specifically binding to a second epitope of the S protein, wherein the first and second epitope binding portions are fused to each other and are not identical to each other. Also provided is a polynucleotide for encoding an antibody or a polypeptide complex and a host cell comprising same, and a method for preparing an antibody or a polypeptide complex. The antibody or polypeptide complex can be used for preventing, treating, diagnosing, and/or detecting coronaviruses.

Description

Bispecific antibodies with neutralizing activity against coronaviruses and uses thereof

This application claims Chinese Application No. 202011300574.2, filed on Nov. 18, 2020, and entitled "Bispecific antibodies with neutralizing activity against coronaviruses and uses thereof" and filed on May 11, 2021, and entitled "For coronavirus Priority of Chinese Application No. 202110512951.7 of "Bispecific Antibody with Virus Neutralizing Activity and Its Use", the content of the above application is incorporated herein by reference in its entirety.

technical field

The present disclosure generally relates to antibodies and uses thereof. More specifically, the present disclosure relates to bispecific antibodies that specifically recognize the coronavirus spike protein, methods of making and uses thereof.

Background technique

Since the outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, coronavirus (CoV) has emerged as an RNA virus that has caused major public health problems. Since the beginning of 2020, the world has focused on the 2019 novel coronavirus (also referred to in this disclosure as "new coronavirus", "2019-nCoV" or "SARS-CoV-2"). The virus can spread from person to person, and patients infected with the virus can present with severe viral pneumonia and respiratory disease.

Due to the continuous mutation of the virus, it is more difficult to expect to obtain herd immunity through vaccines. The re-infection of the new coronavirus in Manaus, Brazil, demonstrates the difficulty of obtaining herd immunity. As early as September 2020, after more than 7 months of the new crown virus raging, the infection rate of the new crown virus in Manaus reached 76%, which is considered to have reached the infection rate of herd immunity. However, in January 2021, the new coronavirus in Manaus was infected again on a large scale, and the strain infected this time was the mutant strain P.1. Scientists speculate that the mutant strain P.1 is more infectious than the ordinary new coronavirus. 50% (Three-quarters attack rate of SARS-CoV-2 in the Brazilian Amazon during a largely unmitigated epidemic, Science 2021Jan 15;371(6526):288-292.doi:10.1126/science.abe9728.).

After more than a year of hard work, a number of vaccines against SARS-CoV-2 have been marketed around the world, such as those developed by Regeneron Pharmaceuticals Inc. (hereinafter referred to as "Regeneron") and Eli Lilly and Company (hereinafter referred to as "Eli Lilly"). Therapeutic monoclonal antibodies are also FDA-approved for emergency use. The new problem faced at this stage is that SARS-CoV-2 is constantly mutating. Due to the decline or failure of vaccine protection caused by virus mutation, and the limited viral epitopes bound by monoclonal antibodies, virus mutants escape and lose neutralization. Effect.

A study published in January 2021 aimed at the SARS-CoV-2 mutant strain B.1.1.7 (hereinafter referred to as "B.1.1.7 mutant strain") that appeared in the United Kingdom and the SARS-CoV-2 mutant strain B. 1.351 (hereinafter referred to as "B.1.351 mutant strain") research shows that the LY-CoV555 and CB6 mAbs developed by Eli Lilly and Shanghai Junshi Biomedical Technology Co., Ltd. (hereinafter referred to as "Junshi") have lost their ability to target B. The neutralizing activity of the 1.351 mutant strain, the monoclonal antibody cocktail developed by Regeneron and Brii Biosciences also decreased the neutralizing activity against the B.1.351 mutant strain by 5-10 times; the sera after immunization of the vaccine developed by Moderna and Pfizer were directed against B. The neutralizing activity of the .1.351 mutant was also decreased by more than 10 times (Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization, bioRxiv 2021 Jan 26; 2021.01.25.428137.doi:10.1101/ 2021.01.25.428137.). Recently, the B.1.617.2 mutant (Delta), which contains mutations in the S1 subunit of the S protein and the Furin protease cleavage site, originally discovered in India, has quickly become a major global drug due to its strong transmission and high viral load. Popular new coronavirus mutants.

Therefore, in view of the constant mutation of SARS-CoV-2 virus, as well as the problems of drug resistance escape, vaccine protection decline or loss that may be caused by mutation, it is of great value to develop drugs that still have therapeutic effects against a variety of mutant strains. In particular, there is an urgent need for patients (especially critically ill patients) who are simultaneously infected with multiple SARS-CoV-2 mutant strains.

SUMMARY OF THE INVENTION

The present disclosure provides a bispecific antibody targeting the new coronavirus spike protein (also known as "Spike protein" or "S protein"), which can effectively target the receptor binding domain of the SARS-CoV-2 virus Spike protein (RBD) mutated various SARS-CoV-2 mutants, widely neutralize various mutants in nature and prevent mutants from escaping, while blocking the binding of Spike protein to its receptor ACE2, effectively neutralizing the new coronavirus and preventing its Infect invading cells.

In a first aspect, the present disclosure provides a polypeptide complex that specifically binds to the coronavirus S protein, the polypeptide complex comprising: (a) a first epitope binding moiety that specifically binds to the coronavirus S protein and (b) a second epitope binding portion comprising a single domain antibody or a VHH fragment thereof that specifically binds to the second epitope of the coronavirus S protein; wherein The first epitope binding portion and the second epitope binding portion are fused to each other, and wherein the first epitope is different from the second epitope.

In some embodiments, the first epitope binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL together form the first epitope that specifically binds to the coronavirus S protein The antigen-binding site of an epitope. In some embodiments, the first epitope binding moiety and the second epitope binding moiety do not compete for epitopes with each other. In some embodiments, the first epitope binding portion comprises a human, humanized, or chimeric antibody or antigen-binding fragment thereof.

In some embodiments, the first epitope binding moiety or VH comprises:

The first heavy chain CDR1 (HCDR1) in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes, the heavy chain variable region shown in SEQ ID NO: 1 The first heavy chain CDR2 (HCDR2) in the amino acid sequence or a variant thereof with no more than 2 amino acid changes, and the first heavy chain CDR3 (HCDR3) in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes.

In some embodiments, the first epitope binding moiety or VL comprises: light chain CDR1 (LCDR1) in the light chain variable region amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof with no more than 2 amino acid changes , the light chain CDR2 (LCDR2) in the light chain variable region amino acid sequence shown in SEQ ID NO:2 or a variant thereof with no more than 2 amino acid changes, and the light chain variable region shown in SEQ ID NO:2 The light chain CDR3 (LCDR3) in the amino acid sequence or a variant thereof with no more than 2 amino acid changes.

In some embodiments, the first HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8; the first HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9; the first HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; the LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; the LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12 and/or the LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.

In some embodiments, the VH comprises or consists of the sequence set forth in SEQ ID NO: 1 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, A sequence of 97%, 98% or 99% identity, and/or the VL comprises or consists of the following sequence: the sequence shown in SEQ ID NO: 2 or has at least 90%, 91%, 92%, 93% therewith , 94%, 95%, 96%, 97%, 98% or 99% identical sequences.

In some embodiments, the first epitope binding portion comprises a heavy chain comprising the VH and a light chain comprising the VL, and wherein the heavy chain comprises or consists of the sequence set forth in SEQ ID NO:22 the sequence shown or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown; and/or the light chain comprises or Consists of the sequence set forth in SEQ ID NO: 23 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:23 sequence.

In some embodiments, the single domain antibody comprises or consists of a VHH fragment.

In some embodiments, the single domain antibody comprises: the second heavy chain CDR1 (HCDR1) in the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7, or a variant thereof with no more than 2 amino acid changes; The second heavy chain CDR2 (HCDR2) in the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7, or a variant thereof with no more than 2 amino acid changes; and set forth in SEQ ID NO: 5, 6 or 7 The second heavy chain CDR3 (HCDR3) in the VHH amino acid sequence or a variant thereof with no more than 2 amino acid changes.

In some embodiments, the single domain antibody comprises: the second heavy chain CDR1, the second heavy chain CDR2 and the second heavy chain CDR3 in the VHH amino acid sequence shown in SEQ ID NO:5; The second heavy chain CDR1, the second heavy chain CDR2 and the second heavy chain CDR3 in the VHH amino acid sequence shown in SEQ ID NO: 7; or the second heavy chain CDR1, the second heavy chain CDR2 in the VHH amino acid sequence shown in SEQ ID NO: 7 and the second heavy chain CDR3.

In some embodiments, the second HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14, 18 or 20; the second HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 or 21 and/or the second HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, 17 or 19.

In some embodiments, the second HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14; the second HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15; and the second HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15; The two HCDR3s comprise or consist of the amino acid sequence shown in SEQ ID NO:17. In some embodiments, the second HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18; the second HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15; and the first HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15; The two HCDR3s comprise or consist of the amino acid sequence shown in SEQ ID NO:19. In some other embodiments, the second HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; the second HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21; and the The second HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

In some embodiments, the single domain antibody or VHH fragment thereof comprises or consists of the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7 or at least 90%, 91%, 92%, Sequences of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In some embodiments, the N-terminus of the second epitope binding moiety is fused to the C-terminus of at least one heavy chain of the first epitope binding moiety; the N-terminus of the second epitope binding moiety fused to the C-terminus of at least one light chain of the first epitope binding moiety; the C-terminus of the second epitope binding moiety to the N-terminus of at least one heavy chain of the first epitope binding moiety fusion; the C-terminus of the second epitope binding moiety is fused to the N-terminus of at least one light chain of the first epitope binding moiety; and/or the second epitope binding moiety comprises at least 2 identical or different VHH fragments fused in tandem to the first epitope binding moiety or separately to the first epitope binding moiety.

In some embodiments, the first epitope binding portion comprises an Fc region. In some embodiments, the Fc region is an IgGl Fc or an IgG4 Fc. In some embodiments, the Fc region is an IgG1 Fc with L234A and L235A or an IgG4 Fc with the S228P mutation.

In some embodiments, the first epitope binding moiety and the second epitope binding moiety are fused to each other via a peptide bond or peptide linker. In some embodiments, the peptide linker has a length of no more than about 30 amino acids. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-45.

In some embodiments, the single domain antibody is a camelid single domain antibody or a humanized single domain antibody.

In some embodiments, the polypeptide complex comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence set forth in SEQ ID NO: 29, 30 or 31 , 97%, 98% or 99% identical sequences.

In some embodiments, the polypeptide complex is a bispecific antibody complex consisting of 2 heavy chains and 2 light chains, the heavy chains consisting of SEQ ID NO: 29, 30 or 31, and the light chain consists of the amino acid sequence shown in SEQ ID NO: 23.

In one aspect, the present disclosure also provides an antibody that specifically binds to the coronavirus S protein, which can be used alone to bind and inhibit the coronavirus S protein, or as an epitope binding part of the polypeptide complex of the present disclosure, As described in detail below.

In one aspect, the present disclosure provides an isolated polynucleotide encoding the antibody or polypeptide complex of the present disclosure. In some embodiments, the polynucleotides of the present disclosure include the sequence set forth in SEQ ID NO:33 and/or the sequence set forth in SEQ ID NO:39, 40, or 41.

In one aspect, the present disclosure provides an isolated vector comprising a polynucleotide as described in the present disclosure.

In one aspect, the present disclosure provides a host cell comprising a polynucleotide or vector as described in the present disclosure.

In one aspect, the present disclosure provides a method of expressing an antibody or polypeptide complex of the present disclosure, the method comprising culturing a host cell of the present disclosure under conditions suitable for expressing the antibody or polypeptide complex , and optionally the antibody or polypeptide complex is recovered from the host cell or from the culture medium.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or polypeptide complex of the present disclosure and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a detection kit comprising the antibody or polypeptide complex of the present disclosure.

In one aspect, the present disclosure provides a method of treating and/or preventing a coronavirus-related disease, eg, a coronavirus infection such as COVID-19, comprising administering to a subject in need thereof an effective amount of an antibody or polypeptide complex of the present disclosure thing.

In one aspect, this document also relates to the use of the antibody or polypeptide complex of the present disclosure in the manufacture of a medicament for the treatment and/or prevention of coronavirus infection in a subject. In some embodiments, the coronavirus is the SARS-CoV-2 virus and the coronavirus infection is COVID-19. In a specific embodiment, the coronavirus is a SARS-CoV-2 mutant.

In one aspect, the present disclosure also relates to the use of the antibody or polypeptide complex of the present disclosure in the preparation of a diagnostic agent or kit for detecting coronavirus or diagnosing coronavirus infection. In some embodiments, the coronavirus is the SARS-CoV-2 virus and the coronavirus infection is COVID-19.

In one aspect, the present disclosure also relates to an antibody or polypeptide complex of the present disclosure for use in the treatment and/or prevention of a coronavirus-related disease, eg, a coronavirus infection such as COVID-19.

In one aspect, the present disclosure provides an in vitro method for detecting coronavirus contamination in the environment, comprising: providing an environmental sample; complexing the environmental sample with the antibody or polypeptide described in the present disclosure or the detection described in the present disclosure kit contacting; and detecting the formation of the complex between the antibody or polypeptide complex described in the present disclosure and the coronavirus S protein. In some embodiments, the coronavirus is the SARS-CoV-2 virus.

In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to the coronavirus S protein, comprising:

(a) heavy chain variable region (VH) and light chain variable region (VL), wherein the VH comprises: the first heavy chain CDR1 in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 ( HCDR1) or its variant with no more than 2 amino acid changes, the first heavy chain CDR2 (HCDR2) in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or its variant with no more than 2 amino acid changes , and the first heavy chain CDR3 (HCDR3) in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes; and wherein the VL comprises: SEQ ID NO: The light chain CDR1 (LCDR1) in the light chain variable region amino acid sequence shown in 2 or its variant with no more than 2 amino acid changes, the light chain in the light chain variable region amino acid sequence shown in SEQ ID NO: 2 CDR2 (LCDR2) or its variants with no more than 2 amino acid changes, and light chain CDR3 (LCDR3) or its variants with no more than 2 amino acid changes in the light chain variable region amino acid sequence shown in SEQ ID NO: 2 body; or

(b) a VHH fragment comprising: the second heavy chain CDR1 (HCDR1) in the VHH amino acid sequence shown in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes; SEQ The second heavy chain CDR2 (HCDR2) in the VHH amino acid sequence set forth in ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes; and the VHH set forth in SEQ ID NO: 5, 6 or 7 The second heavy chain CDR3 (HCDR3) in the amino acid sequence or a variant thereof with no more than 2 amino acid changes.

In a specific embodiment, the antibody is a monoclonal antibody, and its heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:2 amino acid sequence composition.

In some embodiments, the VHH fragment comprises: a second HCDR1 of the amino acid sequence shown in the formula GRFFGSYX ₁ MS, wherein X ₁ is Y, T or V; a second HCDR1 of the amino acid sequence shown in the formula DINTRGX ₂ X ₃ TR A _second HCDR2, wherein X2 is E or I, and _X3 is T or V _; and a second HCDR3 of the amino acid sequence shown in the _formula _{AASX4X5TFX6GRSDPDY} , wherein _X4 is G or P, _X5 is D or _A , and X6 is E or F.

In a specific embodiment, the antibody is a Nanobody, and its VHH fragment consists of the VHH amino acid sequence shown in SEQ ID NO:5.

Description of drawings

Figure 1 depicts the experimental flow of the present disclosure for the production and activity detection of fully human antibodies, nanobodies and bi-epitope bispecific antibodies targeting the S protein of SARS-CoV-2 (2019-nCoV) coronavirus.

Figure 2A shows the binding activity of human ACE2-huFc to S protein RBD-mFc.

Figure 2B shows the binding activity of human ACE2-His to S protein S1-huFc (also called Spike S1-huFc) or S protein RBD-mFc (also called Spike RBD-mFc).

Figure 3A shows the binding ability of the phage outputted by the antibody in the first and second rounds of panning to the S protein RBD-mFc in an ELISA assay, using the VSCM13 helper phage as a negative control.

Figure 3B shows the binding ability of the phage exported by the antibodies in the second and third rounds of panning to the S protein RBD-mFc in an ELISA assay.

Figure 4 shows the results of SDS-PAGE to identify the purity of the antibody after R15-F7 was prepared into a full-length antibody.

Figure 5 shows the results of HPLC-SEC identification of antibody monomer purity after R15-F7 was prepared into a full-length antibody.

Figures 6A-6B show the fluorescence curves and Tm values of the thermal stability of the R15-F7 antibody determined by differential scanning fluorescence (DSF).

Figure 7 shows that the affinity activity of R15-F7 antibody binding to S protein RBD-mFc was determined by ELISA method.

Figure 8A shows a graph of the binding pattern of the affinity activity of the R15-F7 antibody for binding to the S protein RBD-His determined by the Fortebio method.

Figure 8B shows a graph of the binding affinity of R15-F7 to the S protein RBD-His.

Figure 9 shows the results of blocking the binding of S protein RBD-His to receptor protein ACE2 by antibody R15-F7 detected by ELISA method.

Figure 10 shows that antibody R15-F7 blocks the binding of S protein RBD-mFc to ACE2-HEK293 cells detected by FACS method.

Figure 11A shows the binding capacity of the phage outputted by the antibody in the first and second rounds of panning to the S protein RBD-mFc in an ELISA assay, using the VSCM13 helper phage as a negative control.

Figure 11B shows the binding capacity of the phage exported by the antibodies in the second and third rounds of panning to the S protein RBD-mFc in an ELISA assay.

Figure 12 shows the results of SDS-PAGE to identify the purity of the antibody after P14-F8 was prepared into a full-length antibody (VHH-Fc).

Figure 13 shows the results of HPLC-SEC identification of antibody monomer purity after P14-F8 was prepared as a full-length antibody (VHH-Fc).

Figure 14 shows that the affinity activity of P14-F8 antibody binding to antigenic protein RBD was determined by ELISA method.

Figure 15 shows a graph of the binding affinity of P14-F8 to the S protein RBD-His detected by Fortebio.

Figure 16 shows the results of blocking the binding of S protein RBD-His to receptor protein ACE2 by antibody P14-F8 detected by ELISA method.

Figure 17 shows that the antibody P14-F8 blocks the binding of S protein RBD-mFc to ACE2-HEK293 cells by FACS method.

Figures 18A-18B show the determination of the affinity activity of P14-F8 and its humanized antibody binding to the antigenic protein RBD by ELISA method.

Figures 19A-19B show the effect of blocking the binding of the antigenic protein RBD to the receptor ACE2 by ELISA method.

Figure 20 shows that the binding activity of P14-F8-hVH8 and its affinity engineered preferred molecules to S protein RBD-mFc was determined by ELISA method.

Figure 21 shows that P14-F8-hVH8 and its affinity engineered preferred molecules block the binding of S protein RBD-His to ACE2 by ELISA method.

Figure 22 shows the binding activity of the double antibody and the corresponding monoclonal antibody to the S protein RBD-mFc measured by ELISA method.

Figure 23 shows that double antibody and corresponding monoclonal antibody block the binding of S protein RBD-His to ACE2 by ELISA method.

Figures 24A-24G show the determination of the affinity of the double antibody and the corresponding monoclonal antibody for S protein RBD-His by the Fortebio method, wherein Figure 24A is BsAb16, Figure 24B is BsAb17, Figure 24C is BsAb18, Figure 24D is R15-F7, Figure 24E It is P14-F8-43, Figure 24F is P14-F8-35, and Figure 24G is P14-F8-38.

Figures 25A-25C show the detection of double antibodies BsAb16, BsAb17 and BsAb18 and the corresponding monoclonal antibodies R15-F7, P14-F8-35, P14-F8-38, P14-F8-43, and monoclonal antibodies combined with R15-F7 by FACS method +P14-F8-35, R15-F7+P14-F8-38 and R15-F7+P14-F8-43 blocked the binding of S protein RBD-mFc to ACE2-HEK293 cells.

Figures 26A-26M show the detection of double antibodies BsAb16, BsAb17 and BsAb18 and the corresponding mAbs R15-F7, P14-F8-35, P14-F8-38, P14-F8-43, mAb combined with R15-F7 by FACS method The results of non-specific binding of +P14-F8-35, R15-F7+P14-F8-38 and R15-F7+P14-F8-43 to HEK293 cells, in which Figure 26A is a summary of the results, Figure 26B is BsAb16, Figure 26C is BsAb17, Fig. 26D is BsAb18, Fig. 26E is P14-F8-35, Fig. 26F is P14-F8-38, Fig. 26G is P14-F8-43, Fig. 26H is R15-F7, Fig. 26I is R15-F7+P14- F8-35, Figure 26J is R15-F7+P14-F8-38, Figure 26K is R15-F7+P14-F8-43, Figure 26L is an isotype control, and Figure 26M is a cell-only negative control.

Figures 27A-27M show the detection of double antibodies BsAb16, BsAb17 and BsAb18 and the corresponding mAbs R15-F7, P14-F8-35, P14-F8-38, P14-F8-43, mAb combined with R15-F7 by FACS method The results of non-specific binding of +P14-F8-35, R15-F7+P14-F8-38 and R15-F7+P14-F8-43 to Jurkat cells, in which Figure 27A is a summary of the results, Figure 27B is BsAb16, Figure 27C is BsAb17, Fig. 27D is BsAb18, Fig. 27E is P14-F8-35, Fig. 27F is P14-F8-38, Fig. 27G is P14-F8-43, Fig. 27H is R15-F7, Fig. 27I is R15-F7+P14- F8-35, Figure 27J is R15-F7+P14-F8-38, Figure 27K is R15-F7+P14-F8-43, Figure 27L is an isotype control, and Figure 27M is a cell-only negative control.

Figures 28A-28B show the neutralization effect of antibody samples BsAb16, BsAb17, BsAb18, R15-F7 and P14-F8 in neutralizing 2019-nCoV coronavirus, wherein Figure 28A is a pseudovirus neutralization test, and Figure 28B is a true virus neutralization test.

Figures 29A-29F show the ELISA binding activities of double anti-BsAb17 and 39 Spike-RBD or Spike-S1 proteins, with the Spike-RBD wild-type protein as a positive control.

Figures 30A-30F show the ELISA binding activities of mAb R15-F7 and 39 Spike-RBD or Spike-S1 proteins, with the Spike-RBD wild-type protein as a positive control.

Figures 31A-31F show the ELISA binding activities of mAb P14-F8-35 and 39 Spike-RBD or Spike-S1 proteins, with the Spike-RBD wild-type protein as a positive control.

Figures 32A-32C show the effects of double antibody BsAb17, mAb P14-F8-35 and mAb R15-F7 on wild-type SARS-CoV-2 ("131-P3") and South African epidemic mutant ("NF"), respectively neutralizing activity.

Figure 33 shows a schematic diagram of dual-antibody prophylaxis in SARS-CoV-2-infected mice.

Figure 34 shows the survival curve of mice in the administration prophylactic treatment group ("BsAb17P") and the PBS control group ("PBS") after infection with SARS-CoV-2 virus.

Figures 35A-35B show the number of virus copies in the lung (Figure 35A) and bronchial tissue (Figure 35B) of mice in the prophylactic treatment group ("BsAb17P") and the PBS control group.

Figures 36A-36B show the determination of viral load of B.1.351 mutant and mouse-adapted strains in the lung tissue of mice in the antibody prevention + treatment group, the antibody treatment group and the control group.

Figure 37 shows the results of HE staining in the lung tissue of the antibody-treated mice.

Figure 38 shows the results of RNAscope detection of antibody-treated mouse lung tissue.

Detailed ways

While the disclosure may be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that demonstrate the principles of the disclosure. It should be emphasized that the present disclosure is not limited to the specific embodiments illustrated. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this disclosure, the following terms are defined below.

The term "about" when used in conjunction with a numerical value is meant to encompass that numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

The term "and/or" when used in conjunction with two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.

As used herein, the term "comprising" or "comprising" means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps. Herein, when the term "comprising" or "comprising" is used, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers or steps. For example, reference to an antibody variable region that "comprises" a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.

The term "coronaviruses (CoV)" herein refers to viruses belonging to the genus Betacoronavirus of the family Coronaviridae, and the virus particles are spherical or oval in shape and about 60-220 nm in diameter. The virus is a single-stranded positive-stranded RNA (+ssRNA) virus. Of several human pathogenic coronaviruses, most are associated with mild clinical symptoms (SuS, Wong G, Shi W et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends Microbiol 2016;24:490 –502), with two notable exceptions for coronaviruses: SARS-CoV, which caused more than 8,000 human infections and 774 deaths in 37 countries and territories between 2002 and 2003 (Chan-Yeung M, Xu RH. SARS: epidemiology. Respirology 2003; 8(suppl): S9–14); the other is the 2019 novel coronavirus (2019- nCoV), has a strong ability to spread in the population, most infected patients have high fever, some have difficulty breathing, and chest X-ray shows infiltrative lesions in both lungs. The World Health Organization (WHO) recently named the 2019-nCoV SARS-CoV-2. In this article, "2019-nCoV" and "SARS-CoV-2" are used interchangeably.

The term "antibody" is used herein in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) so long as they exhibit the desired antigen binding activity . Antibodies can be intact antibodies (eg, with two full-length light chains and two full-length heavy chain). The monomer of an intact antibody is a tetrapeptide chain molecule formed by connecting two full-length light chains and two full-length heavy chains by disulfide bonds, also known as the monomer of the Ig molecule. Antibody monomers are the basic structures that make up antibodies.

As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) with different antigenic specificities (eg, an isolated antibody or antigen-binding fragment thereof that specifically binds coronavirus S protein is substantially free of specificity Abs that bind to antigens other than the coronavirus S protein). In certain embodiments, the antibody is purified to greater than 95% or 99% purity by, eg, electrophoresis (eg, SDS-PAGE, electrofocusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reaction phase HPLC) to determine.

As used herein, "blocking antibody," "neutralizing antibody," "antibody having neutralizing activity," or "neutralizing antibody" are used interchangeably herein to refer to an antibody that binds to a target antigen or Interacts therewith and prevents the target antigen from binding or associating with a binding partner such as a receptor, thereby inhibiting or blocking biological responses that would otherwise result from the interaction of the target antigen with a binding partner such as a receptor. In the context of the present disclosure, it is meant that the binding of the antibody to the coronavirus S protein results in the inhibition of at least one biological activity of the coronavirus. For example, the neutralizing antibodies of the present disclosure can prevent or block the binding of the coronavirus S protein to ACE2.

An "epitope" or "antigenic determinant" refers to an antigenic determinant that interacts with a specific antigen-binding site called a paratope in the variable region of an antibody molecule. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be formed from contiguous amino acids or discontinuous amino acids joined by the tertiary fold of the protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding typically disappear upon treatment with denaturing solvents. Epitopes typically include at least 3, and more typically at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

The term "antigen-binding fragment" is a portion or segment of an intact or complete antibody having fewer amino acid residues than an intact or complete antibody, which is capable of binding an antigen or competing with an intact antibody (ie, with the intact antibody from which the antigen-binding fragment is derived) bind antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv, single-chain Fv (scFv), single-chain Fab, diabody, single domain antibody or single domain antibody (sdAb, Nanobodies), camelid Ig, Ig NAR, F(ab)' ₃ fragments, bis-scFv, (scFv) ₂ , minibodies, diabodies, tribodies, tetrabodies, disulfide stabilized Fv proteins ("dsFv"). The term also includes genetically engineered forms such as chimeric antibodies (eg, humanized murine antibodies), hybrid antibodies (eg, bispecific antibodies), and antigen-binding fragments thereof. For a more detailed description see also: Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Journal of Immunology, 3rd Edition, WH Freeman & Co., New York, 1997.

The terms "whole antibody", "full length antibody", "complete antibody" and "intact antibody" are used interchangeably herein to refer to a composition comprising at least two heavy chains (HC) interconnected by disulfide bonds and two Light chain (LC) glycoprotein. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Heavy chain constant regions typically consist of 3 (for gamma, alpha and delta: CH1, CH2 and CH3) or 4 domains (for mu and epsilon: CH1, CH2, CH3 and CH4). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region consists of one domain, CL. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma and mu. Mammalian light chains are classified as lambda or kappa. Immunoglobulins comprising alpha, delta, epsilon, gamma and mu heavy chains are classified as immunoglobulin (Ig)A, IgD, IgE, IgG and IgM. Complete antibodies form a "Y" shape. The stem of Y consists of the second and third constant regions of the two heavy chains (and, for IgE and IgM, the fourth constant region) held together, and a disulfide bond (interchain) is formed in the hinge. Heavy chains gamma, alpha, and delta have a constant region consisting of three tandem (in a row) Ig domains, and a hinge region for increased flexibility; heavy chains mu and epsilon have a constant region consisting of four immunoglobulin domains Area. The second and third constant regions are referred to as "CH2 domain" and "CH3 domain", respectively. Each arm of Y includes the variable and first constant regions of a single heavy chain associated with the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

The light and heavy chain variable regions, respectively, comprise "framework" regions interspersed with three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). "Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" (used interchangeably herein with hypervariable regions "HVR"), are the variable domains of antibodies that are hypervariable in sequence and Structurally defined loops ("hypervariable loops") and/or regions containing antigen-contacting residues ("antigen contact points") are formed. The CDRs are mainly responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable domains of antibody heavy chains are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology , 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr/), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures.

However, it should be noted that the CDR boundaries of the variable regions of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. For example, the residue ranges defined by different assignment systems for CDR regions using Kabat and Chothia numbering are shown in Table A below.

Table A. CDR Residue Ranges Defined by Different Assignment Systems

Thus, when referring to the definition of an antibody with a specific CDR sequence as defined in the present disclosure, the scope of the antibody also encompasses antibodies whose variable region sequences comprise the specific CDR sequences, but due to the application of a different scheme (e.g. different assignment system rules or combinations), resulting in their claimed CDR boundaries being different from the specific CDR boundaries defined in this disclosure.

The CDRs of the disclosed antibodies can be manually evaluated to determine boundaries according to any protocol in the art or a combination thereof. Unless otherwise stated, in this disclosure, the term "CDR" or "CDR sequence" encompasses CDR sequences identified in any of the ways described above.

The sequences of the framework regions of different light or heavy chains are relatively conserved within species (eg, humans). The framework regions of antibodies, which are the combined framework regions of the component light and heavy chains, are used to locate and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to epitopes of the antigen. Antibodies with different specificities (ie, different combining sites for different antigens) have different CDRs. Although the CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, eg, by making hybrid antibody-forming cells from fusions of myeloma cells and immune splenocytes. Monoclonal antibodies include humanized monoclonal antibodies.

"Fv" is the smallest antibody fragment containing the complete antigen-binding site. In one example, a double-chain Fv species consists of dimers of one heavy chain variable domain and one light chain variable domain in tight non-covalent association. In the single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker, so that the light and heavy chains can be linked in a manner similar to that of the double-chain Fv species. "Dimeric" structural associations. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. The six HVRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for the antigen) has the ability to recognize and bind antigen, but with lower affinity than the entire binding site.

A Fab fragment contains a heavy chain variable domain and a light chain variable domain and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that several residues are added to the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residues of the constant domains carry free thiol groups. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

The term "specifically binds" or "binding" when referring to antigens/epitopes and antibodies means that the antibody forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen/epitope are well known in the art and include, for example, surface plasmon resonance assays, MSD assays (Estep, P. et al., High throughput solution-based measurement of antibody- antigen affinity and epitope binning, MAbs, 2013.5(2): p.270-278), ForteBio affinity assay (Estep, P et al., High throughput solution based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2) :p.270-8) etc.

In one embodiment, an antibody of the present disclosure that "specifically binds" the coronavirus S protein, eg, a bispecific antibody, as measured in a ForteBio affinity assay, is at least about ^10-8 M, preferably ^10-9 M; more preferably 10 A KD of ^-10 M, further preferably 10 ^-11 M, more preferably 10 ^-12 M binds to the S protein, thereby blocking or inhibiting the binding of the coronavirus S protein to its receptor ACE2 and subsequent membrane fusion.

"Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the binding dissociation equilibrium constant (KD). Affinity can be measured by common methods known in the art, including those known in the art and described herein.

An "affinity matured" antibody is an antibody that has one or more changes in one or more of its HVRs that result in improved affinity of the antibody for the antigen compared to the parent antibody that does not have those changes. In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are prepared by procedures known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describe affinity maturation by mixing VH- and VL-domains. Random mutagenesis of HVR and/or framework residues is described, for example, by Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889 -896 (1992).

When the term "compete" is used in the context of antigen-binding proteins competing for the same epitope (eg, neutralizing antigen-binding proteins or neutralizing antibodies), it means competition between antigen-binding proteins, as determined by the following assay: In the assay, the antigen-binding protein (eg, antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (eg, reduces) the interaction of a reference antigen-binding protein (eg, ligand or reference antibody) with a common antigen (eg, S protein or specific binding of its fragments). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), Sandwich competition assay (see, eg, Stahli et al., 1983, Methods in Enzymology 9:242-253). Typically the assay involves the use of purified antigen bound to a solid surface or cell with either an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the antigen binding protein being tested. Typically the antigen binding protein being tested is present in excess. Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to adjacent epitopes sufficiently close to the binding epitope of the reference antigen-binding protein protein, the two epitopes sterically prevent each other from binding. Additional details regarding methods for determining competitive binding are provided in the Examples herein.

In one embodiment, whether there will be epitope competition between two antibodies can be determined according to the following rules:

Competition percentage=signal value of experimental group/signal value of control group×%

Signal value of the experimental group: Add antibody 1 after immobilizing the antigen, add antibody 2 after the binding of antibody 1 is balanced, and observe the signal value generated by the addition of antibody 2; control group signal value: After immobilizing the antigen, add buffer, buffer and experimental group antibody 1 The incubation time is the same, then add antibody 2, and observe the signal value generated by the addition of antibody 2; among them, "competition percentage < 20%" means "epitope complete competition"; "20% < competition percentage < 60%" means "epitope Partial competition"; "Competition percentage>60%" means "no competition for the epitope at all".

As used herein, the term "variant" refers to a polypeptide or fragment such as a heavy chain variable region or a light chain variable region having at least one,

eg

1, 2 or 3 amino acid changes eg amino acid substitutions, deletions or additions. Modified antigen/epitope binding polypeptides/portions comprising heavy chain or light chain variants substantially retain the biological characteristics of the pre-modified antigen/epitope binding polypeptides/portions. In one embodiment, the antigen/epitope binding polypeptide/portion containing the variant heavy chain variable region or light chain variable region sequence retains 60%, 70%, 80%, 90%, 100% of the antigen binding polypeptide before modification or above biological characteristics. It will be appreciated that each heavy chain variable region or light chain variable region can be modified alone or in combination with another heavy chain variable region or light chain variable region. In one embodiment, the antigen/epitope binding polypeptide/portion of the present disclosure comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94% of the heavy chain variable region amino acid sequence described herein , 95%, 96%, 97%, 98%, or 99% homologous heavy chain variable region amino acid sequences. In one embodiment, the antigen/epitope binding polypeptide/portion of the present disclosure comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94% of the light chain variable region amino acid sequence described herein , 95%, 96%, 97%, 98%, or 99% homologous light chain variable region amino acid sequences. In one embodiment, the antigen/epitope binding polypeptide/portion of the present disclosure comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of a single domain antibody VHH fragment described herein , 95%, 96%, 97%, 98%, or 99% homologous single domain antibody VHH fragments. The percent homology can be over the entire heavy chain variable region and/or the entire light chain variable region, or the percent homology can be limited to the framework regions, while the sequences corresponding to the CDRs are the same as the heavy chain variable region and/or the light chain variable region. The CDRs disclosed herein within the variable regions and/or VHH fragments are 100% identical.

As used herein, the term "CDR variant" refers to a CDR having at least one, eg, 1, 2 or 3 amino acid changes, eg, substitutions, deletions or additions, comprising modified antigen/epitope binding polypeptides/ The portion substantially retains the biological characteristics of the antigen/epitope binding polypeptide/portion prior to modification. In one embodiment, the antigen/epitope binding polypeptide/portion containing the variant CDRs retains at least 60%, 70%, 80%, 90%, 100% or more of the organisms of the antigen/epitope binding polypeptide/portion prior to modification academic characteristics. It is understood that each CDR that can be modified can be modified alone or in combination with another CDR. In one embodiment, the modifications or changes are substitutions, especially conservative substitutions.

"Humanized antibody" refers to a class of engineered antibodies that have CDRs derived from a non-human donor immunoglobulin and the remaining immunoglobulin portion of the humanized antibody is derived from one (or more) human Immunoglobulin. In addition, framework supporting residues can be altered to preserve binding affinity (see, eg, Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10032 (1989), Hodgson et al., Bio/Technology, 9:421 (1991) ). A suitable human acceptor antibody may be an antibody selected by homology to the nucleotide and amino acid sequences of the donor antibody from conventional databases such as the Los Alamos database and the Swiss protein database. Human antibodies characterized by homology (on an amino acid basis) to the framework regions of the donor antibody may be suitable to provide heavy chain constant and/or heavy chain variable framework regions for insertion into the donor CDRs. Suitable acceptor antibodies capable of providing light chain constant or variable framework regions can be selected in a similar manner. It should be noted that acceptor antibody heavy and light chains need not be derived from the same acceptor antibody.

"Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or prepared using any of the techniques for preparing human antibodies as disclosed herein. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be prepared using a variety of techniques known in the art, including phage display libraries.

As known in the art, "polynucleotide" or "nucleic acid" as used interchangeably herein refers to a chain of nucleotides of any length, and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate capable of being incorporated into a chain by DNA or RNA polymerases.

Calculation of sequence identity between sequences is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and

gap weights

16, 14, 12, 10, 8, 6, or 4 and

length weights

1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity. In yet another preferred embodiment, the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and

gap weights

40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences. A particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use the PAM120 weighted remainder table, gap length penalty of 12, gap penalty of 4, using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11-17 ) determines the percent identity between two amino acid sequences or nucleotide sequences. Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.

As used herein, "vector" refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression encapsulated in liposomes Vectors and certain eukaryotic cells, such as producer cells.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably in this disclosure and can include cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as the cell screened or selected in the original transformed cell.

As used herein, "subject", "individual" or "subject" refers to an animal, preferably a mammal, more preferably a human, in need of alleviation, prevention and/or treatment of a disease or disorder such as a viral infection. Mammals also include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice, and rats. The term includes human subjects with or at risk of having a coronavirus infection. In the present disclosure, administering the antibody described in the present disclosure or the pharmaceutical composition or preparation described in the present disclosure to a subject in need thereof refers to administering an effective amount of the antibody or pharmaceutical composition or preparation and the like.

As used in this disclosure, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmacological response, eg, in a tissue, system, animal or human being pursued by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that results in an improved treatment, cure, prevention or alleviation of a disease, disorder or side effect, or reduces the rate of progression of a disease or condition as compared to a corresponding subject not receiving the amount amount. The term also includes within its scope amounts effective to enhance normal physiological function.

In the present disclosure, the term "prophylactic treatment" or "prophylactic treatment", which is used interchangeably with "prophylactic treatment", "prophylactic treatment" and the like, refers to treatment aimed at stopping or delaying the pathological process leading to a disease.

In some embodiments, prophylactic treatment occurs in a trial in which prophylactic treatment is administered, which refers to the administration of a sufficient dose of a therapeutic bispecific to susceptible mice starting 12 hours prior to exposure to a SARS-CoV-2 mutant strain Neutralizing antibodies to protect mice against SARS-CoV-2 mutant virus.

II. Coronaviruses to which the antibodies of the present disclosure are directed, their structure, and their mode of entry into host cells

Coronaviruses (including SARS-CoV and the newly discovered SARS-CoV-2) are spherical single-stranded positive-stranded RNA viruses characterized by a spike protein protruding from the surface of the virion (Barcena, M. et al., Cryo- electron tomography of mouse hepatitis virus: Insights into the structure of the coronavirion. Proc. Natl. Acad. Sci. USA 2009, 106, 582–587). The spherical shape of the virus particles and the spikes make the coronavirus look like a corona under an electron microscope and are named coronaviruses.

Coronaviruses are enveloped viruses (the envelope is derived from the lipid bilayer of the host cell membrane), with mainly viral structural proteins (eg Spike, S), Membrane (M), The viral structure formed by membrane protein (Envelope, E) and nucleocapsid (Nucleocapsid, N)), in which S protein, M protein and E protein are embedded in the viral envelope, and N protein interacts with viral RNA and is located in the virus The core of the particle, forming the nucleocapsid (Fehr, A.R. et al., Coronaviruses: An overview of their replication and pathogenesis. Methods Mol. Biol. 2015, 1282, 1-23). The S protein is a highly glycosylated protein that forms homotrimeric spikes on the surface of virus particles and mediates virus entry into host cells. SARS-CoV-2 is a single-stranded positive-stranded RNA virus with a membrane structure and a size of 80-120nm. The genome length is about 29.9kb. The homology between them is 80%. The open reading frame (ORF) of the viral genome, ORF1a and ORF1b, occupy 2/3 of the genome, and express hydrolytic enzymes and enzymes related to replication and transcription, such as cysteine protease (PLpro) and serine protease (3CLpro) ), RNA-dependent RNA polymerase (RdRp) and helicase (Hel); the latter 1/3 region of the genome is mainly responsible for encoding structural proteins, including spike protein (S), envelope protein (E), membrane protein (M) , nucleocapsid protein (N) and other major structural proteins, in which the N protein wraps the viral genome to form a nucleoprotein complex, the E protein and M protein are mainly involved in the assembly process of the virus, and the S protein is mainly mediated by binding to host cell receptors Virus invasion and determines the host specificity of the virus.

The S protein can be processed by host cell proteases into the S1 subunit (generally comprising amino acids 14-685 of the S protein) and the S2 subunit (generally comprising amino acids 686-1273 of the S protein). The S1 subunit recognizes and binds to host cell surface receptors, while the S2 subunit mediates fusion of the virus with the host cell membrane. After sequence comparison, it was found that the S protein of SARS-CoV-2 virus and SARS-CoV virus had a similarity of 75%. The amino acid residues at positions 442, 472, 479, 487 and 491 of the complex interface distributed in respiratory epithelial cells, lung, heart, kidney and digestive tract are highly conserved. Compared with the S protein of SARS-CoV, at the five sites, only the 491st amino acid of the S protein of SARS-CoV-2 is the same, and the other four amino acids are mutated. Nevertheless, it was found through the prediction of the protein 3D structure that although the 4 key amino acids of the S protein of SARS-CoV-2 were replaced with ACE2, compared with the S protein of SARS-CoV-2, SARS-CoV-2 The three-dimensional structure of the receptor binding domain (RBD) in the S protein of SARS-CoV-2 is almost unchanged, so the S protein of SARS-CoV-2 still has a high affinity for human ACE2. Recent articles (WrappD et al., Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation, Science, 19 February 2020, published online, pii:eabb2507.doi:10.1126/science.abb2507) and (Xiaolong Tian et al., Potent binding of 2019 novel coronavirus spike protein by a SARScoronavirus-specific human monoclonal antibody, Emerging Microbes & Infections, 2020, 9:1, p382-385, DOI: 10.1080/22221751.2020.1729069) found by Fortebio detection, SARS - The affinity (KD) of the S protein of CoV-2 binding to human ACE2 is about 15nM, which is comparable to the affinity of the S protein of SARS-CoV to human ACE2. It can be seen that ACE2 is also a mechanism for SARS-CoV-2 to infect the human body and enter the cell. receptor protein. It is expected that high-affinity neutralizing antibodies targeting the coronavirus S protein and blocking its binding to ACE2 can effectively prevent and treat coronavirus (eg, SARS-CoV-2, SARS-CoV) infection.

In this disclosure, the terms "SARS-CoV-2" or "SARS-CoV-2 virus" are used interchangeably and include SARS-CoV-2 wild-type and mutants. In some embodiments, the GenBank Accession No. of the S protein of SARS-CoV-2 wild-type is QHD43416.1; see also, eg, SEQ ID NO:46. Herein, the amino acid positions in the S protein of the SARS-CoV-2 mutant are based on the amino acid sequence of the S protein of the SARS-CoV-2 wild-type (GenBank Accession No. QHD43416.1, also see, for example, SEQ ID NO: 46) definition.

In some embodiments, the S protein of the SARS-CoV-2 mutant has mutations (eg, amino acid substitutions) and/or deletions compared to the S protein of the SARS-CoV-2 wild-type. For example, the S protein of a SARS-CoV-2 mutant is compared to the wild-type S protein of SARS-CoV-2, e.g. in the receptor binding domain of the S1 subunit (which comprises e.g. 319-532 of the wild-type S protein). amino acids 319-541) and/or other regions (eg, amino acids 14-318 of the wild-type S protein or amino acids 533-685 or the S2 subunit) with deletions and/or mutations.

In some embodiments, the S protein of the SARS-CoV-2 mutant has mutation sites and/or deletions in the S1 subunit and/or the S2 subunit compared to the SARS-CoV-2 wild type. In one embodiment, the mutation site comprises or is selected from one or more of the following: amino acid positions 19, 80, 142, 158, 215, 323, 330, 339, 341, 344, 367, 384, 408, 414 , 417, 435, 439, 444, 445, 446, 450, 452, 453, 455, 458, 475, 476, 477, 478, 483, 484, 485, 486, 490, 493, 501, 518, 519, 614 , 681, 701 and 950. In one embodiment, the mutation site comprises or is selected from one or more of the following: amino acid positions 323, 330, 339, 341, 344, 367, 384, 408, 414, 417, 435, 439, 444 , 445, 446, 450, 452, 453, 455, 458, 475, 476, 477, 478, 483, 484, 485, 486, 490, 493, 501, 518, 519, 614, 681.

In one embodiment, the S protein of the SARS-CoV-2 mutant has a deletion at one or more of the following amino acid positions compared to the SARS-CoV-2 wild type: amino acid positions 69, 70, 156, 157, 241, 242 and 243. In one embodiment, the S protein of the SARS-CoV-2 mutant has deletions at amino acid positions 69 and/or 70 compared to the SARS-CoV-2 wild type. In one embodiment, the S protein of the SARS-CoV-2 mutant has deletions at amino acid positions 69 and 70 (Δ69/70) compared to the SARS-CoV-2 wild type. In one embodiment, the S protein of the SARS-CoV-2 mutant has deletions at amino acid positions 156 and 157 (Δ156/157) compared to the SARS-CoV-2 wild type. In one embodiment, the S protein of the SARS-CoV-2 mutant has deletions at amino acid positions 241, 242 and 243 (Δ241/242/ 243). In one embodiment, the S protein of the SARS-CoV-2 mutant has deletions at the following amino acid positions: (1) amino acid positions 69 and/or 70; (2) amino acid positions 156 and 157; or (3) amino acid positions Locations 241, 242 and 243.

In some embodiments, the S protein of the SARS-CoV-2 mutant has a mutation site in the receptor binding domain of the S1 subunit compared to the SARS-CoV-2 wild type. In one embodiment, the mutation site comprises or is selected from one or more of the following: amino acid positions 323, 330, 339, 341, 344, 367, 384, 408, 414, 417, 435, 439, 444 , 445, 446, 450, 452, 453, 455, 458, 475, 476, 477, 478, 483, 484, 485, 486, 490, 493, 501, 518, 519. More specifically, the mutation site may include or be selected from one or more of those shown in Table 17, eg, mutation site N501Y.

In some embodiments, the S protein of the SARS-CoV-2 mutant has mutation sites at amino acid positions 14-318 and/or 533-685 compared to the SARS-CoV-2 wild type. In a specific embodiment, the mutation site comprises or is selected from one or more of the following: amino acid positions 614, 681. In one embodiment, the mutation site comprises or is selected from one or more of the following: amino acid positions 19, 80, 142, 158, 215, 614 and 681.

In some embodiments, the S protein of the SARS-CoV-2 mutant has a mutation site in the S2 subunit compared to the S protein of the SARS-CoV-2 wild-type. In one embodiment, the mutation sites include or are selected from one or more of the following: amino acid positions 701 and 950.

In a specific embodiment, the S protein of the SARS-CoV-2 mutant comprises the mutation site N501Y. In another specific embodiment, the S protein of the SARS-CoV-2 mutant comprises mutation sites N501Y, E484K and K417N. In another specific embodiment, the S protein of the SARS-CoV-2 mutant comprises mutation sites L452R, T478K and P681R.

In a specific embodiment, the SARS-CoV-2 mutant is SARS-CoV-2 mutant strain B.1.1.7. In a specific embodiment, the S protein of the SARS-CoV-2 mutant strain B.1.1.7 contains the mutation site N501Y compared to the SARS-CoV-2 wild type. In a specific embodiment, the S protein of the SARS-CoV-2 mutant strain B.1.1.7 comprises the mutation site N501Y and deletions of amino acids 69 and 70 (Δ69/70) compared to the SARS-CoV-2 wild type .

In another specific embodiment, the SARS-CoV-2 mutant is SARS-CoV-2 mutant strain B.1.351. In a specific embodiment, the S protein of the SARS-CoV-2 mutant strain B.1.351 comprises mutation sites N501Y, E484K and K417N compared to the SARS-CoV-2 wild type. In a specific embodiment, the S protein of the SARS-CoV-2 mutant strain B.1.351 comprises mutation sites D80A, D215G, K417N, E484K, N501Y, D614G and A701V compared to the SARS-CoV-2 wild type; and Deletion of amino acids 241, 242 and 243 (Δ241/242/243).

In yet another specific embodiment, the SARS-CoV-2 mutant is SARS-CoV-2 mutant strain B.1.617.2. In a specific embodiment, the S protein of the SARS-CoV-2 mutant strain B.1.617.2 comprises mutation sites L452R, T478K and P681R compared to the SARS-CoV-2 wild type. In a specific embodiment, the S protein of the SARS-CoV-2 mutant strain B.1.617.2 comprises mutation sites T19R, R158G, L452R, T478K, D614G, P681R, D950N compared to the SARS-CoV-2 wild type , deletions of amino acids 156 and 157 (Δ156/157); and optional mutation site G142D.

III. Polypeptide complexes against coronavirus S protein

The terms "antibody to coronavirus S protein", "anti-coronavirus S protein antibody", "anti-S protein antibody", "coronavirus S protein antibody", "S protein antibody" or "antibody that binds S protein" are used in the Used interchangeably herein, it refers to an antibody of the present disclosure that is capable of binding coronavirus S protein (eg, SARS-CoV-2 S protein, SARS-CoV S protein) with sufficient affinity such that The antibody can be used as a diagnostic, prophylactic and/or therapeutic agent targeting the coronavirus S protein.

In one aspect, the present disclosure provides a novel polypeptide complex that specifically binds to the coronavirus S protein, the polypeptide complex comprising: (a) a first epitope binding moiety, the first epitope binding moiety comprising A heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL together form an antigen binding site that specifically binds to the first epitope of the coronavirus S protein, and (b) a second An epitope-binding portion comprising a single-domain antibody (sdAb) or a VHH fragment thereof that specifically binds to the second epitope of the coronavirus S protein, wherein the first epitope-binding portion and The second epitope binding moieties are fused to each other, and wherein the first epitope is different from the second epitope. In some embodiments, the first epitope binding moiety and the second epitope binding moiety do not compete for epitopes with each other. In some embodiments, the sdAb is a camelid sdAb or a humanized sdAb. In some embodiments, the first epitope binding moiety comprises a VH-containing heavy chain and a VL-containing light chain. In some embodiments, the second epitope binding moiety is at the heavy chain N-terminus, light chain N-terminus, Fc region C-terminus, heavy chain C-terminus, or light chain C-terminus of the first epitope binding moiety fused to the first epitope binding moiety. In some embodiments, the first epitope binding portion comprises a full-length 4-chain antibody. In some embodiments, the second epitope binding moiety is chemically fused to the first epitope binding moiety. In some embodiments, the second epitope binding moiety is fused to the first epitope binding moiety via a peptide bond or peptide linker. In some embodiments, the peptide linker has a length of no more than about 30 amino acids (such as no more than any of about 25, 20, or 15). In some embodiments, the first antigen-binding fragment comprises an Fc region, such as an IgGl Fc or IgG4 Fc.

The inventors of the present application unexpectedly found that the polypeptide complexes that specifically bind to different epitopes of the coronavirus S protein can specifically bind the coronavirus S protein with high affinity. Compared with the single epitope that specifically binds the coronavirus S protein alone, the polypeptide complex of the present disclosure binds the coronavirus S protein in a synergistic manner and blocks the binding of the coronavirus S protein to ACE2. In some embodiments, the polypeptide complexes of the present disclosure can be used to prevent and/or treat coronavirus-infected individuals.

The polypeptide complexes of the present disclosure have at least two epitopes/antigen-binding moieties that can specifically bind to at least two different epitopes on the coronavirus S protein. The polypeptide complexes of the present disclosure can be symmetric or asymmetric. For example, the polypeptide complexes of the present disclosure may comprise one or two copies of a first epitope/antigen binding moiety, and one to eight copies of a second epitope/antigen binding moiety.

First epitope/antigen binding moiety

The terms "epitope binding portion" and "antigen binding portion" are used interchangeably herein. In some embodiments, the first epitope binding portion comprises an antigen binding site that binds the first epitope of the coronavirus S protein. In some embodiments, the first epitope binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL together form a first epitope that specifically binds to the coronavirus S protein the antigen-binding site. In some embodiments, the first epitope binding moiety is a full-length antibody or an antigen-binding fragment thereof such as F(ab) ₂ .

In some embodiments, in the first epitope binding portion of the present disclosure, the heavy chain variable region (VH) may comprise: the first heavy chain in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 1 CDR1 (HCDR1) or its variant with no more than 2 amino acid changes; the first heavy chain CDR2 (HCDR2) in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or its variant with no more than 2 amino acid changes and/or the first heavy chain CDR3 (HCDR3) in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes.

In some embodiments, in the first epitope binding portion of the present disclosure, the light chain variable region (VL) may comprise: the light chain CDR1 ( LCDR1) or a variant thereof with no more than 2 amino acid changes; light chain CDR2 (LCDR2) or a variant thereof with no more than 2 amino acid changes in the light chain variable region amino acid sequence shown in SEQ ID NO: 2; and /or light chain CDR3 (LCDR3) in the light chain variable region amino acid sequence shown in SEQ ID NO: 2 or a variant thereof with no more than 2 amino acid changes.

In some embodiments, amino acid changes can be additions, deletions or substitutions of amino acids, eg, amino acid changes are conservative amino acid substitutions.

In some embodiments, the first HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8; the first HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9; and/or the Said first HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10.

In some embodiments, the LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11; the LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12; and/or the LCDR3 comprises or It consists of the amino acid sequence shown in SEQ ID NO: 13.

In some embodiments, the first epitope binding portion of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or consists of the sequence of SEQ ID NO: 1 or the same Sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, and/or light chain variable regions Comprising or consisting of the sequence of SEQ ID NO: 2 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or 99% identical sequences. In some embodiments, the amino acid changes do not occur in the CDR regions.

In some embodiments, the first epitope binding portion of the present disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the sequence set forth in SEQ ID NO: 22 or at least 90%, 91% thereof , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences; and/or the light chain comprises or consists of the following sequence: the sequence shown in SEQ ID NO:23 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith. In some embodiments, the amino acid changes do not occur in the CDR regions.

In some embodiments, the first epitope binding portion of the present disclosure comprises an Fc region. In some embodiments, the Fc region is from an IgG, eg, IgGl, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is from IgGl or IgG4. In some embodiments, the Fc region is from human IgGl or human IgG4. In some more specific embodiments, the Fc region is an IgG1 Fc with L234A and L235A or an IgG4 Fc with the S228P mutation.

In some embodiments of the present disclosure, amino acid changes described herein include amino acid substitutions, insertions, or deletions. In some embodiments, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another neutral amino acid replace. Exemplary substitutions are shown in Table B below:

Table B. Exemplary Amino Acid Substitutions

原始残基original residue	示例性取代Exemplary substitution	优选的取代Preferred substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Asp、Lys；ArgGln; His; Asp, Lys; Arg	GlnGln
Asp(D)Asp(D)	Glu；AsnGlu; Asn	GluGlu
Cys(C)Cys(C)	Ser；AlaSer; Ala	SerSer
Gln(Q)Gln(Q)	Asn；GluAsn;Glu	AsnAsn
Glu(E)Glu(E)	Asp；GlnAsp;Gln	AspAsp
Gly(G)Gly(G)	AlaAla	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile(I)	Leu，Val；Met；Ala；Phe；正亮氨酸Leu, Val; Met; Ala; Phe; Norleucine	LeuLeu
Leu(L)Leu(L)	正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr	TyrTyr
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	Val；SerVal; Ser	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; Norleucine	LeuLeu

In some embodiments, the amino acid changes described in the present disclosure occur in regions outside the CDRs (eg, in FRs). In some embodiments, the amino acid changes described in the present disclosure occur in the Fc region. In some embodiments, antibodies or epitope binding moieties are provided comprising an Fc domain comprising one or more mutations that enhance or weaken the antibody or epitope binding moiety, eg, at acidic pH compared to neutral pH Binding of FcRn receptors. For example, the present disclosure includes an anti-coronavirus S protein antibody or an epitope binding portion that binds an epitope of the coronavirus S protein containing a mutation in the CH2 or CH3 region of the Fc domain, wherein the one or more mutations increase the Affinity to FcRn in acidic environments (eg, in endosomes with pH in the range of about 5.5 to about 6.0). This mutation can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example: positions 250 (eg E or Q), 250 and 428 (eg L or F), 252 (eg L/Y/F/W or T), 254 Modification of position (e.g. S or T) and position 256 (e.g. S/R/Q/E/D or T); or position 428 and/or 433 (e.g. H/L/R/S/P/Q or K) ) and/or 434 (e.g. A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or 250 and/or 428; or 307 or 308 ( For example, modifications at positions 308F, V308F) and 434. In one embodiment, the modifications include 428L (eg, M428L) and 434S (eg, N434S) modifications; 428L, 259I (eg, V259I) and 308F (eg, V308F) modifications; 433K (eg, H433K) and 434 (eg, 434Y) modifications; 252, 254 and 256 (eg 252Y, 254T and 256E) modifications; 250Q and 428L modifications (eg T250Q and M428L); and 307 and/or 308 modifications (eg 308F or 308P). In yet another embodiment, the modification includes 265A (e.g., D265A) and/or 297A (e.g., N297A) modifications. For example, the present disclosure includes anti-coronavirus S protein antibodies or epitope binding portions comprising an Fc domain comprising one (set) or pairs (set) of mutations selected from the group consisting of: 250Q and 248L (eg, T250Q and M248L); 252Y, 254T and 256E (e.g. M252Y, S254T and T256E); 428L and 434S (e.g. M428L and N434S); 257I and 311I (e.g. P257I and Q311I); 257I and 434H (e.g. P257I and N434H); 376V and 434H (eg D376V and N434H); 307A, 380A and 434A (eg T307A, E380A and N434A); and 433K and 434F (eg H433K and N434F). In one embodiment, the present disclosure includes an anti-coronavirus S protein antibody or epitope binding portion comprising an Fc domain comprising the S108P mutation in the hinge region of IgG4 to facilitate dimer stabilization. Any possible combination of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are included within the scope of the present disclosure.

In some embodiments, the coronavirus S protein antibodies or epitope binding portions provided herein are altered to increase or decrease the degree of glycosylation thereof. Addition or deletion of glycosylation sites to the coronavirus S protein antibody or epitope binding portion can be conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When the coronavirus S protein antibody or epitope binding portion comprises an Fc region, the carbohydrates linked to the Fc region can be altered. In some applications, modifications to remove unwanted glycosylation sites may be useful, such as removal of fucose moieties to improve antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277 :26733). In other applications, galactosylation modifications can be made to modulate complement-dependent cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the coronavirus S protein antibodies or epitope binding portions provided herein to generate Fc region variants to enhance, for example, the coronaviruses of the present disclosure. Effectiveness of viral S protein antibodies in preventing and/or treating coronavirus infection. In some embodiments, native sequence Fc regions suitable for use in the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4, preferably human IgGl. In some embodiments, ADCC and CDC effects are removed or reduced by mutating L234A and L235A of human IgGl Fc.

"Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig binds to certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and macrophages) The presence of Fc receptors (FcRs) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. To assess ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, such as those described in US Pat. Nos. 5,500,362 or 5,821,337.

"Complement-dependent cytotoxicity" or "CDC" refers to lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (appropriate subclasses) that bind to their cognate antigens. To assess complement activation, a CDC assay can be performed, eg, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Antibody variants with altered Fc region amino acid sequences and increased or decreased Clq binding capacity are described in US Pat. No. 6,194,551 B1 and WO 99/51642. The contents of those patent publications are specifically incorporated herein by reference. See also, Idusogie et al., J. Immunol. 164:4178-4184 (2000).

In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to the coronavirus S protein, which can be used alone or as an epitope binding part of the polypeptide complex of the present disclosure for preventing coronavirus infection and /or treatment of coronavirus-infected individuals.

In some embodiments, the antibodies of the present disclosure that specifically bind to the coronavirus S protein comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) may comprise : Heavy chain CDR1 (HCDR1) in the heavy chain variable region amino acid sequence shown in SEQ ID NO:1 or a variant thereof with no more than 2 amino acid changes; heavy chain variable region amino acids shown in SEQ ID NO:1 The heavy chain CDR2 in the sequence (HCDR2) or a variant thereof with no more than 2 amino acid changes; and/or the heavy chain CDR3 (HCDR3) in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof. A variant with more than 2 amino acid changes; and/or the light chain variable region (VL) may comprise: the light chain CDR1 (LCDR1) or its light chain variable region amino acid sequence shown in SEQ ID NO: 2 A variant with no more than 2 amino acid changes; the light chain CDR2 (LCDR2) in the light chain variable region amino acid sequence shown in SEQ ID NO: 2 or a variant thereof with no more than 2 amino acid changes; and/or SEQ ID The light chain CDR3 (LCDR3) in the amino acid sequence of the light chain variable region shown in NO: 2 or a variant thereof with no more than 2 amino acid changes. In some further embodiments, the HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8; the HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9; and/or the HCDR3 Consists of or consists of the amino acid sequence shown in SEQ ID NO: 10. In some further embodiments, the LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11; the LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12; and/or the LCDR3 Comprising or consisting of the amino acid sequence shown in SEQ ID NO:13.

In some further embodiments, the antibody that specifically binds the coronavirus S protein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or consists of the following sequence: the sequence of SEQ ID NO: 1 or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and/or the light chain may The variable region comprises or consists of the sequence of SEQ ID NO: 2 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Sequences that are 98% or 99% identical. In some embodiments, the amino acid changes do not occur in the CDR regions.

In some further embodiments, the antibody that specifically binds to the coronavirus S protein of the present disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the following sequence: the sequence shown in SEQ ID NO: 22 or has at least a A sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; and/or the light chain comprises or consists of the following sequence: SEQ ID NO: 23 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the amino acid changes do not occur in the CDR regions.

In some further embodiments, the antibody of the present disclosure that specifically binds to the coronavirus S protein is the R15-F7 antibody of the present disclosure.

Second epitope binding moiety

In some embodiments, the polypeptide complexes of the present disclosure comprise (a) a first epitope binding moiety and (b) a second epitope binding moiety comprising a second epitope binding moiety that specifically binds to the coronavirus S A single domain antibody or VHH fragment thereof to the second epitope of the protein.

In some embodiments, the second epitope binding portion comprises a VHH fragment that specifically binds to the coronavirus S protein. In some embodiments, the VHH fragments are native, humanized and/or affinity matured.

The terms "single domain antibody", "single domain antibody" or "sdAb" refer to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). The sdAb alone was able to bind to the antigen, but not to the corresponding CDR-containing polypeptide. In some cases, the sdAb is engineered from a camelid HCAb, and its heavy chain variable domain is referred to herein as "VHH." Camelid sdAbs are among the smallest known antigen-binding antibody fragments (see, eg, Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh - Ghassabeh et al. Nanomedicine (Lond), 8:1013-26 (2013)).

The single domain antibodies disclosed herein can be prepared by one of skill in the art according to methods known in the art or any future methods. For example, VHHs can be obtained using methods known in the art, such as by immunizing camelid animals and obtaining therefrom VHHs that bind to and neutralize the target antigen, or by cloning the disclosed molecules using molecular biology techniques known in the art The VHH library was then selected by using phage display. In some embodiments, the single domain antibodies of the present disclosure are produced naturally in camelids, ie, produced by immunizing camelids with the coronavirus S protein or fragments thereof using the techniques described herein for other antibodies.

In some embodiments, single domain antibodies are obtained by immunizing llamas or alpacas with the desired antigen and subsequently isolating mRNA encoding the heavy chain antibody. Through reverse transcription and polymerase chain reaction, gene libraries containing millions of clones of single domain antibodies are generated. Screening techniques such as phage display and ribosome display aid in the identification of antigen-binding clones. In phage display, an antibody library is synthesized on phage, the library is screened with an antigen of interest or an antibody-binding portion thereof, and an antigen-binding phage is isolated, from which immunoreactive fragments can be obtained. Methods for preparing and screening such libraries are well known in the art, and kits for generating phage display libraries are commercially available (eg, Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and StratageneSurfZAP™ Phage Display Kit, Cat. No. 240612). Still other methods and reagents can be used to generate and screen antibody display libraries (see, eg, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)). When the most efficient clones are identified, their DNA sequences can be optimized, eg, by affinity maturation or humanization, to prevent the body's immune response against the antibody. Thus, the single domain antibodies of the present disclosure can be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding the naturally occurring VHH domain; (3) By "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by a naturally occurring VHH domain from any animal species, particularly mammalian species, such as from humans "Camelization" of VH domains, or by expression of nucleic acids encoding such camelized VH domains; (5) "camelization" by "domain antibodies" or "dAbs" (see e.g. Ward et al., 1989, Nature 341:544-546), or by expressing a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (7) by using for nucleic acid synthesis and/or (8) by any combination of the foregoing. Suitable methods and techniques for performing the foregoing will be apparent to those skilled in the art based on the disclosure herein, and include, for example, the methods and techniques described in greater detail below. Single domain antibodies are typically produced by PCR cloning of variable domain libraries into phage display vectors from cDNAs of blood, lymph node or spleen lymphocytes obtained from immunized animals. Antigen specificity is typically selected by panning the corresponding library on immobilized antigens (eg, antigens coated on the plastic surface of test tubes, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the cell surface) Sexual single domain antibody. The affinity of sdAbs can be improved by mimicking this strategy in vitro, for example by site-directed mutagenesis of CDR regions and under increased stringency conditions (higher temperature, high or low salt concentration, high or low pH and low antigen concentration) for immobilized antigen Further panning was performed (Wesolowski et al., Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol (2009) 198:157-174). Methods for preparing VHHs that specifically bind antigens or epitopes are described in the references, see e.g.: R. van der Linden et al, Journal of Immunological Methods, 240 (2000) 185-195; Li et al, J Biol Chem., 287(2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8(12), pp. 2645, 17 June, 2009 and WO94/04678.

In some embodiments, the polypeptide complexes of the present disclosure comprise at least one epitope binding moiety comprising an sdAb. Exemplary sdAbs include, but are not limited to, heavy chain variable domains (e.g., VHH or VNAR) from heavy chain antibodies only, binding molecules that naturally lack light chains, single domains derived from conventional 4-chain antibodies ( such as VH or VL), humanized heavy chain-only antibodies, and human sdAbs produced from transgenic mice or rats expressing human heavy chain segments. Any sdAb known in the art or developed by the inventors can be used to construct the polypeptide complexes of the present application. The sdAbs can be derived from any species including, but not limited to, mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit and cow. Single domain antibodies contemplated herein also include naturally occurring sdAb molecules from species other than camelid and shark.

In some embodiments, the sdAbs are derived from naturally occurring single-domain antigen-binding molecules, which are referred to as light-chain-deficient heavy-chain antibodies (also referred to herein as "heavy-chain-only antibodies"). Such single domain molecules are disclosed, for example, in WO 94/04678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For clarity, variable domains derived from heavy chain molecules that naturally lack light chains are referred to herein as VHHs to distinguish them from the conventional VHs of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in camelid species, eg, camels, llamas, llamas, dromedaries, alpacas, and guanaco. Species other than Camelidae can produce heavy chain molecules that naturally lack light chains, and such VHHs are within the scope of this application.

VHH molecules from llamas are about 10 times smaller than IgG molecules, are single polypeptides and can be very stable, resistant to extreme pH and temperature conditions. Furthermore, it is resistant to the action of proteases, which is not the case with conventional antibodies. Furthermore, in vitro expression of VHHs yields high yields of correctly folded functional VHHs. In addition, antibodies raised in alpacas can recognize epitopes other than those recognized by antibodies produced in vitro using antibody libraries or via immunization of mammals that are not alpacas (see, For example, WO9749805). Thus, polypeptide complexes comprising one or more VHH domains can interact with targets more efficiently than conventional antibodies. Because VHHs are known to bind into "abnormal" epitopes such as cavities or grooves, the affinity of polypeptide complexes comprising such VHHs may be more suitable for therapeutic treatment than conventional multispecific polypeptides.

In some embodiments, the sdAbs are derived from variable regions of immunoglobulins present in cartilaginous fish. For example, sdAbs can be derived from immunoglobulin isotypes called novel antigen receptors (NARs) present in shark serum. Methods of making single domain molecules ("IgNARs") derived from the variable regions of NARs are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

In some embodiments, sdAbs are recombinant, CDR-grafted, humanized, camelized, deimmunized, and/or generated in vitro (eg, by phage display selection). In some embodiments, the sdAb is a human sdAb produced by a transgenic mouse or rat expressing a human heavy chain fragment. See, eg, US20090307787A1, US Patent 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794. In some embodiments, the sdAb is affinity matured.

sdAbs comprising VHH domains can be humanized to have human-like sequences. In some embodiments, the FR regions of the VHH domains used herein comprise amino acid sequence identity to at least about any of the following: 50%, 60%, 70%, 80%, 90%, 95% to the human VH framework regions % or more. An exemplary class of humanized VHH domains is characterized in that the VHH carries an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine at position 45 according to the Kabat numbering, such as L45 acid, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine or glutamine, and carry tryptophan at position 103. Thus, polypeptides belonging to this class show high amino acid sequence homology to the human VH framework regions, and the polypeptides can be administered directly to humans, but undesired immune responses are not thereby expected, and are not further humanized burden.

Another exemplary class of humanized camelid sdAbs has been described in WO 03/035694 and contains hydrophobic FR2 residues typically found in conventional antibodies of human origin or other species, but which are This loss of hydrophilicity is compensated by charged arginine residues that replace the conserved tryptophan residues present in the VH from diabodies. Thus, peptides belonging to these two types show high amino acid sequence homology to the human VH framework regions, and the peptides can be administered directly to humans, but undesired immune responses are not expected therefrom, and are not further humanized burden.

In some embodiments, the polypeptide complex comprises a naturally occurring sdAb or VHH fragment or derivative thereof, such as a camelid sdAb or VHH fragment thereof, or a humanized sdAb or VHH fragment thereof derived from a camelid sdAb. In some embodiments, the sdAb is obtained from a llama. In some embodiments, the sdAbs are further engineered to remove sequences not normally found in human antibodies (such as CDR regions or CDR-FR junctions).

In some embodiments, the polypeptide complexes of the present disclosure comprise a second epitope binding moiety comprising an sdAb or VHH fragment thereof with appropriate affinity for a coronavirus S protein epitope. For example, the affinity of the sdAb or its VHH fragment can affect the affinity of the polypeptide complex for the coronavirus S protein, which may further affect the efficacy of the polypeptide complex. In some embodiments, the sdAb or VHH fragment thereof binds its epitope with high affinity. A high affinity sdAb or VHH fragment thereof binds its epitope with a dissociation constant (Kd) in the low nanomolar ( ^10-9 M) range, such as no more than about any of the following: 5nM, 4nM, 3nM, 2nM, 1nM , 0.5nM, 0.2nM, 0.1nM, 0.05nM, 0.02nM, 0.01nM, 5pM, 2pM, 1pM or less.

In some embodiments, the sdAb or VHH fragment thereof of the present disclosure comprises: the second heavy chain CDR1 (HCDR1 ) in the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7 or a variation of no more than 2 amino acids thereof Variants; the second heavy chain CDR2 (HCDR2) in the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes; and SEQ ID NO: 5, 6 or 7 The second heavy chain CDR3 (HCDR3) in the indicated VHH amino acid sequence or a variant thereof with no more than 2 amino acid changes. The second HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, 18 or 20; the second HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15 or 21; and/or the Said second HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, 17 or 19. In some further embodiments, the second HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14; the second HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15; and the The second HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; the second HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; the second HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18 the amino acid sequence shown in: 15; and the second HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; or the second HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20 The second HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; and the second HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16.

In some embodiments, the sdAb or VHH fragment thereof of the present disclosure comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14, 18 or 20; HCDR2 comprising or consisting of SEQ ID NO: 15 or The amino acid sequence shown in 21 consists of; HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, 17 or 19.

In some embodiments, the sdAbs of the present disclosure or VHH fragments thereof comprise or consist of or have at least 80%, 85%, 90%, 91%, or at least 80%, 85%, 90%, 91% of the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7. Sequences of %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In some embodiments, the amino acid changes do not occur in the CDRs of the VHH fragment.

In some embodiments, the sdAb or VHH fragment thereof of the present disclosure comprises:

i) CDR1 of the amino acid sequence _of the formula _GRFFGSYX1 MS, wherein X1 is Y, T or V;

ii) CDR2 of an amino acid sequence of the formula DINTRGX ₂ X ₃ TR, wherein X ₂ is E or I, and X ₃ is T or V; and/or

iii) CDR3 of an amino acid sequence represented by the formula AASX ₄ X ₅ TFX6GRSDPDY, wherein X ₄ is G or P, X ₅ is D or A, and X ₆ is E or F.

In one aspect, the sdAbs of the present disclosure can be used alone or as an epitope-binding portion of a polypeptide complex of the present disclosure for the prevention of coronavirus infection and/or the treatment of coronavirus-infected individuals.

In some embodiments, the sdAbs of the present disclosure are used alone to prevent and/or treat coronavirus-infected individuals. In some embodiments, the present disclosure provides an sdAb comprising the VHH amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6 or 7 (preferably, SEQ ID NO: 5, 6 or 7) or A sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the present disclosure provides an sdAb comprising or consisting of the sequence set forth in SEQ ID NO: 24, 25, 26, 27 or 28 (preferably, SEQ ID NO: 26, 27 or 28) The amino acid sequence shown or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown. In some embodiments, the amino acid changes do not occur in the CDRs of the VHH fragment. In some embodiments, the sdAb of the present disclosure is the single domain antibody P14-F8, P14-F8-hVH8, P14-F8-35, P14-F8-38, or P14-F8-43 described herein.

fusion polypeptide

In some embodiments, the first epitope binding portion and the second epitope binding portion of the polypeptide complexes of the present disclosure are fused to each other (ie, covalently linked). Accordingly, the polypeptide complexes of the present disclosure comprise one or more fusion polypeptides.

As used herein, the term "fusion" means that the first epitope binding portion and the second epitope binding portion of the polypeptide complexes of the present disclosure are directly or indirectly linked together by means of covalent linkage. In some embodiments, fusion includes, but is not limited to, direct fusion via a peptide bond or non-peptide bond or indirect fusion via a linker such as a peptide linker. Those skilled in the art will appreciate that direct or indirect linkages known in the art or that may be discovered in the future that can be used to link polypeptides together can be used in the "fusions" of the present disclosure.

The first epitope binding moiety and the second epitope binding moiety can be directly linked by a single chemical bond, such as a peptide bond, or via a peptide linker. The second epitope binding moiety may be fused at the N-terminus or the C-terminus of any (including each) polypeptide of the first epitope binding moiety, or may be fused at any (including each) polypeptide of the first epitope binding moiety ) at an internal position of the polypeptide, such as at the N-terminus of the Fc region in the heavy chain of the first epitope binding moiety. Fusion polypeptides can be obtained recombinantly or chemically. In some embodiments, the C-terminus of the second epitope binding moiety is fused to the N-terminus of any (including each) polypeptide of the first epitope binding moiety via a chemical bond (such as a peptide bond) or a peptide linker. In some embodiments, the N-terminus of the second epitope binding moiety is fused to the C-terminus of any (including each) polypeptide of the first epitope binding moiety via a chemical bond (such as a peptide bond) or a peptide linker. In some embodiments, the second epitope binding moiety is fused to the first epitope binding moiety via a chemical bond that is not a peptide involving the backbone chemical group of the amino acid.

In some embodiments, the first epitope binding portion comprises a single chain antibody fragment comprising VH and VL. In some embodiments, the first epitope binding moiety comprises a scFv. In some embodiments, the polypeptide complex comprises a fusion polypeptide comprising, in the N-terminal to C-terminal direction: a second epitope binding portion comprising an sdAb or VHH fragment thereof, an optional peptide linker, a VH domain and a VL domain; in some embodiments, the polypeptide complex comprises a fusion polypeptide comprising, in the N-terminal to C-terminal direction: a second epitope binding portion comprising an sdAb or VHH fragment thereof, Optional peptide linkers, VL domains and VH domains. In some embodiments, the polypeptide complex comprises a fusion polypeptide comprising, in the N-terminal to C-terminal direction: a VH domain, a VL domain, an optional peptide linker, and a sdAb or VHH fragment thereof comprising The second epitope binding moiety. In some embodiments, the polypeptide complex comprises a fusion polypeptide comprising, in the N-terminal to C-terminal direction: a VL domain, a VH domain, an optional peptide linker, and a sdAb or VHH fragment thereof comprising The second epitope binding moiety.

In some embodiments, the polypeptide complex comprises an scFv that specifically recognizes a first epitope, and a first sdAb or VHH fragment thereof that specifically recognizes a second epitope and a second sdAb or VHH fragment thereof, wherein the first The sdAb and the second sdAb can be the same or different. In some embodiments, the C-terminus of the first sdAb or VHH fragment thereof is fused to the N-terminus of the scFv, and the N-terminus of the second sdAb or VHH fragment thereof is fused to the C-terminus of the scFv. In some embodiments, the first sdAb or VHH fragment thereof is in tandem with the second sdAb or VHH fragment thereof, and the N-terminus of the tandem fragment is fused to the C-terminus of the scFv. In some embodiments, the first sdAb or VHH fragment thereof is in tandem with the second sdAb or VHH fragment thereof, and the C-terminus of the tandem fragment is fused to the N-terminus of the scFv.

In some embodiments, the first epitope binding moiety comprises a VH domain-containing heavy chain and a VL domain-containing light chain. In some embodiments, the heavy chain further comprises one or more heavy chain constant domains, eg, CH1, CH2, CH3, and CH4, and/or a hinge region (HR). In some embodiments, the light chain further comprises a light chain constant domain (CL). In some embodiments, the N-terminus of the second epitope binding moiety is fused to the C-terminus of the heavy chain. In some embodiments, the C-terminus of the second epitope binding moiety is fused to the N-terminus of the heavy chain. In some embodiments, the N-terminus of the second epitope binding moiety is fused to the C-terminus of the light chain. In some embodiments, the C-terminus of the second epitope binding moiety is fused to the N-terminus of the light chain. In some embodiments, the polypeptide complex comprises a first polypeptide comprising, from N-terminus to C-terminus: a heavy chain, an optional peptide linker, and a second polypeptide comprising an sdAb or VHH fragment thereof an epitope binding portion; and a second polypeptide comprising a light chain. In some embodiments, the polypeptide complex comprises a first polypeptide comprising, from N-terminus to C-terminus: a second epitope binding moiety comprising an sdAb or VHH fragment thereof, an optional peptide linker and a heavy chain; and a second polypeptide comprising a light chain. In some embodiments, the polypeptide complex comprises a first polypeptide comprising, from N-terminus to C-terminus: a light chain, an optional peptide linker, and a second polypeptide comprising an sdAb or VHH fragment thereof an epitope binding portion; and a second polypeptide comprising a heavy chain. In some embodiments, the polypeptide complex comprises a first polypeptide comprising, from N-terminus to C-terminus: a second epitope binding moiety comprising an sdAb or VHH fragment thereof, an optional peptide linker and a light chain; and a second polypeptide comprising a heavy chain.

In some embodiments, the first epitope binding portion comprises a full-length antibody consisting of two heavy chains and two light chains. In some embodiments, the full-length antibody is a full-length monoclonal antibody, which consists of two identical heavy chains and two identical light chains. In some embodiments, the polypeptide complex comprises two identical first polypeptides each comprising, from the N-terminus to the C-terminus: a heavy chain, an optional peptide linker, and an sdAb or its a second epitope binding portion of a VHH fragment; and two identical second polypeptides, each of the second polypeptides comprising a light chain. In some embodiments, the polypeptide complex comprises two identical first polypeptides each comprising from N-terminus to C-terminus: a second epitope binding portion comprising an sdAb or VHH fragment thereof, an optional peptide linker and a heavy chain; and two identical second polypeptides each comprising a light chain. In some embodiments, the polypeptide complex comprises two identical first polypeptides each comprising, from the N-terminus to the C-terminus: a light chain, an optional peptide linker, and an sdAb or its a second epitope binding portion of a VHH fragment; and two identical second polypeptides, each comprising a heavy chain. In some embodiments, the polypeptide complex comprises two identical first polypeptides each comprising from N-terminus to C-terminus: a second epitope binding portion comprising an sdAb or VHH fragment thereof, an optional peptide linker and a light chain; and two identical second polypeptides comprising the heavy chain.

In some embodiments, the polypeptide complex comprises: (a) a full-length antibody consisting of two heavy chains and two light chains, wherein the full-length antibody specifically recognizes the first epitope; (b) the specificity A first sdAb or VHH fragment thereof and a second sdAb or VHH fragment thereof recognizing the second epitope, wherein the first sdAb and the second sdAb may be the same or different. In some embodiments, the C-terminus of the first sdAb or VHH fragment thereof is fused to the N-terminus of each heavy chain, and the N-terminus of the second sdAb or VHH fragment thereof is fused to the N-terminus of each heavy chain C-terminal fusion. In some embodiments, the C-terminus of the first sdAb or VHH fragment thereof is fused to the N-terminus of one heavy chain, and the C-terminus of the second sdAb or VHH fragment thereof is fused to the N-terminus of the other heavy chain - End fusion. In some embodiments, the N-terminus of the first sdAb or VHH fragment thereof is fused to the C-terminus of one heavy chain, and the N-terminus of the second sdAb or VHH fragment thereof is fused to the C-terminus of the other heavy chain - End fusion. In some embodiments, the C-terminus of the first sdAb or VHH fragment thereof is fused to the N-terminus of one light chain, and the C-terminus of the second sdAb or VHH fragment thereof is fused to the N-terminus of the other light chain - End fusion. In some embodiments, the N-terminus of the first sdAb or VHH fragment thereof is fused to the C-terminus of one light chain, and the N-terminus of the second sdAb or VHH fragment thereof is fused to the C-terminus of the other light chain - End fusion. In some embodiments, the first sdAb or VHH fragment thereof is tandem with the second sdAb or VHH fragment thereof, and the N-terminus of the tandem fragment is fused to the C-terminus of the heavy or light chain. In some embodiments, the first sdAb or VHH fragment thereof is tandem with the second sdAb or VHH fragment thereof, and the C-terminus of the tandem fragment is fused to the N-terminus of the heavy or light chain.

In some embodiments, in the polypeptide complex, the first epitope binding moiety comprises a Fab or Fab' and the second epitope binding moiety comprises an sdAb or VHH fragment thereof. In some embodiments, the N-terminus of the second epitope binding moiety is fused to the C-terminus of the Fab or Fab' heavy chain. In some embodiments, the N-terminus of the second epitope binding moiety is fused to the C-terminus of the Fab or Fab' light chain. In some embodiments, the C-terminus of the second epitope binding moiety is fused to the N-terminus of the Fab or Fab' heavy chain. In some embodiments, the C-terminus of the second epitope binding moiety is fused to the N-terminus of the Fab or Fab' light chain.

In some embodiments, the polypeptide complex comprises a Fab or Fab' that specifically recognizes a first epitope, and a first sdAb or VHH fragment thereof that specifically recognizes a second epitope and a second sdAb or VHH fragment thereof, Wherein the first sdAb and the second sdAb can be the same or different. In some embodiments, the C-terminus of the first sdAb or VHH fragment thereof is fused to the N-terminus of a Fab or Fab' heavy chain, and the N-terminus of the second sdAb or VHH fragment thereof is fused to the Fab or Fab' N-terminus 'C-terminal fusion of the heavy chain. In some embodiments, the C-terminus of the first sdAb or VHH fragment thereof is fused to the N-terminus of the Fab or Fab' light chain, and the N-terminus of the second sdAb or VHH fragment thereof is fused to the Fab or Fab' light chain 'C-terminal fusion of the light chain. In some embodiments, the N-terminus of the first sdAb or VHH fragment thereof is fused to the C-terminus of a Fab or Fab' light chain, and the N-terminus of the second sdAb or VHH fragment thereof is fused to the Fab or Fab' light chain 'C-terminal fusion of the heavy chain. In some embodiments, the N-terminus of the first sdAb or VHH fragment thereof is fused to the C-terminus of the Fab or Fab' heavy chain, and the N-terminus of the second sdAb or VHH fragment thereof is fused to the Fab or Fab' heavy chain 'C-terminal fusion of the light chain. In some embodiments, the first sdAb or VHH fragment thereof is in tandem with the second sdAb or VHH fragment thereof, and the N-terminus of the tandem fragment is fused to the C-terminus of the Fab or Fab' heavy or light chain. In some embodiments, the first sdAb or VHH fragment thereof is in tandem with the second sdAb or VHH fragment thereof, and the C-terminus of the tandem fragment is fused to the N-terminus of the Fab or Fab' heavy or light chain.

In some embodiments, in a polypeptide complex, the first epitope binding moiety comprises (Fab) ₂ or (Fab') ₂ and the second epitope binding moiety comprises an sdAb or VHH fragment thereof. In some embodiments, the N-terminus of the second epitope binding moiety is fused to the C-terminus of one of the heavy chains of (Fab) ₂ or (Fab') ₂ . In some embodiments, the N-terminus of the second epitope binding moiety is fused to the C-terminus of one of the light chains of (Fab) ₂ or (Fab') ₂ . In some embodiments, the C-terminus of the second epitope binding moiety is fused to the N-terminus of one of the heavy chains of (Fab) ₂ or (Fab') ₂ . In some embodiments, the C-terminus of the second epitope binding moiety is fused to the N-terminus of one of the light chains of (Fab) ₂ or (Fab') ₂ .

In some embodiments, the polypeptide complex comprises (Fab) ₂ or (Fab') ₂ that specifically recognizes a first epitope, and a first sdAb or VHH fragment thereof that specifically recognizes a second epitope and a second An sdAb or VHH fragment thereof, wherein the first sdAb and the second sdAb may be the same or different. In some embodiments, the C-terminus of the first sdAb or VHH fragment thereof is fused to the N-terminus of one heavy chain of (Fab) ₂ or (Fab') ₂ , and the second sdAb or VHH fragment thereof is fused to the C-terminus of one of the heavy chains of (Fab) ₂ or (Fab') ₂ . In some embodiments, the C-terminus of the first sdAb or VHH fragment thereof is fused to the N-terminus of one light chain of (Fab) ₂ or (Fab') ₂ , and the second sdAb or VHH fragment thereof The N-terminus of (Fab) ₂ or (Fab') ₂ is fused to the C-terminus of one of the light chains of (Fab')2. In some embodiments, the N-terminus of the first sdAb or VHH fragment thereof is fused to the C-terminus of a light chain of (Fab) ₂ or (Fab') ₂ , and the second sdAb or VHH fragment thereof is fused to the C-terminus of one of the heavy chains of (Fab) ₂ or (Fab') ₂ . In some embodiments, the N-terminus of the first sdAb or VHH fragment thereof is fused to the C-terminus of one of the heavy chains of (Fab) ₂ or (Fab') ₂ , and the second sdAb or VHH fragment thereof The N-terminus of (Fab) ₂ or (Fab') ₂ is fused to the C-terminus of one of the light chains of (Fab')2. In some embodiments, the first sdAb or VHH fragment thereof is in tandem with the second sdAb or VHH fragment thereof, and the N-terminus of the tandem fragment is tandem with one of the heavy chains of (Fab) ₂ or (Fab') ₂ or C-terminal fusion of the light chain. In some embodiments, the first sdAb or VHH fragment thereof is in tandem with the second sdAb or VHH fragment thereof, and the C-terminus of the tandem fragment is attached to one of the heavy chains of (Fab) ₂ or (Fab') ₂ or N-terminal fusion of the light chain.

In some embodiments, the antibody or polypeptide complex of the present disclosure binds mammalian coronavirus S protein, eg, human coronavirus S protein, simian coronavirus S protein. For example, an antibody or polypeptide complex of the present disclosure specifically binds to one or more epitopes (eg, linear or conformational epitopes) on the coronavirus S protein.

In some embodiments, the present disclosure provides a polypeptide complex comprising or having at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence set forth in SEQ ID NO: 29, 30 or 31 , 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences.

In further embodiments, the present disclosure provides a polypeptide complex, wherein the polypeptide complex is a bispecific antibody complex consisting of 2 heavy chains and 2 light chains The heavy chain comprises or consists of or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence set forth in SEQ ID NO: 29, 30 or 31 , 96%, 97%, 98% or 99% identical sequences, and the light chain comprises or consists of or has at least 80%, 85%, 90%, 91%, or the amino acid sequence shown in SEQ ID NO: 23 Sequence composition of %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In some embodiments, the amino acid changes do not occur in the CDRs of the VHH fragment. In further embodiments, the polypeptide complexes of the present disclosure are bispecific antibody complexes BsAbl6, BsAbl7, or BsAbl8 described herein.

Peptide linker

The polypeptide complexes described herein may comprise one or more peptide linkers between the first epitope binding moiety and the second epitope binding moiety. In some embodiments, the first epitope binding site and the second epitope binding moiety are fused directly to each other without a peptide linker therebetween.

The epitope binding portions of the polypeptide complexes can be fused to each other via a peptide linker. The peptide linkers linking the different epitope binding moieties can be the same or different. Each peptide linker can be individually optimized. Peptide linkers can be of any suitable length. In some embodiments, the peptide linker is at least about any of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more amino acids in length. In some embodiments, the length of the peptide linker is any of the following: about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, or about 10 amino acids to about 30 amino acids in length.

Peptide linkers can have naturally occurring sequences or non-naturally occurring sequences. For example, sequences derived only from the hinge region of an antibody heavy chain can be used as linkers. See, eg, WO1996/34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G)n (n is an integer of at least 1), glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGGS)n, (GGGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker comprises the amino acid sequence of GGGGSGGGS (SEQ ID NO:42) or GGGGSGGGGSGGGGS (SEQ ID NO:43). In some embodiments, the peptide linker comprises the hinge region of an IgG, such as the hinge region of human IgGl. In some embodiments, the peptide linker comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO:44). In some embodiments, the peptide linker comprises a modified sequence derived from the hinge region of IgG, such as the hinge region of human IgGl. For example, one or more cysteines in the hinge region of an IgG can be substituted with serine. In some embodiments, the peptide linker comprises the amino acid sequence EPKSSDKTHTSPPSP (SEQ ID NO:45).

IV. Nucleic acids of the present disclosure and host cells comprising the same

In one aspect, the present disclosure provides nucleic acids encoding the antibody or polypeptide complexes herein that specifically bind to the coronavirus S protein, or any chain or functional fragment thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells) or other cells suitable for the production of antibody or polypeptide complexes. In another embodiment, the host cell is prokaryotic. For example, a nucleic acid of the present disclosure comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-7 and 22-31, or encoding an amino acid sequence selected from any of SEQ ID NOs: 1-7 and 22-31 An amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the indicated amino acid sequence nucleic acid. In some embodiments, the nucleic acid of the present disclosure comprises the sequence set forth in any one of SEQ ID NOs: 32-41.

In one embodiment, the nucleic acid of the present disclosure comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 23, 29, 30, and 31, or encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 29, 30 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of 31 The amino acid sequence of a nucleic acid. In some embodiments, the nucleic acid of the present disclosure comprises the sequence set forth in SEQ ID NO: 33, 39, 40, or 41.

The present disclosure also encompasses nucleic acids that hybridize under stringent conditions to nucleic acids that encode polypeptide sequences having one or more amino acid substitutions (eg, conservative substitutions), deletions, or insertions compared to nucleic acids that include encoding selected A nucleic acid that encodes a nucleic acid sequence selected from the amino acid sequence shown in any one of SEQ ID NOs: 1-7 and 22-31; A nucleic acid whose sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the nucleic acid sequence of the amino acid sequence.

The present disclosure also encompasses nucleic acids that hybridize under stringent conditions to nucleic acids that encode polypeptide sequences having one or more amino acid substitutions (eg, conservative substitutions), deletions, or insertions compared to nucleic acids that include encoding selected A nucleic acid that encodes a nucleic acid sequence selected from the amino acid sequence shown in any one of SEQ ID NOs: 23, 29, 30 and 31; A nucleic acid whose sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the nucleic acid sequence of the amino acid sequence.

In one embodiment, one or more vectors are provided comprising the nucleic acids or polynucleotides of the present disclosure. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). In some embodiments, host cells comprising the expression vectors of the present disclosure are provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for producing antibodies. Suitable host cells include prokaryotic microorganisms such as E. coli. Host cells can also be eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as insect cells and the like. Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered for growth in suspension can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells ( CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NSO, P3X63 and Sp2/0, etc. For a review of suitable mammalian host cell lines for protein production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cells are CHO cells or 293 cells. Once the expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this, eg, protoplast fusion, calcium phosphate precipitation, electroporation, transduction of retroviruses, viral transfection, biolistic, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are grown in culture medium and screened for appropriate activity. Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on methods known in this specification and in the prior art, depending on the particular expression vector used and Mammalian host cell modification or optimization. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide prototrophy, biocidal resistance (eg, antibiotics), or heavy metal (eg, copper) resistance, etc. to an auxotrophic host. The selectable marker gene can be introduced into the same cell by direct ligation to the DNA sequence to be expressed or by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements can include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

V. Production and Purification of Antibody or Polypeptide Complexes of the Disclosure

In one embodiment, the present disclosure provides a method of making an antibody or polypeptide complex of the present disclosure, wherein the method comprises culturing a complex comprising an antibody or polypeptide encoding the antibody or polypeptide under conditions suitable for expression of the antibody or polypeptide complex. The nucleic acid of the complex or the host cell of the expression vector comprising the nucleic acid, and optionally the isolation of the antibody or polypeptide complex. In some embodiments, the method further comprises recovering the antibody or polypeptide complex from the host cell (or host cell culture medium).

For recombinant production of an antibody or polypeptide complex of the present disclosure, a nucleic acid encoding the disclosed antibody or polypeptide complex is first obtained and inserted into a vector for further cloning and/or expression in host cells. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes capable of binding specifically to nucleic acids encoding the antibody or polypeptide complexes of the present disclosure.

Antibody or polypeptide complexes of the present disclosure prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art. The purity of the antibody or polypeptide complexes of the present disclosure can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

VI. Activity Assays for Antibody or Polypeptide Complexes of the Disclosure

The antibody or polypeptide complexes provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art. In one aspect, the antibody or polypeptide complexes of the present disclosure are tested for their antigen-binding activity, eg, by known methods such as ELISA, Western blotting, and the like. Binding to the coronavirus S protein can be determined using methods known in the art. In some embodiments, the binding of the antibody or polypeptide complexes of the present disclosure to the coronavirus S protein is assayed using SPR or biofilm interferometry. The present disclosure also provides assays for identifying biologically active antibody or polypeptide complexes. Biological activities can include, for example, blocking binding to cell surface ACE2.

VII. DRUG COMBINATIONS AND PHARMACEUTICAL FORMULATIONS

In some embodiments, the present disclosure provides compositions comprising any of the antibody or polypeptide complexes described herein, preferably the compositions are pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier such as a pharmaceutical excipient. In one embodiment, a composition (eg, a pharmaceutical composition) comprises a pharmaceutically acceptable carrier of the present disclosure, in combination with one or more other therapeutic agents (eg, an anti-infective active agent, a small molecule drug). Anti-infective active agents and small molecule drugs suitable for the present disclosure can be any anti-infective active agents, small molecule drugs used to treat, prevent or alleviate coronavirus infection in subjects, including but not limited to remdesivir, Bavirin, oseltamivir, zanamivir, hydroxychloroquine, interferon-α2b, analgesics, azithromycin, and corticosteroids. In the context of the present disclosure, coronavirus infection includes infection caused by coronaviruses, including but not limited to SARS-CoV-2, SARS-CoV.

In some embodiments, the pharmaceutical compositions or formulations of the present disclosure comprise suitable pharmaceutically acceptable carriers such as pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers suitable for use in the present disclosure can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerin , propylene, glycol, water, ethanol, etc. See also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago, for the use of excipients and their uses. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin. Compounds containing an antibody or polypeptide of the present disclosure can be prepared by mixing an antibody or polypeptide complex of the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. ed. (1980)). The pharmaceutical formulations or compositions described herein are preferably in the form of lyophilized formulations or aqueous solutions. The pharmaceutical compositions or formulations of the present disclosure may also contain more than one active ingredient required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it would be desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators, and the like. The active ingredients are suitably combined in amounts effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or polypeptide complexes of the present disclosure in the form of shaped articles such as films or microcapsules.

VIII. Combination products or kits

In some embodiments, the present disclosure also provides a combination product comprising at least one antibody or polypeptide complex of the present disclosure, or further comprising one or more other therapeutic agents (eg, anti-infective active agents, small molecule drugs or immunomodulators, etc.).

In some embodiments, two or more components of a combination product of the present disclosure may be administered to a subject in combination sequentially, separately, or simultaneously.

In some embodiments, the present disclosure also provides kits comprising the antibody or polypeptide complexes, pharmaceutical compositions, or combination products of the present disclosure, and optional package inserts directing administration. In some embodiments, the present disclosure also provides pharmaceutical articles of manufacture comprising the antibody or polypeptide complexes, pharmaceutical compositions, combination products of the present disclosure, optionally, the pharmaceutical articles further comprising a package insert directing administration.

IX. Prophylactic and/or Therapeutic Uses of the Antibody or Polypeptide Complexes of the Disclosure

The present disclosure provides a method for preventing a coronavirus-related disease or condition such as a coronavirus infection (preferably COVID-19) in a subject, comprising administering to the subject an antibody or polypeptide complex of the present disclosure. Subjects at risk for coronavirus-related disease include subjects who have been in contact with an infected person or have been exposed to the coronavirus in some other way. Administration of a prophylactic agent can be administered prior to the development of symptoms characteristic of a coronavirus-related disease in order to arrest the disease, or alternatively delay the progression of the disease.

The present disclosure also provides methods of treating a coronavirus-related disease, such as a coronavirus infection, preferably COVID-19, in a patient. In one embodiment, the method involves administering to a patient suffering from the disease an antibody, antibody combination or multispecific antibody of the present disclosure that neutralizes the coronavirus.

In some embodiments, a method of treating a coronavirus infection in a patient is provided, the method comprising administering an antibody or polypeptide complex of the present disclosure. In a preferred embodiment, two of the antibody or polypeptide complexes of the present disclosure are administered to the patient together. In some embodiments, the antibody or polypeptide complexes of the present disclosure can cross-neutralize human and zoonotic coronavirus isolates. In some embodiments, the antibody or polypeptide complex of the present disclosure is administered within the first 24 hours after coronavirus infection.

The pharmaceutical compositions of the present disclosure can be administered to a subject in need thereof in vivo by various routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intratracheal, Oral, intraperitoneal, intradermal, topical, transdermal and intrathecal or by inhalation. The pharmaceutical compositions of the present disclosure can be formulated into solid, semi-solid, liquid or gaseous formulations; including but not limited to tablets, capsules, powders, granules, ointments, solutions, injections, inhalants, and aerosols. Appropriate formulations and routes of administration can be selected depending on the intended application and treatment regimen. The frequency of administration can be determined and adjusted during treatment. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, sustained continuous release formulations of the pharmaceutical compositions of the present disclosure for use in therapy may be suitable. Those skilled in the art will appreciate that appropriate dosages may vary from patient to patient. Determining the optimal dose generally involves balancing the level of therapeutic benefit against any risks or harmful side effects. The dose level selected will depend on a variety of factors including, but not limited to, the activity of the particular antibody or polypeptide complex, route of administration, time of administration, rate of clearance, duration of treatment, other concomitant drugs, the severity of the disorder, and Species, patient's gender, age, weight, condition, general health and previous medical history, etc. The amount and route of administration of the compound is ultimately at the discretion of the physician, veterinarian or clinician, but the dosage is generally selected to achieve local concentrations at the site of action to achieve the desired effect without causing substantial deleterious or adverse side effects. In general, the CL antibody or polypeptide complexes of the present disclosure can be administered in various dosage ranges. In some embodiments, the antibody or polypeptide complexes provided herein can be administered at about 0.01 mg/kg to about 100 mg/kg (eg, about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg ) at a therapeutically effective dose. In certain of these embodiments, the antibody or polypeptide complex is administered at a dose of about 50 mg/kg or less, and in certain of these embodiments, the dose is 10 mg/kg or less, 5 mg/kg or lower, 1 mg/kg or lower, 0.5 mg/kg or lower, or 0.1 mg/kg or lower. In certain embodiments, the dose administered may vary over the course of treatment. For example, in certain embodiments, the initially administered dose may be higher than the subsequently administered dose. In certain embodiments, the administered dose may vary over the course of treatment depending on the subject's response. The frequency of administration can be determined by one of skill in the art, eg, based on the considerations of the attending physician based on the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like. In certain preferred embodiments, a course of treatment involving an antibody or polypeptide complex of the present disclosure will comprise multiple doses of the selected drug administered over a period of weeks or months. More specifically, the antibody or polypeptide complexes of the present disclosure can be administered daily, every two days, every four days, every week, every ten days, every two weeks, every three weeks, or at longer intervals. In this regard, it will be appreciated that the dosage may be varied or the interval adjusted based on patient response and clinical practice.

X. METHODS AND COMPOSITIONS FOR THE DIAGNOSIS AND DETECTION OF CORONAVIRUS

In some embodiments, any antibody or polypeptide complex provided herein can be used to detect the presence of a coronavirus in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic beads complexed with antibody molecules, ELISA assays.

In one embodiment, an antibody or polypeptide complex that specifically binds to the coronavirus S protein for use in a diagnostic or detection method is provided. In another aspect, a method of detecting the presence of coronavirus in a biological sample or contamination of the environment with coronavirus is provided. In certain embodiments, the method comprises detecting the presence of the coronavirus S protein in a biological sample or environment. In certain embodiments, the method comprises contacting a biological sample or environmental sample with an antibody or polypeptide complex as described herein under conditions that allow the antibody or polypeptide complex to bind to the coronavirus S protein, and detecting Whether a complex is formed between the antibody or polypeptide complex and the coronavirus S protein. The formation of a complex indicates the presence of a coronavirus. The method can be in vitro or in vivo.

Exemplary diagnostic assays for coronavirus include, for example, contacting a sample obtained from a patient with an antibody or polypeptide complex of the present disclosure, wherein the antibody or polypeptide complex of the present disclosure is labeled with a detectable label or reporter molecule or used as a capture ligand. body to selectively isolate coronaviruses from patient samples. Alternatively, unlabeled antibodies or polypeptide complexes of the present disclosure can be used in diagnostic applications in combination with a second antibody that is itself detectably labeled. Detectable labels or reporter molecules can be radioisotopes such as 3H, 14C, 32P, 35S or 125I; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine, or enzymes such as alkaline phosphatase, beta -Galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure coronavirus in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in coronavirus diagnostic assays according to the present disclosure include any biological sample obtainable from a patient that contains detectable amounts of the coronavirus spike protein or fragments thereof under normal or physiological conditions. In some embodiments, the biological sample is blood, serum, throat swabs, lower respiratory tract samples (eg, tracheal secretions, tracheal aspirates, bronchoalveolar lavage fluid), or other samples of biological origin. In general, coronavirus spike protein levels will be measured in specific samples obtained from healthy patients (eg, patients unaffected by coronavirus-related disease) to initially establish baseline or standard coronavirus levels. This baseline level of coronavirus can then be compared to levels of coronavirus measured in samples obtained from individuals suspected of having a coronavirus-related condition or symptoms associated with the condition. The antibody or polypeptide complex specific for the coronavirus spike protein may contain no other labels, or it may contain an N-terminal or C-terminal label. In one embodiment, the label is biotin. In binding assays, the position of the label (if present) determines the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin will be oriented so that the C-terminal portion of the peptide is away from the surface.

Embodiments of the present invention can also be enumerated as follows:

1. a polypeptide complex that specifically binds to the S protein of a coronavirus, the polypeptide complex comprising:

(a) a first epitope binding portion comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region together form a specific an antigen-binding domain that binds the first epitope of the coronavirus S protein, and

(b) a second epitope binding portion comprising a single domain antibody or a VHH fragment thereof that specifically binds to the second epitope of the coronavirus S protein,

wherein the first epitope binding moiety and the second epitope binding moiety are fused to each other, and

wherein the first epitope is different from the second epitope.

2. The polypeptide complex according to item 1, wherein the first epitope binding moiety and the second epitope binding moiety do not compete for epitopes with each other.

3. The polypeptide complex according to item 1, wherein the first epitope binding moiety comprises a human antibody, a humanized antibody or a chimeric antibody or an antigen-binding fragment thereof.

4. The polypeptide complex according to item 2, wherein the first epitope binding moiety comprises a human antibody, a humanized antibody or a chimeric antibody or an antigen-binding fragment thereof.

5. The polypeptide complex according to any one of items 1-4, wherein

The heavy chain variable region comprises:

The first heavy chain CDR1 in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes,

The first heavy chain CDR2 in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes, and

The first heavy chain CDR3 in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes; and/or

The light chain variable region comprises:

The light chain CDR1 in the light chain variable region amino acid sequence shown in SEQ ID NO:2 or a variant thereof with no more than 2 amino acid changes,

The light chain CDR2 in the light chain variable region amino acid sequence shown in SEQ ID NO: 2 or a variant thereof with no more than 2 amino acid changes, and

The light chain CDR3 in the light chain variable region amino acid sequence shown in SEQ ID NO: 2 or a variant thereof with no more than 2 amino acid changes.

6. The polypeptide complex according to item 5, wherein the first heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; the first heavy chain CDR2 comprises or consists of SEQ ID NO: The amino acid sequence shown in 9 consists of; the first heavy chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; the light chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; The light chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; and/or the light chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.

7. The polypeptide complex according to any one of items 1-4 and 6, wherein:

The heavy chain variable region comprises or consists of the following sequence: the sequence shown in SEQ ID NO: 1 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% thereof , 98% or 99% identical sequences, and/or

The light chain variable region comprises or consists of the following sequence: the sequence shown in SEQ ID NO: 2 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% thereof , 98% or 99% identical sequences.

8. The polypeptide complex according to item 5, wherein:

9. The polypeptide complex according to any one of 1-4, 6 and 8, wherein the first epitope binding moiety comprises a heavy chain comprising the heavy chain variable region and a variable region comprising the light chain. The light chain of the variable region, and wherein the heavy chain comprises or consists of the sequence shown in SEQ ID NO: 22 or has at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98% or 99% identity; and/or the light chain comprises or consists of the sequence shown in SEQ ID NO: 23 or has at least 90%, 91%, 92 Sequences of %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

10. The polypeptide complex of any one of items 1-4, 6 and 8, wherein the single domain antibody comprises or consists of a VHH fragment.

11. The polypeptide complex according to any one of items 1-4, 6 and 8, wherein the single domain antibody comprises:

The second heavy chain CDR1 in the VHH amino acid sequence shown in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes;

The second heavy chain CDR2 in the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes; and

The second heavy chain CDR3 in the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes.

12. The polypeptide complex according to item 11, wherein the second heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, 18 or 20; the second heavy chain CDR2 comprises or consists of and/or the second heavy chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, 17 or 19.

13. The polypeptide complex according to item 12, wherein:

The second heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14; the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; and the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; The chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

The second heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; and the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; The chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; or

The second heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20; the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; and the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; The chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:16.

14. The polypeptide complex according to any one of items 1-4, 6, 8, 12 and 13, wherein the single domain antibody or VHH fragment thereof comprises or consists of the following sequences: SEQ ID NOs: 5, 6 or the VHH amino acid sequence shown in 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

15. The polypeptide complex according to any one of items 1-4, 6, 8, 12 and 13, wherein the N-terminus of the second epitope binding moiety and at least the N-terminus of the first epitope binding moiety C-terminal fusion of one heavy chain.

16. The polypeptide complex of item 14, wherein the N-terminus of the second epitope binding moiety is fused to the C-terminus of at least one heavy chain of the first epitope binding moiety.

17. The polypeptide complex according to any one of items 1-4, 6, 8, 12 and 13, wherein the N-terminus of the second epitope binding moiety and at least the N-terminus of the first epitope binding moiety C-terminal fusion of one light chain.

18. The polypeptide complex of item 14, wherein the N-terminus of the second epitope binding moiety is fused to the C-terminus of at least one light chain of the first epitope binding moiety.

19. The polypeptide complex according to any one of items 1-4, 6, 8, 12 and 13, wherein the C-terminus of the second epitope binding moiety and at least the first epitope binding moiety N-terminal fusion of one heavy chain.

20. The polypeptide complex of item 14, wherein the C-terminus of the second epitope binding moiety is fused to the N-terminus of at least one heavy chain of the first epitope binding moiety.

21. The polypeptide complex according to any one of items 1-4, 6, 8, 12 and 13, wherein the C-terminus of the second epitope binding moiety and at least the first epitope binding moiety N-terminal fusion of one light chain.

22. The polypeptide complex of item 14, wherein the C-terminus of the second epitope binding moiety is fused to the N-terminus of at least one light chain of the first epitope binding moiety.

23. The polypeptide complex according to any one of items 1-4, 6, 8, 12 and 13, wherein the second epitope binding moiety comprises at least 2 identical or different VHH fragments, the VHH fragments The first epitope binding moiety is fused in tandem or separately to the first epitope binding moiety.

24. The polypeptide complex according to item 14, wherein the second epitope binding moiety comprises at least 2 identical or different VHH fragments that are in tandem with the first epitope binding moiety fused or separately to the first epitope binding moiety.

25. The polypeptide complex according to any one of 16, 18, 20 and 22, wherein the second epitope binding moiety comprises at least 2 identical or different VHH fragments, the VHH fragments being in tandem fused to the first epitope binding moiety or separately to the first epitope binding moiety.

26. The polypeptide complex of any one of items 1-4, 6, 8, 12, 13, 16, 18, 20, 22 and 24, wherein the first epitope binding moiety comprises an Fc region.

27. The polypeptide complex according to item 26, wherein the Fc region is an IgGl Fc or an IgG4 Fc.

28. The polypeptide complex according to item 26, wherein the Fc region is an IgG1 Fc with L234A and L235A or an IgG4 Fc with the S228P mutation.

29. The polypeptide complex of any one of items 1-4, 6, 8, 12, 13, 16, 18, 20, 22, 24, 27 and 28, wherein the first epitope binding moiety and The second epitope binding moieties are fused to each other via peptide bonds or peptide linkers.

30. The polypeptide complex of item 29, wherein the peptide linker has a length of no more than about 30 amino acids.

31. The polypeptide complex according to item 30, wherein the peptide linker comprises a peptide selected from the group consisting of (G)n, (GS)n, (GSGGS)n, (GGGS)n, (GGGGS)n and SEQ ID NO: The group consisting of amino acid sequences shown in 42-45, wherein n is an integer of at least 1.

32. The polypeptide complex of any one of items 1-4, 6, 8, 12, 13, 16, 18, 20, 22, 24, 27, 28, 30 and 31, wherein the single domain antibody It is a camelid single-domain antibody or a humanized single-domain antibody.

33. The polypeptide complex according to item 32, wherein the polypeptide complex comprises or has at least 90%, 91%, 92%, 93%, Sequences of 94%, 95%, 96%, 97%, 98% or 99% identity.

34. The polypeptide complex of any one of items 1-4, 6, 8, 12, 13, 16, 18, 20, 22, 27, 28, 30, 31 and 33, wherein the polypeptide complex is a bispecific antibody complex consisting of 2 heavy chains and 2 light chains, the heavy chains consisting of the amino acid sequence set forth in SEQ ID NO: 29, 30 or 31, and The light chain consists of the amino acid sequence shown in SEQ ID NO: 23.

35. An isolated polynucleotide encoding the polypeptide complex of any one of items 1-34.

36. The polynucleotide according to item 35, comprising the sequence shown in SEQ ID NO:33 and the sequence shown in SEQ ID NO:39, 40 or 41.

37. An isolated vector comprising the polynucleotide of item 35 or 36.

38. A host cell comprising the polynucleotide according to item 35 or 36 or the vector according to item 37.

39. A method of expressing the polypeptide complex according to any one of items 1 to 34, the method comprising culturing the host cell according to item 38 under conditions suitable for expressing the polypeptide complex, and optionally recovering the polypeptide complex of any one of items 1 to 34 from the host cell or from the culture medium.

40. A pharmaceutical composition comprising the polypeptide complex according to any one of items 1 to 34 and a pharmaceutically acceptable carrier.

41. A detection kit comprising the polypeptide complex according to any one of items 1 to 34.

42. Use of the polypeptide complex according to any one of items 1 to 34 in the manufacture of a medicament for the treatment and/or prevention of coronavirus infection in a subject.

43. The use according to item 42, wherein the coronavirus is the SARS-CoV-2 virus and the coronavirus infection is COVID-19.

44. Use of the polypeptide complex according to any one of items 1 to 34 in the preparation of a diagnostic agent or kit for detecting coronavirus or diagnosing coronavirus infection.

45. The use according to item 44, wherein the coronavirus is the SARS-CoV-2 virus and the coronavirus infection is COVID-19.

46. A method for in vitro detection of coronavirus contamination in an environment, comprising: providing an environmental sample; making the environmental sample and the polypeptide complex according to any one of items 1-34 or the detection of item 41 contacting the kit; and detecting the formation of the complex between the polypeptide complex of any one of items 1-34 and the coronavirus S protein.

47. The method of item 46, wherein the coronavirus is the SARS-CoV-2 virus.

48. An antibody or an antigen-binding fragment thereof that specifically binds to coronavirus S protein, comprising:

(a) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: the first heavy chain CDR1 in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 or any other region thereof The variant with more than 2 amino acid changes, the first heavy chain CDR2 in the heavy chain variable region amino acid sequence shown in SEQ ID NO:1 or a variant with no more than 2 amino acid changes, and the variant shown in SEQ ID NO:1 The first heavy chain CDR3 or a variant thereof with no more than 2 amino acid changes in the heavy chain variable region amino acid sequence shown; and wherein the light chain variable region comprises: the light chain variable region shown in SEQ ID NO:2 The light chain CDR1 in the amino acid sequence of the variable region or its variant with no more than 2 amino acid changes, the light chain CDR2 in the light chain variable region amino acid sequence shown in SEQ ID NO:2 or its variant with no more than 2 amino acid changes A variant, and the light chain CDR3 in the light chain variable region amino acid sequence shown in SEQ ID NO:2 or a variant thereof with no more than 2 amino acid changes; or

(b) a VHH fragment comprising: the second heavy chain CDR1 in the VHH amino acid sequence shown in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes; SEQ ID NO: The second heavy chain CDR2 in the VHH amino acid sequence shown in 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes; and the second heavy chain CDR2 in the VHH amino acid sequence shown in SEQ ID NO: 5, 6 or 7 Heavy chain CDR3 or a variant thereof with no more than 2 amino acid changes.

49. The antibody or antigen-binding fragment thereof according to item 48,

wherein the first heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; the first heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9; the first heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9; The chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; the light chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; the light chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12 the amino acid sequence shown; and/or the light chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; or

Wherein the VHH fragment comprises: the second heavy chain CDR1 of the amino acid sequence shown in the formula GRFFGSYX1MS, wherein X1 is Y, T or V; the second heavy chain CDR2 of the amino acid sequence shown in the formula DINTRGX2X3TR, wherein X2 is E or I, and X3 is T or V; and the second heavy chain CDR3 of the amino acid sequence shown in the formula AASX4X5TFX6GRSDPDY, wherein X4 is G or P, X5 is D or A, and X6 is E or F.

50. The antibody or antigen-binding fragment thereof according to item 48 or 49, wherein the antibody is a monoclonal antibody, and its heavy chain variable region is made up of the amino acid sequence shown in SEQ ID NO: 1, and the light chain The variable region consists of the amino acid sequence shown in SEQ ID NO:2.

51. The antibody or antigen-binding fragment thereof according to item 48 or 49, wherein the antibody is a Nanobody, and the VHH fragment consists of the VHH amino acid sequence shown in SEQ ID NO:5.

52. Use of the antibody or antigen-binding fragment thereof according to any one of items 48 to 51 in the manufacture of a medicament for the treatment and/or prevention (eg prophylactic treatment) of a coronavirus infection in a subject.

53. The use according to item 52, wherein the coronavirus is the SARS-CoV-2 virus and the coronavirus infection is COVID-19.

54. The use according to item 45 or 53, wherein the SARS-CoV-2 virus is a SARS-CoV-2 wild-type or a SARS-CoV-2 mutant.

55. The use according to item 54, wherein the S protein of the SARS-CoV-2 mutant has a mutation site or deletion in the S1 subunit compared to the SARS-CoV-2 wild type.

56. The use according to item 55, wherein the S protein of the SARS-CoV-2 mutant has a mutation site in the receptor binding domain of the S1 subunit compared to the SARS-CoV-2 wild type.

57. The use according to item 54, wherein the wild-type S protein of SARS-CoV-2 is the S protein of GenBank accession number QHD43416.1.

58. The use according to item 56, wherein the mutation site comprises or is selected from one or more of the following: amino acid positions 323, 330, 339, 341, 344, 367, 384, 408, 414, 417 , 435, 439, 444, 445, 446, 450, 452, 453, 455, 458, 475, 476, 477, 483, 484, 485, 486, 490, 493, 501, 518, 519.

59. The use according to item 55, wherein the S protein of the SARS-CoV-2 mutant has mutations at amino acid positions 14-318 and 533-685 compared to the SARS-CoV-2 wild-type point.

60. The use according to item 59, wherein the mutation site comprises or is selected from one or more of the following: amino acid positions 614, 681.

61. The use according to item 55, wherein the mutation sites comprise or are selected from one or more of those shown in Table 17.

62. The use according to item 61, wherein the S protein of the SARS-CoV-2 mutant comprises the mutation site N501Y.

63. The use according to item 62, wherein the S protein of the SARS-CoV-2 mutant comprises mutation sites N501Y, E484K and K417N.

64. The use according to item 55, wherein the S protein of the SARS-CoV-2 mutant has a deletion at amino acid position 69 and/or amino acid position 69 compared to the SARS-CoV-2 wild type 70.

65. The use according to item 54, wherein the SARS-CoV-2 mutant is SARS-CoV-2 mutant strain B.1.1.7.

66. The use according to item 54, wherein the SARS-CoV-2 mutant is SARS-CoV-2 mutant strain B.1.351.

beneficial effect

The antibody or polypeptide complex of the present disclosure can effectively inhibit the infection of coronaviruses, and is expected to be an effective drug for the prevention and treatment of such coronaviruses. The antibody or polypeptide complex of the present disclosure is capable of binding to the coronavirus S protein with a binding dissociation equilibrium constant KD of less than about 1 nM, eg, about 0.8 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM. The antibody or polypeptide complexes of the present disclosure are also capable of blocking the binding of the coronavirus S protein to the isolated ACE2 protein with an IC50 of less than about 1 nM, eg, less than about 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.15 nM, 0.12 nM or binding to the cell surface expressed receptor ACE2. The antibody or polypeptide complex of the present disclosure has a high affinity for the coronavirus S protein, eg, has a binding EC50 of less than 0.1 nM, eg, less than 0.05 nM, or even as low as 0.04 nM.

In addition, the antibodies or polypeptides of the present disclosure also have high thermal stability, eg, up to a melting temperature Tm of 50°C, 55°C, 65°C or above, eg, 70 or 72°C above.

More surprisingly, the polypeptide complex of the present disclosure further significantly improves the effect of inhibiting the coronavirus S protein in a synergistic manner, for example, significantly improves the Kd binding to the coronavirus S protein, compared to the single polypeptide or the combined polypeptide. values (Table 12) and IC50 values for blocking the binding of the coronavirus S protein to the cell surface expressed receptor ACE2 (Table 13).

In particular, not only for wild-type strains, but also for mutant strains (including but not limited to UK (mutant B.1.1.7), South Africa (including mutant B) containing the N501Y mutation site, and not only for wild-type strains .1.351) and Brazilian (including mutant P.1) circulating mutant strains, as well as Indian circulating strains (including mutant B.1.617.2) that contain mutations in both the S1 subunit and the Furin protease cleavage site showed a clear activity advantage.

Example

The disclosure, generally described herein, will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure. These examples are not intended to indicate that the experiments below are all or only experiments performed.

Example 1 Preparation of coronavirus S protein antigen and preparation of ACE2 for detection

The following antigens and ACE2 proteins were used in the examples: S protein RBD-His (319Arg-532Asn), S protein S1-huFc (14Gln-685Arg), human ACE2-huFc (18Gln-740Ser), human ACE2-His (18Gln- 740Ser), S protein RBD-mFc (purchased from Sinobio, 40592-V05H), the specific preparation methods of the first four proteins are as follows.

1.1 Plasmid construction

Each protein sequence was obtained from NCBI, wherein the human ACE2 sequence was obtained from NCBI Gene ID: 59272, and the S protein sequence was obtained from NCBI Gene ID: 43740568. The protein sequences were obtained according to the above amino acid fragment positions, and were converted into gene sequences by GenScript. Biotechnology Co., Ltd. performed the gene synthesis of the target fragment. Each target fragment was amplified by PCR, and then constructed into a eukaryotic expression vector pcDNA3.3-TOPO (Invitrogen) by homologous recombination for subsequent recombinant protein expression.

1.2 Plasmid preparation

The constructed recombinant protein expression vectors were transformed into Escherichia coli SS320, cultured overnight at 37°C, and then plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids with For eukaryotic expression.

1.3 Expression and purification of each protein

S protein RBD-His (319Arg-532Asn), S protein S1-huFc (14Gln-685Arg), human ACE2-huFc (18Gln-740Ser) and human ACE2-His (18Gln-740Ser) were all expressed by Expi293 transient expression system (ThermoFisher) , A14635) expression, the specific method is as follows:

On the day of transfection, confirm that the cell density is about 4.5×10 ⁶ to 5.5×10 ⁶ viable cells per ml, and the cell viability is >95%. At this time, adjust the cells to the final concentration with fresh Expi293 expression medium pre-warmed at 37°C for 3 x 10 ⁶ cells per ml. Dilute the target plasmid with Opti-MEMTM pre-cooled at 4°C (add 1 μg plasmid to 1 mL Opti-MEMTM ^{), and dilute the ExpiFectamine TM} ²⁹³ reagent with Opti-MEMTM at the same time, then mix the two in equal volumes and mix by gently pipetting to prepare a The ExpiFectamine ^TM 293 reagent/plasmid DNA mixture was incubated at room temperature for 10-20 min, slowly added to the prepared cell suspension while gently shaking, and finally placed in a cell culture shaker at 37°C, 8% CO ₂ cultured under conditions.

ExpiFectamine ^{TM 293 Transfection Enhancer 1 and ExpiFectamine TM} ²⁹³ Transfection Enhancer 2 were added within 18-22 h after transfection, and the flask was placed in a shaker at 32°C and incubated under 5% CO ₂ conditions. After 5-7 days of transfection, the cell expression supernatant was centrifuged at 15000g for 10min, and the obtained Fc-tagged protein expression supernatant was affinity purified with MabSelectSuRe LX (GE, 17547403), and then washed with 100mM sodium acetate (pH3.0). The target protein was removed, followed by neutralization with 1M Tris-HCl; the obtained His-tagged protein expression supernatant was subjected to affinity purification with Ni Smart Beads 6FF (Changzhou Tiandi Renren Biotechnology Co., Ltd., SA036050), and then eluted with a gradient concentration of imidazole target protein. The eluted proteins were respectively replaced into PBS buffer by ultrafiltration concentration tubes (Millipore, UFC901096). After identification by SDS-PAGE and activity identification, they were frozen at -80°C until use.

1.4 Quality detection of each protein

The S protein S1-huFc (also known as Spike-S1-huFc; or S1-huFc) and the S protein RBD-mFc (also known as Spike-RBD-mFc; or RBD-mFc) prepared in 1.3 above were combined with human ACE2- huFc, human ACE2-His were assayed for mutual binding by ELISA assay and the results are shown in Figures 2A and 2B. As shown in Figure 2A, human ACE2-huFc and RBD-mFc had good binding activities. As shown in Figure 2B, S protein S1-huFc and S protein RBD-mFc had good binding activities to human ACE2-His, respectively, and the activities were comparable.

Example 2 Construction and screening of natural human antibody phage display library

In this example, an antibody gene phage display library was constructed, and the recombinant S protein RBD-mFc (ie, Spike-RBD-mFc, Sinobio, 40592-V05H) was used as the screening antigen to screen the library, and multiple An antibody molecule that specifically binds to the S protein RBD-mFc.

2.1 Construction of human antibody gene library

Take 15 mL of Ficoll-Paque density gradient separation solution (purchased from GE, catalog number: 17144003S) and slowly add it into a 50 mL centrifuge tube. Tilt the centrifuge tube and slowly add 15 mL of normal human blood collected along the tube wall in batches, so that the Ficoll-Paque density gradient separation solution and the normal human blood maintain a clear separation interface. Centrifuge the 50 mL centrifuge tube containing the blood and separation solution at about 15° C. for 20 min, wherein the centrifuge is set to 400 g, the acceleration is 3, and the deceleration is 0 parameters. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll-Paque liquid. PBMC cell layer. The plasma mixture in the upper layer was carefully aspirated with a sterile Pasteur pipette, and then PBMCs were aspirated with a new sterile Pasteur pipette to obtain isolated PBMCs. The isolated PBMCs were first rinsed twice with PBS, then centrifuged at 1500 rpm for 10 min at 4°C, and finally resuspended with 1.5 mL of PBS, and counted by a cell counter (CountStar, CountStar Altair).

Total RNA was extracted from isolated PBMC cells by conventional methods. The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa company, catalog number: 6210A). Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers were designed at the front end of the V region and the back end of the first constant region of the heavy chain and light chain, respectively (Li Xiaolin, Large-capacity non-immune human-derived Fab phage antibody Library construction and preliminary screening, "China Union Medical University" master's thesis, June 2007), the heavy chain variable region gene fragments and light chain variable region gene fragments of the antibody were obtained after PCR. After the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody are recovered, the fragments containing the light chain and heavy chain variable regions of the antibody are amplified by fusion PCR method. Next, the PCR product and the phage display vector were digested, recovered and ligated. The ligation product was recovered by a recovery kit (Omega, catalog number: D6492-02). For specific materials and methods, see Li Xiaolin's paper above. Finally, it was transformed into competent E. coli SS320 (Lucigen, MC1061 F) by electroporation (Bio-Rad, MicroPulser), and the transformed E. coli SS320 bacterial solution was coated on ampicillin-resistant 2-YT solid Plates (solid plates are made up of 1.5% tryptone, 1% yeast extract, 0.5% NaCl and 1.5% agar in g/mL by mass volume).

2.2 Calculation of antibody gene pool

The transformed Escherichia coli SS320 bacterial solution was inoculated with antibiotic-free 2YT medium at a volume of 1:50, cultured at 37°C and 220 rpm for 1.5-2 hours until the OD600 reached 0.5-0.6, and then taken out to room temperature. The bacterial solution was added to a 96-well round-bottom dilution plate at 90 μL/well, and each bacterial solution sample was subjected to 10-fold gradient dilution, with a total of 12 dilution gradients. Use an 8-channel 10 μL range pipette for the diluted sample, and pipette 2 μL of the liquid into the 2YT with carbenicillin and tetracycline concentrations of 50 μg/mL and 50 μg/mL in the order of dilution gradient from low to high (hereinafter also referred to as 2YT). (C+/T+2YT for short) plate, placed upright for 5 min and then placed upside down at 37°C for overnight incubation. The colony growth was observed the next day, and the pool capacity was calculated. The calculation method of storage capacity is as follows, starting from row A, and labelled as

row

1, 2, 3, 4, 5, 6, 7, 8 to row X in sequence. First select the counting hole, first select the counting hole with the number of clones in the range of 3-20 clones, get the number of rows X, and count the number of clones in the corresponding hole n, the calculation formula is 5 × 100 × 10X × n, after calculating , to obtain an antibody gene library with a capacity of 3×10 ¹¹ cfu per milliliter of bacterial liquid, that is, 3×10 ¹¹ antibody genes.

2.3 Preparation of antibody gene phage display library

Based on the capacity of the antibody gene library, pipette 50 OD (1 OD is 5×10 ⁸ cfu) of the fully human antibody gene library into fresh 2-YT liquid medium, so that the initial OD value is 0.1. The resultant was placed in a shaker at 37° C., 220 rpm and cultured to the logarithmic growth phase (OD600=about 0.6), and then VSCM13 helper phage was added at a number 50 times the number of bacteria (that is, the multiplicity of infection (MOI) was about 50). (Purchased from Stratagene), mixed well, left to stand for 30 min, and then cultured in a shaker at 220 rpm for 1 hour. Subsequently, the culture was centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, and the culture medium was replaced with 50 μg/mL carbenicillin/40 μg/mL kanamycin double-resistant 2-YT medium (hereinafter also referred to as 2-YT medium). C+/K+ 2-YT medium), and continued to cultivate overnight at 30°C, 220 rpm. The next day, the bacterial solution was centrifuged at 13000g for 10min, and after collecting the supernatant, 20% PEG/NaCl (prepared by PEG6000 with a volume concentration of 20% and 2.5M NaCl) was added to make the final concentration of PEG/NaCl 4%. And placed on ice for 1 hour, then centrifuged at 13000g for 10min, and the precipitated phage was washed with PBS and stored for subsequent phage screening.

2.4 Screening of antibody gene phage display library

2.4.1 Screening of antibody gene phage display library by magnetic bead method

Magnetic bead screening is based on labeling S protein RBD-mFc with biotin, and then combining with streptavidin-conjugated magnetic beads, incubating and washing the antigen-binding magnetic beads and antibody gene phage display library and elution of the panning process. Typically 3-4 rounds of panning are performed, whereby the specific monoclonal antibodies against the antigen can be enriched in large quantities. In this example, biotin-labeled S protein RBD-mFc was used for phage display library screening, and after 3 rounds of panning, a primary screening of monoclonal antibodies against S protein RBD-mFc was performed.

The specific implementation method of antibody screening is as follows:

The biotin-labeled S protein RBD-mFc was first incubated with streptavidin-coupled magnetic beads, so that the biotin-labeled S protein RBD-mFc was bound to the magnetic beads. The magnetic beads bound to S protein RBD-mFc were incubated with the constructed phage library for 2 h at room temperature. After washing with PBST for 6-8 times, the non-specifically adsorbed phage was removed, and Trypsin (Gibco, 25200072) was added and mixed gently and reacted for 20 min to elute the specifically bound antibody-displayed phage. Subsequently, the log-phase SS320 cells (Lucigen, MC1061 F) were infected with the eluted phage and allowed to stand for 30 min, then cultured at 220 rpm for 1 h, and then VSCM13 helper phage was added and allowed to stand for 30 min, and continued at 220 rpm. The cells were cultured for 1 h, centrifuged and replaced with C+/K+2-YT medium, and the resulting phages were used for the next round of panning.

2.4.2 Screening of antibody gene phage display library by immunotube method

The purpose of both the immunotube method and the magnetic bead method is to enrich the specific antibodies against the antigen, and they are two experimental methods that complement and validate each other.

The principle of immune tube screening is to coat the S protein RBD-mFc on the surface of the immune tube with high adsorption, by adding the phage-displayed antibody library to the immune tube and incubating with the antigen protein adsorbed on the surface of the immune tube, washing and During the elution panning process, after 2-4 rounds of panning, the specific monoclonal antibodies against the antigen are finally enriched.

The specific implementation method is as follows:

In the first round of screening, 1 mL of 100 μg/mL S protein RBD-mFc was added to the immune tube, and it was coated overnight at 4°C. The coating solution was discarded the next day, and 5% milk in PBS was added to block for 2 h. After washing twice with PBS, the constructed phage library of fully human antibody gene was added, incubated for 2 h, washed with PBS for 8 times, and then washed with PBST for 2 times to remove non-specifically bound phage. Then 0.8 mL of 0.05% EDTA trypsinization solution was added to the immunotube for elution of phages that specifically bind to the target antigen. Then infect log phase SS320 cells (Lucigen, 60512-1), let stand at 37°C for 30 min, then culture at 220 rpm for 1 h, add VSCM13 helper phage, let stand for 30 min, and continue to culture at 220 rpm for 1 h , centrifuged and replaced into C+/K+2-YT medium, and continued to culture overnight at 30°C and 220rpm. Phages were pelleted the next day for subsequent 2-4 rounds of screening. Usually, the antigen coating concentration used for the second, third and fourth rounds of phage screening decreases sequentially, 30 μg/mL, 10 μg/mL and 3 μg/mL, respectively; in addition, the PBS washing strength is also gradually increased. The PBS elution times were 12 times, 16 times and 20 times respectively.

ELISA was performed on the eluted phage pools in each round to evaluate the effect of enrichment, and 10 clones were randomly selected from the phage pools screened in each round for sequence analysis. The results are shown in Figures 3A and 3B, from which it can be seen that each round has better enrichment, and the best enrichment is 3rd-4 (ie, sample No. 4 in the third round of panning).

The results showed that the enrichment of antibody sequences was obvious after the third round of screening. Therefore, the clones obtained in the third round were selected for the screening of positive clones by ELISA.

2.5 Selection of single clones

After a total of four rounds of screening, clones obtained in the third round were selected for ELISA screening of positive clones by ELISA. Finally, a total of 88 positive clones that could bind to S protein RBD-mFc were screened out of 2304 clones. After sequencing analysis, ELISA binding and FACS blocking detection at the Fab level, the sequences of multiple clones were selected to construct a full-length antibody for further experiments, wherein the antibody with clone number R15-F7 is the preferred molecule of the present invention.

Example 3 Construction, expression and purification of full-length candidate antibody

In this example, the R15-F7 antibody obtained in Example 2 was constructed as a human IgG1 subtype, wherein the light chain was of the kappa subtype, and the antibody type was a fully human antibody.

3.1 Plasmid construction

The antibody light and heavy chain variable region fragments were obtained by PCR amplification from the Fab antibody-containing strains obtained by screening. By homologous recombination method, it was respectively constructed into the modified eukaryotic expression vector plasmid pcDNA3.3-TOPO (Invitrogen) containing light and heavy chain constant region fragments to form a complete antibody light and heavy chain full-length gene. The sequences shown in SEQ ID NOs: 1-2, 8-13, 22-23 and 32-33 are the variable region amino acid sequence, CDR amino acid sequence, and antibody full-length amino acid sequence of the heavy chain and light chain of the antibody R15-F7, respectively and the full-length nucleotide sequence of the antibody.

3.2 Plasmid preparation

The constructed vectors containing the full-length genes of the light and heavy chains of the antibody were transformed into Escherichia coli SS320 respectively, and cultured at 37°C overnight. Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody light and heavy chain plasmids for eukaryotic expression.

3.3 Expression and purification of antibodies

The candidate antibody R15-F7 was expressed by the ExpiCHO transient expression system (Thermo Fisher, A29133) as follows:

On the day of transfection, confirm that the cell density is about 7×10 ⁶ to 1×10 ⁷ viable cells/mL, and the cell viability is >98%. The concentration was ⁶ x 106 cells/mL. Dilute the target plasmid with OptiPRO ^TM SFM pre-cooled at 4°C (add 1 μg of plasmid to 1 mL of the medium), and at the same time, dilute ^{ExpiFectamine} CHO with OptiPRO ^TM SFM, then mix the two in equal volumes and mix them by gentle pipetting. into ExpiFectamine ^TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5 min, slowly add it to the prepared cell suspension, shake gently at the same time, and finally place it in a cell culture shaker at 37°C, 8% CO ₂ cultured under conditions.

18-22h after transfection, ExpiCHO ^TM Enhancer and ExpiCHO ^TM Feed were added to the culture medium, and the flask was placed in a shaker at 32° C. and 5% CO ₂ to continue culturing. On day 5 post-transfection, add the same volume of ExpiCHO ^™ Feed slowly while gently mixing the cell suspension. After 7-15 days of transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10min, and the obtained supernatant was affinity purified with MabSelectSuRe LX (GE, 17547403), and then with 100mM sodium acetate (pH3.0). The protein of interest was eluted, followed by neutralization with 1M Tris-HCl, and finally the resulting protein was displaced into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).

3.4 Determination of antibody concentration

The concentration of the purified antibody protein was measured by a verified ultra-micro spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300), and the value obtained by dividing the measured A280 value by the theoretical extinction coefficient of the antibody was used as a follow-up study. After passing the quality inspection, aliquot and store at -80°C.

Example 4 Identification of physicochemical properties of candidate antibody

In this example, the relative molecular weight and purity of the candidate antibody R15-F7 were detected by SDS-PAGE, SEC-HPLC and DSF.

4.1 Identification of candidate antibodies by SDS-PAGE

4.1.1 Sample solution preparation

Preparation of non-reducing solution: candidate antibody, control antibody and quality control substance IPI (abbreviation of Ipilimumab, used as a quality control substance for physical and chemical properties such as SDS-PAGE, SEC-HPLC, etc.) 1 μg was added to 5× SDS loading buffer and 40 mM iodoacetamide were heated in a dry bath at 75 °C for 10 min, cooled to room temperature, centrifuged at 12,000 rpm for 5 min, and the supernatant was taken.

Preparation of reducing solution: 2 μg of candidate antibody, control antibody and quality control substance IPI were added to 5×SDS loading buffer and 5mM DTT, heated in a dry bath at 100°C for 10min, cooled to room temperature, centrifuged at 12000rpm for 5min, and the supernatant was taken.

4.1.2 Experimental process

Bis-tris 4-15% gradient gel (purchased from GenScript), electrophoresis at constant voltage 110V, when Coomassie brilliant blue migrates to the bottom of the gel, stop running, take out the gel piece and place it in Coomassie brilliant blue staining solution for 1-2 hours, Discard the staining solution, add the de-staining solution, replace the de-staining solution 2-3 times as needed, and store in deionized water after de-staining until the gel background is transparent.

4.1.3 Experimental results

The results are shown in Figure 4. The results show that the bands of the candidate antibody R15-F7 and the quality control product IPI non-reducing gel are about 150kD, and the bands of the reducing gel are about 55kD and 25kD, respectively, which are in line with the expected size and purity. More than 95%, the purity of the candidate antibody R15-F7 in this batch of samples is 95.40%.

4.2 Purity identification of candidate antibody monomers

In this example, the monomeric purity of candidate antibody R15-F7 was detected by SEC-HPLC.

4.2.1 Material Preparation

Mobile phase: 150mmol/L phosphate buffer, pH 7.4.

Sample preparation: Candidate antibody, control antibody and quality control IPI were diluted to 0.5 mg/mL with mobile phase solution.

4.2.1 Experimental process

An Agilent HPLC 1100 chromatographic column (XBridge BEH SEC 3.5 μm, 7.8 mm I.D.×30 cm, Waters) flow rate was set to 0.8 mL/min, the injection volume was 20 μL, and the VWD detector wavelengths were 280 nm and 214 nm. Inject blank solution, IPI quality control solution and sample solution in sequence.

4.2.3 Experimental results

The percentages of high molecular weight polymers, antibody monomers and low molecular weight substances in the sample were calculated according to the area normalization method, and the results are shown in Figure 5. The results showed that the monomeric purity of candidate antibody R15-F7 in this batch of samples was 99.5%.

4.3 Thermal stability test of candidate antibodies

Differential scanning fluorimetry (DSF) can provide information about the stability of protein structure according to the fluorescence change process in the protein map, detect the conformational change of the protein, and obtain the melting temperature (Tm) of the protein.

In this example, the DSF method was used to detect the Tm value of the candidate antibody R15-F7.

4.3.1 Experimental process

Prepare antibody solution, 0.2 mg/mL, 19 μL/well, set three parallel wells in a 96-well plate (Nunc) for each test article, and use PBS and IPI as reference, then add 1 μL concentration to each well For 100× SYPRO orange dye, mix it with a pipette and prepare for the machine. The thermal stability test of the sample was carried out by ABI 7500FAST RT-PCR instrument, the test type was selected as melting curve, the continuous mode was used, the scanning temperature range was 25-95 °C, the heating rate was 1%, and the 25 °C was equilibrated for 5 minutes. Data were collected during the heating process and reported. Select "ROX" for the group, "None" for the quenching group, and the reaction volume is 20 μL. The temperature corresponding to the first peak and valley of the first derivative of the melting curve is determined as the melting temperature Tm of the candidate antibody. The peak diagram is shown in the figure. 6A-6B, the results show that the candidate antibody R15-F7 has good thermal stability.

4.3.2 Experimental results

The experimental results are shown in Table 1. The results show that the thermal stability of the candidate antibody R15-F7 is about 72°C in this batch of samples, which has good thermal stability.

Table 1 Melting temperatures of candidate and control antibodies

抗体名称Antibody name	Tm(℃)Tm(℃)
R15-F7R15-F7	72.6072.60

Example 5 Affinity determination of candidate antibody

In this example, the affinity activity of candidate antibody R15-F7 for 2019-nCoV coronavirus S protein was detected by ELISA and Fortebio methods.

5.1 Detection of the affinity activity of candidate antibodies based on ELISA

On a 96-well ELISA plate, the recombinant S protein RBD-mFc was coated, 2 μg/mL, 30 μL/well, overnight at 4°C. The next day, the plate was washed three times with PBST and then blocked with 5% nonfat milk for 2 hours. After washing the plate three times with PBST, serially diluted candidate antibody R15-F7 or negative control antibody IPI was added and incubated for 1 hour. After that, after washing 3 times with PBST, 100 μL/well of HRP-labeled anti-human Fc secondary antibody (Abeam, ab98624 (goat anti-human IgG Fc (HRP) pre-adsorbed antibody) diluted at 1:5000) was added and incubated for 1 h. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpectreMax 190). The results are shown in FIG. 7 . The results showed that the candidate antibody R15-F7 showed better S protein affinity, and the binding EC50 value was about 0.05nM.

5.2 Detection of the affinity of candidate antibodies based on Fortebio

In this example, the FortebioBLItz instrument was used to detect the affinity of the candidate antibody R15-F7 with the 2019-nCoV coronavirus S protein RBD-His.

5.2.1 Material Preparation

Weigh 10 g of BSA, measure 5 mL of Tween 20, add it to 1000 mL of 10×PBS, mix well to make 10×KB buffer. Store in aliquots after filtering. Pipette 0.1mL of 0.1M glycine solution with pH=2.0 into 0.9mL of ultrapure water, and mix well to prepare sensor regeneration buffer. The S protein RBD-His as the antigen was diluted to 10 μg/mL with 10×KB, and the antibody was diluted with 2-fold gradient with 10×KB, which were 62.5, 31.25, and 15.63 nM in sequence.

5.2.2 Experimental Procedure

Under dark conditions, use 10×KB buffer to pre-wet the sensor (Anti-Penta-HIS, HIS1K, Fortebio, CA), start testing the sample plate (GreinierBio, PN655209) after at least 10 min, and proceed according to the preset program after the test is correct. First, the antigen S protein RBD-His was used for binding for 300 s. After the binding was completed, the sensor was equilibrated in 10×KB buffer for 30 s. The antigen-bound sensor was transferred to different concentrations of antibody diluents for binding for 300 s. After the signal was stabilized, the transfer was performed again. In 10×KB buffer, the dissociation time was 900s, and finally KD, Kon and Koff were obtained by fitting the binding and dissociation data of different concentrations of antibodies. The antigen-antibody binding pattern and the binding dissociation curve are shown in Figure 8A and Figure 8B, respectively.

5.2.3 Analysis of results

The results are shown in Table 2. The affinity of candidate antibody R15-F7 for binding to S protein RBD is about 5.69 nM, which is excellent.

Table 2 Determination of antibody affinity for 2019-nCoV S protein RBD-His based on Fortebio equipment

抗体名称Antibody name	KD(M)KD(M)	kon(1/Ms)kon(1/Ms)	koff(1/s)koff(1/s)
R15-F7R15-F7	5.69E-095.69E-09	1.60E+051.60E+05	9.12E-049.12E-04

Example 6 Detection of blocking function of candidate antibody

In this example, the ELISA method and the FACS method were used to detect the effect of the candidate antibody R15-F7 on blocking the binding of the 2019-nCoV coronavirus S protein RBD-His to the receptor ACE2 at the protein level and the cellular level, respectively.

6.1 Detection of blocking activity of candidate antibodies based on ELISA method

96-well plates were coated with human ACE2-huFc protein, 8 μg/mL, 30 μL/well, overnight at 4°C. The next day, the 96-well plate was washed three times with PBST and then blocked with 5% skim milk for 2 h. Then, the candidate antibody R15-F7 was serially diluted and premixed with biotin-labeled S protein RBD-His for 1.0 h in advance. After blocking and plate washing, it was transferred to a 96-well ELISA plate and incubated for 1 h. After washing 3 times with PBST, 100 μL/well of 1:5000 diluted secondary antibody NeutrAvidin-HRP (Thermofisher, 31001) was added and incubated for 1 h. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to stop the reaction, and the plate was read by a microplate reader (Molecular Devices, SpecterMax 190) at OD450. Data analysis was performed using a sigmoid dose-response model within Prism ^™ software (GraphPad). The results are shown in Figure 9, using the calculated IC50 value (defined as the concentration of antibody required to reduce the binding of the viral S protein to ACE2 by 50%) as an indicator of blocking potency, the candidate antibody R15-F7 was calculated to have 0.96 nM The IC50 value of R15-F7 indicates that the candidate antibody R15-F7 has excellent ability to block the binding of viral S protein to the isolated ACE2 protein.

6.2 Detection of blocking activity of candidate antibodies based on FACS method

In this example, the activity of the candidate antibody to block the binding of the viral S protein RBD domain to the cell surface expressed receptor ACE2 was evaluated based on the FACS method. The human ACE2-HEK293 cell line used in this example belongs to the stable human ACE2 transfected cell line.

6.2.1 Preparation of ACE2-HEK293 cells

Construction of a plasmid expressing the full length of human ACE2 (18Gln-805Phe): A DNA fragment containing human ACE2 protein was synthesized by gene synthesis technology and cloned into an expression vector. Introduced into E. coli by transformation. After picking a single clone of E. coli, the correct plasmid clone was obtained by sequencing, and the plasmid was extracted and sequenced again to confirm. Electroporation: HEK293 cells were cultured using Gibco's DMEM serum-free medium (Cat. No. 12634010). One day before electroporation, the cells were passaged to 2×10 ⁵ /mL, and the next day, the constructed plasmid was introduced into HEK293 cells using Invitrogen’s electroporation kit (Cat. The electroporated cells were transferred to DMEM medium and placed in a 37°C cell incubator for 48 h. Cell plating after electroporation: The electroporated HEK293 cells were plated into a 96-well plate at 1000 cells/well, puromycin with a final concentration of 2 μg/mL was added, and placed in a carbon dioxide incubator at 37 °C for culture, and 2 μg was added after 14 days. /mL of puromycin medium. Clonal selection, cell expansion and FACS identification: Pick single cell clones grown in 96-well plates, transfer them to 24-well plates for further expansion, and then identify the cell lines that have successfully transfected human ACE2 by FACS.

6.2.2 Experimental Procedure

FACS buffer (1X PBS+2% FBS) was prepared, the candidate antibody R15-F7 and the control antibody were serially diluted with FACS buffer, and 100 μL of the antibody dilution was added to each well in a 96-well round bottom plate. The S protein RBD-mFc was also diluted to 1 μg/mL with FACS buffer, 100 μL was added to the corresponding 96-well plate, and the 96-well plate was placed at 4°C and incubated for 1 h.

The ACE2-HEK293 cells that were passaged 2-4 times and in good growth condition were used for the experiment. After trypsinization, the cells were resuspended, and the supernatant was removed by centrifugation at 4°C and 300 g. Subsequently, the cells were resuspended in FACS buffer, and the cells were counted. The density was adjusted to 2×10 ⁶ cells/mL, 100 μL per well was added to a new 96-well round bottom plate, centrifuged at 4° C., 300 g, and the supernatant was removed. Add 180 μL of the pre-incubated antibody/S protein RBD-mFc mixture to the cells corresponding to the corresponding positions of the 96-well plate, mix by gently pipetting with a drain gun, and incubate at 4°C for 30 min.

The incubated cell mixture was centrifuged at 4°C and 300g to remove the supernatant, then 200 μL of FACS buffer was added to the corresponding wells to resuspend the cells, and the supernatant was removed by centrifugation at 4°C and 300g; this step was repeated twice. PE-labeled anti-mouse IgG-Fc flow antibody (Jackson, 115-115-164) was prepared at 1:200 as a secondary antibody dilution using FACS buffer, and 200 μL/well of this antibody was added to the corresponding cells using a drain gun. Resuspend the cells with the secondary antibody diluent and gently pipetting, and then place the cells at 4°C for 30 min in the dark. After the incubation, centrifuge the cells at 4°C and 300 g to remove the supernatant, add FACS buffer to resuspend the cells, and repeat the process. Step twice. Finally, it was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

6.2.3 Experimental results

The experimental results are shown in Table 3 and FIG. 10 . The results showed that the IC50 value of the candidate antibody R15-F7 for blocking the binding of the RBD domain of the viral S protein to the receptor ACE2 expressed on the cell surface was about 0.32 nM, which had a good blocking effect. The candidate antibody R15-F7 showed dose-dependent receptor binding blocking activity.

Table 3 R15-F7 candidate antibody IC50

抗体名称Antibody name	IC50(nM)IC50(nM)
R15-F7R15-F7	0.320.32

Example 7 Functional detection of candidate antibody neutralizing 2019-nCoV coronavirus

In this example, the anti-CD20 antibody Rituxmab (Rituxmab) was used as a negative isotype control (Isotype IgG) to test and evaluate the neutralizing effect of the candidate antibody R15-F7 on the 2019-nCoV coronavirus. The binding of the 2019-nCoV coronavirus S protein to the receptor ACE2 on the cell surface is the first step for the virus to infect host cells. The Vero E6 cells used in this example belong to the green monkey kidney cell line and naturally express ACE2. Because the sequence homology between green monkey ACE2 and human ACE2 reaches 95%, Vero E6 cells are often used for in vitro efficacy evaluation of anti-coronavirus drug candidates, so Vero E6 cells are used for the virus neutralization activity assay of candidate antibodies.

7.1 Material Preparation

7.1.1 Preparation of cell culture medium

In MEM medium (Invitrogen, 41500-034), add glutamine (final concentration 2 mM), penicillin (final concentration 100 U/mL), streptomycin (final concentration 100 μg/mL), inactivated FBS (final concentration 10 %).

7.1.2 Preparation of virus medium

In MEM medium (Invitrogen, 41500-034), glutamine (final concentration 2 mM), penicillin (final concentration 100 U/ml), streptomycin (final concentration 100 μg/mL), inactivated FBS (final concentration 5) were added %).

7.1.3 Prepare Antibody Diluent

Dilute the test antibody and negative isotype control (Isotype IgG) in serum-free medium with an initial concentration of 60 μg/mL, 2-fold serial dilution, and a total of 12 dilutions (prepared antibody concentrations are 60, 30, 15, 7.5 , 3.75, 1.875, 0.938, 0.469, 0.234, 0.117, 0.059, 0.029 μg/mL). Take 50 μL of each antibody concentration into a 96-well cell culture plate, and set up 3 replicate wells.

7.2 Experimental Procedure

7.2.1 Virus and antibody mixed incubation

The 2019-nCoV virus (BetaCoV/Beijing/IMEBJ01/2020, whose genome serial number is GWHACAX01000000) was obtained, diluted in serum-free MEM, and infected Vero E6 cells. Six days after infection, the 50% tissue culture (in this example, cells) infectious dose (TCID50) was calculated using the Karber method.

An equal volume of 50 μL of the 2019-nCoV virus (BetaCoV/Beijing/IMEBJ01/2020, diluted in serum-free MEM) containing 100 TCID50 was added to each antibody dilution, mixed well, and incubated at room temperature for 60 min. At this time, the final antibody concentration was: 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.234, 0.117, 0.059, 0.029, 0.015 μg/mL.

7.2.2 Infection of cells with virus and antibody cocktails

Collect freshly cultured Vero E6 cells, use the virus medium (5% FBS-MEM) prepared in Example 7.1.2 to prepare a cell concentration of 2 × 10 ⁵ cells/mL, and add 100 μl to the culture of the above virus-containing antibody mixture Plate and mix well, place in a 35°C, 5% _CO2 cell incubator.

7.2.3 Observation of cytopathic changes and calculation of neutralizing effect of antibodies

The cell growth was observed under the microscope on the 2nd day after inoculation, and the cytopathic effect (CPE) was observed and recorded on the 4th day. The final judgment result on the 6th day.

CPE grading standard: "+" means less than 25% of cells show CPE; "++" means more than 25%, less than 50% of cells show CPE; "+++" means 50%-70% of cells show CPE; "++++" means that more than 75% of cells have CPE.

Judgment standard: the antibody group itself has no obvious cytotoxicity, the normal cell control group with only medium is shown as cell growth, and the virus control group with only virus is shown as CPE up to ++++.

The neutralization end point of the antibody to the virus was calculated by Karber method (the antibody dilution was converted into logarithm), that is, the highest dilution concentration of the antibody that could protect 50% of cells from infection by the 100 TCID50 challenge virus solution was the titer of the antibody.

7.2.4 Experimental results

The experimental results are shown in Table 4. The results showed that the candidate antibody R15-F7 could significantly inhibit the infection of Vero E6 cells by the 2019-nCoV virus, and its antibody titer was 8.30 ng/mL, or 55.36 pM, which protected 50% of the cells from infection with 100 TCID50 virus fluid.

Table 4 Neutralization of 2019-nCoV by antibodies

抗体名称Antibody name	抗病毒滴度(ng/mL)Antiviral titer (ng/mL)	抗病毒滴度(pM)Antiviral titer (pM)
R15-F7R15-F7	8.308.30	55.3655.36
同种型IgGIsotype IgG	无中和活性No neutralizing activity	无中和活性No neutralizing activity

Example 8 Construction and screening of camel-derived natural nanobody phage display library

In this example, a nanobody gene phage display library was constructed, and the recombinant 2019-nCoV coronavirus RBD protein (ie, S protein RBD-mFc) was used as the screening antigen to screen the library, and multiple specific binding 2019 - Nanobody against the RBD protein of nCoV coronavirus.

8.1 Construction of the gene library of camel-derived nanobodies

Take 15 mL of Ficoll-Paque density gradient separation solution (purchased from GE, catalog number: 17144003S) and slowly add it into a 50 mL centrifuge tube. Tilt the centrifuge tube and slowly add 15 mL of the collected unimmunized alpaca blood along the tube wall in batches, so that the Ficoll-Paque density gradient separation solution and the alpaca blood maintain a clear separation interface. Centrifuge the 50 mL centrifuge tube containing the blood and separation solution at about 15° C. for 20 min, wherein the centrifuge is set to 400 g, the acceleration is 3, and the deceleration is 0 parameters. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll-Paque liquid. PBMC cell layer. The upper plasma mixture was carefully aspirated with a sterile Pasteur pipette, and then PBMCs were aspirated with a new sterile Pasteur pipette to obtain isolated PBMCs. The isolated PBMCs were first rinsed twice with PBS, then centrifuged at 1500 rpm for 10 min at 4°C, and finally resuspended in 1.5 mL of PBS, and counted by a cell counter (CountStar, CountStar Altair).

Total RNA was extracted from isolated PBMC cells by conventional methods. The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa company, catalog number: 6210A). Based on the situation of the VHH antibody germline, degenerate primers were designed at the front end of the V region and the middle of the second constant region (CH2) of the VHH antibody. After PCR amplification, the VHH-CH2 fragment and the VH-CH1-CH2 fragment of the antibody were obtained. The length difference of the fragments, the PCR products were identified by agarose gel electrophoresis, and the VHH-CH2 fragments were recovered. The recovered VHH-CH2 fragment, by the method of secondary PCR, adopts the forward and reverse primers of the amplification of VHH, with VHH-CH2 as a template to amplify the VHH antibody fragment (Sabir JS, El-Domyati FM et al. Construction. of

camelids VHH repertoire in phage display-based library. C R Biol. 2014 Mar 20;337(4):244-249.doi:10.1016/j.crvi.2014.02.004). Next, the PCR product and the phage display vector were digested, recovered and ligated, and the ligated product was recovered by a recovery kit (Omega, catalog number: D6492-02). For specific materials and methods, please refer to Li Xiaolin's paper above. Finally, it was transformed into competent E. coli SS320 (Lucigen, MC1061 F) by electroporation (Bio-Rad, MicroPulser), and the transformed E. coli SS320 was coated on ampicillin-resistant 2-YT solid Plate (solid plate is prepared by 1.5% tryptone, 1% yeast extract, 0.5% NaCl, 1.5% agar, by mass volume g/mL).

8.2 Calculation of antibody gene pool

row

8.3 Preparation of antibody gene phage display library

Based on the capacity of the antibody gene library, pipette 50 OD (1 OD is 5×10 ⁸ cfu) of the camel-derived nanobody gene library into fresh 2-YT liquid medium, so that the initial OD value is 0.1. The resultant was placed in a shaker at 37° C., 220 rpm and cultured to the logarithmic growth phase (OD600=about 0.6), and then VSCM13 helper phage was added at a number 50 times the number of bacteria (that is, the multiplicity of infection (MOI) was about 50). (Purchased from Stratagene), mixed well, left to stand for 30 min, and then cultured in a shaker at 220 rpm for 1 hour. Subsequently, the culture was centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, and the culture medium was replaced with 50 μg/mL carbenicillin/40 μg/mL kanamycin double-resistant 2-YT medium (hereinafter also referred to as 2-YT medium). C+/K+ 2-YT medium), and continued to cultivate overnight at 30°C, 220 rpm. The next day, the bacterial solution was centrifuged at 13000g for 10min, and after collecting the supernatant, 20% PEG/NaCl (prepared by PEG6000 with a volume concentration of 20% and 2.5M NaCl) was added to make the final concentration of PEG/NaCl 4%. And placed on ice for 1 hour, then centrifuged at 13000g for 10min, and the precipitated phage was washed with PBS and stored for subsequent phage screening.

8.4 Screening of phage display libraries for antibody genes

8.4.1 Screening of antibody gene phage display library by magnetic bead method

Magnetic bead screening is based on labeling S protein RBD-mFc with biotin, and then combining with streptavidin-conjugated magnetic beads, incubating and washing the antigen-binding magnetic beads and antibody gene phage display library and elution of the panning process, usually through 3-4 rounds of panning, whereby the specific monoclonal antibodies against the antigen can be enriched in large quantities. In this example, biotin-labeled S protein RBD-mFc was used for phage display library screening, and after 3 rounds of panning, a primary screening of monoclonal antibodies against S protein RBD-mFc was performed.

The specific implementation method of antibody screening is as follows:

The biotin-labeled S protein RBD-mFc was first incubated with streptavidin-coupled magnetic beads, allowing the biotin-labeled RBD protein to bind to the magnetic beads. The magnetic beads bound to RBD protein and the constructed phage library were incubated at room temperature for 2 h. After washing with PBST for 6-8 times, the non-specifically adsorbed phage was removed, and Trypsin (Gibco, 25200072) was added and mixed gently and reacted for 20 min to elute the specifically bound antibody-displayed phage. Subsequently, the log-phase SS320 cells (Lucigen, MC1061 F) were infected with the eluted phage and allowed to stand for 30 min, then cultured at 220 rpm for 1 h, and then VSCM13 helper phage was added and allowed to stand for 30 min, and continued at 220 rpm. The cells were cultured for 1 h, centrifuged and replaced with C+/K+2-YT medium, and the resulting phages were used for the next round of panning.

8.4.2 Screening of antibody gene phage display library by immunotube method

The specific implementation method is as follows:

In the first round of screening, 1 mL of 100 μg/mL S protein RBD-mFc was added to the immune tube, and the cells were coated overnight at 4°C. The coating solution was discarded the next day, and PBS with 5% milk was added to block for 2 h, and rinsed with PBS for two days. After 200 alpacas, the constructed phage library of nanobody genes was added, incubated for 2 h, washed with PBS for 8 times, and then washed with PBST for 2 times to remove non-specifically bound phage, and then added to the immune tube. Add 0.8 mL of 0.05% EDTA trypsin digestion solution to elute the phage that specifically binds to the target antigen, and then infect log-phase SS320 cells (Lucigen, 60512-1), and let stand at 37°C for 30 min , then cultured at 220rpm for 1h, then added VSCM13 helper phage, let stand for 30min, continued to culture at 220rpm for 1h, centrifuged and replaced into C+/K+2-YT medium, and continued at 30℃, 220rpm Incubate overnight. Phages were pelleted the next day for subsequent 2-4 rounds of screening. Usually, the antigen coating concentration used for the second, third and fourth rounds of phage screening decreases sequentially, to 30 μg/mL, 10 μg/mL and 3 μg/mL, respectively; in addition, the PBS washing strength is also gradually increased. The PBS elution times were 12 times, 16 times and 20 times respectively.

ELISA was performed on the eluted phage pools in each round to evaluate the effect of enrichment, and 10 clones were randomly selected from the phage pools screened in each round for sequence analysis. The enrichment results are shown in Figure 11A and Figure 11B.

The results showed that the antibody sequences were significantly enriched after the third round of screening, with better enrichment in each round, and the best enrichment was 3rd-1 (ie, sample No. 1 in the third round of panning). Therefore, clones obtained in the third round were selected for positive clone screening by ELISA.

8.5 Selection of single clones

After a total of four rounds of screening, clones obtained in the third round were selected for ELISA screening of positive clones by ELISA. After sequencing analysis, ELISA binding and FACS blocking detection at the Fab level, the sequences of multiple clones were selected to construct a full-length antibody (VHH-Fc) for further experiments. Antibodies to P14-F8 are preferred molecules of the invention.

Example 9 Construction, expression and purification of full-length candidate camel-derived nanobodies

In this example, the antibody with clone number P14-F8 obtained in Example 8 was constructed as VHH-Fc of human IgG1 subtype. Antibody preparation was performed for physicochemical properties and functional analysis of antibodies.

9.1 Plasmid construction

The antibody VHH fragments were obtained by PCR amplification from the VHH nanobody-containing strains obtained by screening, and constructed on the eukaryotic expression vector plasmid pcDNA3.3-TOPO (Invitrogen) containing heavy chain constant region fragments by homologous recombination. The complete VHH-Fc gene, the sequences shown in SEQ ID NO: 3, 14-16, 24 and 34 are the heavy chain variable region (VHH) amino acid sequence, CDR amino acid sequence, fusion Fc (VHH- Fc) antibody full-length amino acid sequence and full-length nucleotide sequence.

9.2 Plasmid preparation

9.3 Expression and purification of antibodies

9.4 Determination of antibody concentration

9.2-9.4 For specific experimental operations, refer to "Plasmid preparation, antibody expression and purification, and antibody concentration determination" in Example 3.

Example 10 Identification of physicochemical properties of candidate camel-derived nanobodies

In this example, the relative molecular weight and purity of the candidate antibody P14-F8 were detected by SDS-PAGE, SEC-HPLC and DSF.

10.1 Identification of candidate antibodies by SDS-PAGE

10.1.1 Experimental Procedure

For specific experimental operations, refer to "4.1 Identification of Candidate Antibodies by SDS-PAGE" in Example 4.

10.1.2 Experimental results

The results are shown in Figure 12. The results showed that the bands of the candidate antibody P14-F8 and the quality control IPI non-reducing gel were about 80kD and 150kD, respectively, and the bands of the reducing gel were about 40kD, 55kD, and 25kD, respectively, which were in line with the expected size. The purity of P14-F8 was 90.30 %.

10.2 Purity identification of candidate antibody monomers

In this example, the monomeric purity of candidate antibody P14-F8 was detected by SEC-HPLC.

10.2.1 Material preparation and implementation process

For the specific experimental operation, refer to "4.2 Identification of the Purity of Candidate Antibody Monomers" in Example 4.

10.2.2 Experimental results

The percentages of high molecular weight polymers, antibody monomers and low molecular weight substances in the sample were calculated according to the area normalization method, and the results are shown in Figure 13. The results showed that the monomeric purity of the candidate antibody P14-F8 was 88.73%, and there was a high-molecular aggregate accounting for 11.37%, as indicated by the arrow in Fig. 13 . The antibody molecules are subsequently modified and optimized.

10.3 Thermal stability test of candidate antibodies

Differential scanning fluorimetry (DSF) can provide information about the stability of protein structure according to the fluorescence change process in the protein map, detect the conformational change of the protein, and obtain the melting temperature (Tm) of the protein. In this example, the DSF method was used to detect the Tm value of the candidate antibody P14-F8.

10.3.1 Experimental Procedure

For specific experimental operations, refer to "4.3 Detection of Thermal Stability of Candidate Antibodies" in Example 4.

10.3.2 Experimental results

The experimental results are shown in Table 5. The results showed that the thermal stability of the candidate antibody P14-F8 was not ideal, and the subsequent modification was carried out to improve the thermal stability. _Tm1 and _Tm2 are the 2 melting temperatures of the antibody detected in the thermal stability assay, respectively.

Table 5 Melting temperature of candidate antibodies

抗体名称Antibody name	T _m1(℃) T _m1 (°C)	T _m2(℃) T _m2 (°C)
P14-F8P14-F8	44.6244.62	65.0765.07

Example 11 Affinity determination of candidate camel-derived nanobodies

In this example, the affinity activity of candidate antibody P14-F8 to 2019-nCoV coronavirus S protein RBD-mFc was detected by ELISA and Fortebio methods.

11.1 ELISA-based detection of affinity activity of candidate antibodies

For the specific experimental operation, refer to "5.1 Detection of Affinity Activity of Candidate Antibodies Based on ELISA" in Example 5. The results are shown in Figure 14. The results showed that the EC50 value of P14-F8 binding to S protein RBD-mFc was about 0.09nM, and the affinity was better. The candidate antibody P14-F8 showed better S protein RBD-mFc affinity activity.

11.2 Detection of the affinity of candidate antibodies based on Fortebio

In this example, the FortebioBLItz instrument was used to detect the affinity of the candidate antibody P14-F8 with the 2019-nCoV coronavirus S protein RBD-His.

11.2.1 Material preparation and experimental procedure

For the specific experimental operation, refer to "5.2 Detection of Affinity of Candidate Antibodies Based on Fortebio" in Example 5. Antibodies were diluted at 10×KB into a serial concentration gradient of 62.5, 31.25, 15.63 nM, and the results are shown in FIG. 15 and Table 6.

11.2.3 Analysis of results

The results showed that the binding affinity of candidate antibody P14-F8 to S protein RBD-His was 18.2nM.

Table 6 Determination of antibody affinity for 2019-nCoV S protein RBD-His based on Fortebio equipment

抗体名称Antibody name	KD(M)KD(M)	kon(1/Ms)kon(1/Ms)	koff(1/s)koff(1/s)
P14-F8P14-F8	1.82E-081.82E-08	1.38E+051.38E+05	2.51E-032.51E-03

Example 12 Detection of blocking function of candidate camel-derived nanobodies

In this example, the ELISA method and the FACS method were used to detect the effect of the candidate camel-derived antibody P14-F8 on blocking the binding of the 2019-nCoV coronavirus S protein RBD-His to the receptor ACE2 at the protein level and the cellular level, respectively.

12.1 Detection of blocking activity of candidate antibodies based on ELISA method

For specific experimental operations and calculations, refer to "6.1 Detection of blocking activity of candidate antibodies based on ELISA method" in Example 6. The results are shown in Figure 16, the candidate antibody P14-F8 has an IC50 value of 1.89 nM, indicating that the candidate antibody P14-F8 has an excellent ability to block the binding of the viral S protein RBD-His to the isolated ACE2 protein.

12.2 Detection of blocking activity of candidate antibodies based on FACS method

In this example, the binding activity of the candidate antibody to block the RBD domain of the viral S protein and the receptor ACE2 was evaluated based on the FACS method. The ACE2 used in this example is stably transfected with ACE2-HEK293 cells.

For the specific experimental procedure of ACE2-HEK293 cell line preparation and FACS operation, refer to "6.2 Detection of blocking activity of candidate antibodies based on FACS method" in Example 6. The results are shown in Figure 17. The results show that the IC50 value of antibody P14-F8 for blocking the binding of the viral S protein RBD domain to ACE2 expressed on the cell surface is about 0.47nM, which has a good blocking effect.

Example 13 Functional detection of candidate camel-derived nanobodies to neutralize 2019-nCoV coronavirus

In this example, the anti-CD20 antibody Rituxmab (Rituxmab) was used as a negative isotype control (Isotype IgG) to test and evaluate the neutralizing effect of the candidate antibody P14-F8 on the 2019-nCoV coronavirus. The binding of the 2019-nCoV coronavirus S protein to the receptor ACE2 on the cell surface is the first step for the virus to infect host cells. The Vero E6 cells used in this example belong to the green monkey kidney cell line and naturally express ACE2.

13.1 Material preparation and experimental procedure

For specific experimental operations and calculations, refer to Example 7 "Functional Detection of Neutralization of 2019-nCoV Coronavirus by Candidate-derived Antibodies".

13.2 Experimental Results

The experimental results are shown in Table 7. The results showed that the candidate antibody P14-F8 could inhibit the infection of Vero E6 cells by 2019-nCoV virus, and its antibody titer for protecting 50% of cells from infection with 100TCID50 virus fluid was 1.17 μg/mL, or 14.58 nM.

Table 7 Neutralizing effect of P14-F8 antibody on 2019-nCoV coronavirus

抗体名称Antibody name	抗病毒滴度(μg/mL)Antiviral titer (μg/mL)	抗病毒滴度(nM)Antiviral titer (nM)
P14-F8P14-F8	1.171.17	14.5814.58
同种型IgGIsotype IgG	无中和活性No neutralizing activity	无中和活性No neutralizing activity

Example 14 Humanization of candidate camel-derived nanobodies

In this example, in order to reduce the immunogenicity that may be caused by the camel-derived nanobody P14-F8, the framework region (Framework) of the VHH of the nanobody was subjected to humanized mutation design, and a humanized antibody was obtained through back mutation.

14.1 Nanobody Humanization Transformation Process

Compare the antibody sequence of P14-F8 with the human antibody germline gene (germline) database to find 1-3 germline genes with high homology to P14-F8, and take into account the germline gene Germline medicine The IGHV3-23 germline gene Germline was finally selected, and a total of 11 non-human sites in the P14-F8 framework region were counted. Homology modeling was performed on P14-F8, and the homology modeling referenced the Nanobody Result Model of the PDB database (http://www.rcsb.org/). Combined with the structural model of P14-F8 and the situation of non-human sites, a combined back mutation design was carried out. The back mutation design avoided the introduction of potential post-translational modification sites. A total of 11 antibody sequences with different degrees of humanization were designed for P14-F8. . Through physicochemical properties and ELISA affinity blocking detection, for specific detection methods, refer to "4. Identification of Physicochemical Properties of Candidate Antibodies" and "5. Affinity Determination of Candidate Antibodies" in Examples 4-5.

14.2 Results of Nanobody Humanization Transformation

Table 8 shows the degree of humanization of the 9 designed humanized antibodies; Figure 18A and Figure 18B and Figure 19A and Figure 19B show the affinity blocking effect of the humanized antibodies. The degree of humanization of P14-F8-hVH8 reached 98.39%, and its physicochemical properties and affinity blocking effects were better than those of the parent. The sequences shown in SEQ ID NOs: 4, 14-16, 25 and 35 are the heavy chain variable region amino acid sequence, CDR amino acid sequence, and full-length amino acid sequence of the antibody fused to Fc (VHH-Fc) of antibody P14-F8-hVH8, respectively and full-length nucleotide sequences.

Table 8 Statistics of the number of non-humanization sites in the framework region of P14-F8 antibody and the degree of humanization

克隆编号clone number	人源化比例Humanized ratio	非人源位点数量Number of non-human loci
P14-F8-ParentalP14-F8-Parental	91.13％91.13%	1111
P14-F8-hVH1P14-F8-hVH1	91.94％91.94%	1010
P14-F8-hVH2P14-F8-hVH2	91.94％91.94%	1010
P14-F8-hVH3P14-F8-hVH3	92.74％92.74%	99
P14-F8-hVH4P14-F8-hVH4	92.74％92.74%	99
P14-F8-hVH5P14-F8-hVH5	93.55％93.55%	88
P14-F8-hVH6P14-F8-hVH6	93.55％93.55%	88
P14-F8-hVH7P14-F8-hVH7	96.77％96.77%	44
P14-F8-hVH8P14-F8-hVH8	98.39％98.39%	22
P14-F8-hVH9P14-F8-hVH9	100.00％100.00%	00

Example 15 Affinity maturation of candidate camel-derived nanobodies

In this example, the affinity maturation transformation of the humanized Nanobody P14-F8-hVH8 is mainly described. The affinity, blocking effect and virus neutralizing effect of P14-F8 were evaluated in the previous Examples 11-13. Since the affinity, blocking and virus neutralization effects of P14-F8 are not as good as those of R15-F7 compared with the R15-F7 molecule (refer to Example 5-7 for R15-F7 data), the humanized P14 -F8-hVH8 undergoes affinity modification to improve the affinity and blocking effect of the modified antibody, thereby improving the function of the molecule to neutralize the virus. Affinity engineering methods are based on phage display technology, including library design and construction, screening, functional verification of candidate molecules, and molecular selection.

15.1 Affinity Maturation Library Design and Construction

The antibody engineering library is designed to mutate the CDR region of the antibody. The mutation method includes a single-point saturation mutation and a 2-3 point continuous mutation strategy. The mutations of different CDRs are combined to construct a mutation combinatorial library.

The specific library building method is as follows: first, synthesizing primers containing point mutations (synthesis company: Jinweizhi Biotechnology Co., Ltd.); secondly, using P14-F8-hVH8 to be transformed as a PCR amplification template, amplifying CDRs containing design mutations Sequence, through the method of bridging PCR, the fragments containing different mutations are combined, and the combined complete VHH antibody is inserted into the nanobody phage display vector by enzymatic ligation, and electroporation, storage capacity calculation and phage library preparation are performed. For details, please refer to the operation process. Example 8 Library Construction Section.

15.2 Screening of Affinity Maturation Libraries

For the specific operation method of library screening, please refer to the library screening part in Example 8. The sea selection, primary screening, affinity sorting and sequence analysis of the library were carried out, and 42 clones expressing VHH supernatants were selected for affinity sorting. Binding affinity ranking and sequence analysis data, 18 preferred candidate antibodies were selected for sample preparation and functional screening.

15.3 Preparation of candidate molecules for affinity modification, evaluation of physicochemical properties, and detection of affinity and blocking effect

Preparation of 18 candidate antibodies, evaluation of physicochemical properties, and detection of affinity and blocking effect. For detailed operation methods, see Examples 3-6.

15.4 Selection of candidate molecules for affinity engineering

The data for the evaluation of various indicators of the candidate molecules for affinity modification are shown in the following table. According to the physicochemical properties, affinity and effect of blocking RBD-ACE2 binding of the antibodies, P14-F8-35, P14-F8-38 and P14-F8-43 nanobodies were selected as candidate molecules for double antibody construction. The data summary is shown in Table 9, and the ELISA affinity ranking and blocking results are shown in Figure 20 and Figure 21. The results show that the preferred engineered antibody has better affinity activity and blocking activity than the parent molecule P14-F8-hVH8. The sequences shown in SEQ ID NOs: 5-7, 14-21, 26-28 and 36-38 are the heavy chain variable region amino acid sequences of antibodies P14-F8-35, P14-F8-38 and P14-F8-43, respectively , CDR amino acid sequence, full-length amino acid sequence and full-length nucleotide sequence of the antibody fused to Fc (VHH-Fc).

Table 9 Evaluation data of candidate molecules for affinity modification

抗体编号Antibody number	P14-F8-hVH8P14-F8-hVH8	P14-F8-43P14-F8-43	P14-F8-35P14-F8-35	P14-F8-38P14-F8-38
等电点Isoelectric point	6.736.73	7.167.16	7.167.16	7.167.16
消光系数Extinction coefficient	1.551.55	1.521.52	1.551.55	1.521.52
分子量(kD)Molecular weight (kD)	8080	8080	8080	8080
SDS-PAGE(％)SDS-PAGE (%)	90.390.3	96.196.1	96.996.9	92.492.4
HPLC-SEC(％)HPLC-SEC (%)	88.7088.70	99.4399.43	99.4199.41	99.1799.17
DSF(T _m℃) DSF(T _m °C)	47.8047.80	62.3262.32	56.1556.15	57.7857.78
Fortebio KD(nM)Fortebio KD(nM)	1.62E-081.62E-08	2.63E-092.63E-09	5.12E-095.12E-09	1.75E-091.75E-09
ka(1/Ms)ka(1/Ms)	1.57E+051.57E+05	1.30E+051.30E+05	1.47E+051.47E+05	1.23E+051.23E+05
kd(1/s)kd(1/s)	2.54E-032.54E-03	3.42E-043.42E-04	7.51E-047.51E-04	2.15E-042.15E-04
ELISA亲和EC50(nM)ELISA Affinity EC50 (nM)	0.5090.509	0.0440.044	0.0420.042	0.0200.020
ELISA阻断IC50(nM)ELISA blocking IC50 (nM)	0.1400.140	0.7510.751	0.4730.473	0.3360.336

Example 16 Human antibody and camelid nanobody binding epitope analysis

In this example, in order to clarify the difference between the R15-F7 and P14-F8 antibody epitopes for the double-antibody combination, based on the Fortebio method, the FortebioBLItz instrument was used to determine the antibody epitopes of R15-F7 and P14-F8.

16.1 Experimental Procedure

Under dark conditions, use 10×KB buffer to pre-wet the sensor (Anti-Penta-HIS, HIS1K, Fortebio, CA), and start testing the sample plate (GreinierBio, PN655209) after at least 10 min, and proceed according to the preset program after the test is correct. Immobilized antigen S protein RBD-His, 300s, equilibrated in 10×KB buffer for 30s and bound with 200nM primary antibody for 300s, after the signal stabilized, recorded its signal intensity, and then transferred to 200nM secondary antibody solution , and observe its binding strength. At this time, if the binding signal value is significantly enhanced on the basis of the first antibody, it indicates that the binding epitopes of the two antibodies are different. If the binding signal value is not enhanced compared with the first antibody, it indicates that the two antibodies The binding epitopes of the two antibodies are the same.

16.2 Analysis of Results

16.2.1 Result calculation method

Determine whether there will be epitope competition between the two antibody quality tests according to the following rules:

Signal value of the experimental group: After immobilizing the antigen S protein RBD-His, add antibody 1. After the binding of antibody 1 is balanced, add antibody 2, and observe the signal value generated by adding antibody 2;

Signal value of control group: After immobilizing the antigen S protein RBD-His, add buffer, the incubation time of the buffer and antibody 1 in the experimental group is the same, then add antibody 2, and observe the signal value generated by the addition of antibody 2;

Perfect competition of epitopes: competition percentage < 20%;

Partial competition of epitopes: 20% < competition percentage < 60%;

No competition for epitopes at all: percent competition >60%.

16.2.2 Experimental Results

The experimental results are shown in Table 10. The results showed that the candidate antibodies R15-F7 and P14-F8 had completely different binding epitopes of RBD protein, while P14-F8 and R15-F7 themselves had a strong competitive effect. Therefore, these two antibodies can bind to different antigenic epitopes after the combination of double antibodies, and the preparation and functional verification of double antibodies will be carried out in the following examples.

Table 10 Antibody binding epitope competition analysis

抗体编号Antibody number	R15-F7R15-F7	P14-F8P14-F8
R15-F7R15-F7	12％12%	113％113%
P14-F8P14-F8	104％104%	15％15%

Example 17 Design and construction, expression and purification of diabodies (bi-epitope bispecific antibodies)

In this example, the three nanobodies P14-F8-35, P14-F8-38 and P14-F8-43 obtained by humanization and affinity maturation of R15-F7 and P14-F8 were double-antibody antibodies. Design and preparation.

17.1 Design of Double Antibody

The design of the double antibody relies on the fully human antibody R15-F7 containing light and heavy chains and the nanobodies P14-F8-35, P14-F8-38 and P14-F8-43 obtained after humanization and affinity maturation. Make combinations. The selected molecular pattern is that the nanobody is constructed at the C-terminus of the R15-F7 heavy chain, the IgG subtype of the double antibody molecule is human IgG1, and the ADCC effect and CDC effect are removed by mutating L234A and L235A of Fc (Hezareh M, Parren PW). et al. Effects function activities of a panel of mutants of a broadly neutralizing antibody again human immunodeficiency virus type 1.J Virol. 2001;75(24):12161-12168.doi:10.1128/JVI.75.24.12161-12168.2001).

17.2 Plasmid Construction

Using R15-F7 and P14-F8-35, P14-F8-38 and P14-F8-43 mAb sequences as template sequences, the heavy chain of R15-F7 and P14-F8-43, P14-F8- 35 and P14-F8-38 VHH sequences, the sequences containing R15-F7 heavy chain and VHH were constructed on the eukaryotic expression vector plasmid pcDNA3.3-TOPO (Invitrogen) by homologous recombination method to form a complete double antibody heavy chain. The chain full-length genes are BsAb16, BsAb17 and BsAb18, respectively. The sequences shown in SEQ ID NOs: 29-31, 23, 33 and 39-41 are the amino acid and nucleotide sequences of the full-length heavy chain and full-length light chain of the antibodies BsAb16, BsAb17 and BsAb18, respectively.

17.3 Plasmid preparation

The constructed vectors containing the full-length genes of the double-antibody BsAb16, BsAb17 and BsAb18 light and heavy chains were transformed into Escherichia coli SS320 respectively, and cultured at 37°C overnight. Plasmid extraction was carried out using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody light and heavy chain plasmids for eukaryotic expression.

17.4 Expression, purification and concentration determination of antibodies

The candidate double antibodies BsAb16, BsAb17 and BsAb18 were expressed by the ExpiCHO transient expression system (Thermo Fisher, A29133). For the specific operation method, refer to Example 3.

Example 18 Detection of physicochemical properties of double antibody

In this example, BsAb16, BsAb17 and BsAb18 were detected by SDS-PAGE, HPLC-SEC and DSF. For the specific operation method, refer to Example 4. The test results data are shown in Table 11. The results showed that the physical and chemical properties of the three double antibodies were in line with the standard of conventional antibodies. _Tm1 and _Tm2 are the 2 melting temperatures of the antibody detected in the thermal stability assay, respectively.

Table 11 Summary of physical and chemical properties of double-antibody testing data

Example 19 Affinity determination of double antibody

In this example, the candidate double antibodies BsAb16, BsAb17 and BsAb18 and the corresponding mAbs R15-F7 and P14-F8-43, P14-F8-35 and P14-F8-38 were detected by ELISA and Fortebio against 2019- Affinity activity of nCoV coronavirus S protein.

19.1 Detection of Affinity Activity of Candidate Antibodies Based on ELISA

For the specific experimental method, refer to "Detection of Affinity Activity of Candidate Antibodies Based on ELISA" in Example 5. The data are detailed in Figure 22. The results showed that the affinity of R15-F7 monoclonal antibody was the best based on ELISA, which may be because part of the double-antibody binding epitope was hidden due to the antigen coating on the ELISA plate. Subsequently, the affinity was further determined by Fortebio and the function of the double antibody was evaluated by blocking experiments and virus neutralization experiments.

19.2 Fortebio-based Affinity Detection of Candidate Antibodies

For the specific experimental method, refer to "Detection of Affinity of Candidate Antibodies Based on Fortebio" in Example 5. The data summary is shown in Table 12, and the Fortebio detection chart is shown in Figure 24A-24G. The results showed that the affinity of the double antibodies BsAb16, BsAb17 and BsAb18 was significantly better than that of the corresponding mAbs R15-F7 and P14-F8-43/35/38. The affinity of the double antibody molecule to the antigen is higher than that of the corresponding monoclonal antibody molecule, which is mainly manifested in the slower dissociation rate koff (1/s).

Table 12 Fortebio detects the affinity of candidate antibodies to S protein RBD

抗体名称Antibody name	KD(M)KD(M)	kon(1/Ms)kon(1/Ms)	koff(1/s)koff(1/s)
BsAb16BsAb16	3.80E-103.80E-10	1.75E+051.75E+05	6.64E-056.64E-05
BsAb17BsAb17	6.24E-106.24E-10	1.65E+051.65E+05	1.03E-041.03E-04
BsAb18BsAb18	7.28E-107.28E-10	1.56E+051.56E+05	1.14E-041.14E-04
R15-F7R15-F7	5.89E-095.89E-09	1.55E+051.55E+05	9.13E-049.13E-04
P14-F8-43P14-F8-43	3.06E-093.06E-09	1.26E+051.26E+05	3.86E-043.86E-04
P14-F8-35P14-F8-35	5.56E-095.56E-09	1.39E+051.39E+05	7.75E-047.75E-04
P14-F8-38P14-F8-38	1.99E-091.99E-09	1.22E+051.22E+05	2.43E-042.43E-04

Example 20 Determination of blocking function of double antibody

In this example, the ELISA method and the FACS method were used to detect the candidate double antibodies BsAb16, BsAb17 and BsAb18 and the corresponding mAbs R15-F7, P14-F8-35, P14-F8- 38 and P14-F8-43 block the effect of 2019-nCoV coronavirus S protein RBD binding to ACE2.

20.1 Detection of blocking activity of candidate antibodies based on ELISA

For the specific experimental method, refer to "Detection of blocking activity of candidate antibodies based on ELISA method" in Example 6. The data are detailed in Figure 23. The results showed that the blocking effect of double antibody molecules BsAb17 and BsAb18 was better than that of monoclonal antibody molecules, and the blocking effect of BsAb16 and R15-F7 was similar.

20.2 Detection of blocking activity of candidate antibodies based on FACS method

In this example, the binding activity of the candidate antibody to block the viral S protein RBD-mFc and the receptor ACE2 was evaluated based on the FACS method. The human ACE2-HEK293 cell line used in this example belongs to the stable human ACE2 transfected cell line.

For the specific experimental procedure of ACE2-HEK293 cell line preparation and FACS operation, refer to "6.2 Detection of blocking activity of candidate antibodies based on FACS method" in Example 6. Figures 25A-25C show the dual antibodies BsAb16, BsAb17 and BsAb18 and the corresponding mAbs R15-F7, P14-F8-35, P14-F8-38, P14-F8-43, mAbs in combination with R15-F7+P14-F8 -35, R15-F7+P14-F8-38 and R15-F7+P14-F8-43 blocked the binding of S protein RBD-mFc to ACE2-HEK cells. The results showed that the blocking effect of the three double antibodies was better than that of the monoclonal antibody or the combination of two monoclonal antibodies. Table 13 provides blocking IC50 values.

Table 13 IC50 of candidate antibodies blocking the binding of RBD protein to ACE2-HEK293 cells

抗体名称Antibody name	BsAb17BsAb17	R15-F7R15-F7	P14-F8-35P14-F8-35	R15-F7+P14-F8-35R15-F7+P14-F8-35
IC50(nM)IC50(nM)	0.12270.1227	0.22610.2261	0.29070.2907	0.28230.2823
抗体名称Antibody name	BsAb16BsAb16	R15-F7R15-F7	P14-F8-43P14-F8-43	R15-F7+P14-F8-43R15-F7+P14-F8-43
IC50(nM)IC50(nM)	0.16570.1657	0.22610.2261	0.27380.2738	0.26840.2684
抗体名称Antibody name	BsAb18BsAb18	R15-F7R15-F7	P14-F8-38P14-F8-38	R15-F7+P14-F8-38R15-F7+P14-F8-38
IC50(nM)IC50(nM)	0.1670.167	0.22610.2261	0.27170.2717	0.30340.3034

Example 21 Detection of non-specific binding of double antibody to cells

In this example, based on the FACS method, the candidate antibody and the human cell line HEK293 and Jurkat without 2019-nCoV coronavirus Spike protein expression were evaluated for non-specific binding, and this method was used to preliminarily evaluate the specificity of antibody and antigen binding. The tested antibodies include candidate double antibodies BsAb16, BsAb17 and BsAb18, corresponding mAbs R15-F7 and P14-F8-35, P14-F8-38, P14-F8-43, and mAbs in combination with R15-F7+P14- F8-35, R15-F7+P14-F8-38 and R15-F7+P14-F8-43.

21.1 Experimental procedure

Human HEK293 and Jurkat cells in exponential growth phase were collected, centrifuged at 300 g to remove the supernatant, the cells were resuspended in the prepared FACS buffer, counted, and the density of the cell suspension was adjusted to 2×10 ⁶ /mL. Subsequently, HEK293 and Jurkat cells were added to a 96-well round bottom plate at 100 μL per well, and the supernatant was removed by centrifugation at 300 g. Add 4-fold serial dilutions of candidate antibody and negative control antibody dilution to the corresponding wells respectively. The antibody concentration in the first well is 800nM, and the cells are blown evenly with a blow gun and incubated at 4°C for 30min. Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL of FACS buffer to the corresponding wells and resuspend the cells using a drain gun; repeat twice, centrifuge at 300 g to remove the supernatant; add PE-labeled anti-human-IgG- Fc flow antibody (Abeam, 98596), the cells were blown evenly with a drain gun and placed at 4° C. for 30 min, and the supernatant was removed by centrifugation at 300 g. Subsequently, FACS buffer was added and the cells were resuspended. After repeating twice, FACS buffer was added to the wells at 200 μL per well to resuspend the cells. Finally, it was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

21.2 Experimental Results

Based on FACS data, the mean fluorescence intensity (MFI) of antibody and cell binding and the proportion of positive cells were analyzed under the condition of the highest antibody concentration of 800nM. And the isotype control was 120.0 μg/ml, the nanomab was 64.0 μg/ml, and the secondary antibody combination was 184.0 μg/ml. According to the average fluorescence intensity analysis, the average fluorescence intensity of non-specific binding of each double antibody and monoclonal antibody to cells under high concentration conditions is similar to that of the control antibody. Under the condition of high concentration of antibody, the proportion of non-specific binding of antibody to cells is up to 3.24% in HEK293 and 1.44% in Jurkat cells, which is within the experimental error range, so the non-specific binding of candidate double antibody to cells almost negative. The corresponding data statistics are shown in Table 14, and the experimental data results are shown in Fig. 26A-Fig. 26M and Fig. 27A-Fig. 27M.

Table 14 Nonspecific binding of candidate antibodies to HEK293 and Jurkat cells

Example 22 Functional detection of double antibodies in neutralizing 2019-nCoV coronavirus pseudovirus at the cellular level

In this example, the neutralizing effect of candidate BsAb16, BsAb17 and BsAb18 and corresponding monoclonal antibodies against the 2019-nCoV coronavirus pseudovirus was tested.

22.1 Material Preparation

Take out the reagents (trypsin, DMEM complete medium) stored at 2-8°C and equilibrate at room temperature for more than 30min; inactivate the serum (or plasma) to be tested in a water bath at 56°C for 30min, centrifuge at 6000g for 3min, and then put the The serum was transferred to a 1.5mL centrifuge tube for use; the serum was diluted with DMEM complete medium (1% double antibody, 20mM HEPES, 10% FBS) (usually the initial dilution was 1:30, and then the dilution was 3 times. The serum to be tested was doubling dilution in a gradient, with a total of 6 dilutions); the pseudovirus was diluted to 1.33×10 ⁴ TCID50/mL with DMEM complete medium for use.

22.2 Experimental procedure

Serum and pseudovirus were co-incubated, and serum of different dilutions was added to a 96-well plate with a volume of 100 μL/well. Each dilution was made 3 replicate wells, and then the diluted pseudovirus was added to the wells containing serum. Add volume of 50 μL/well, and make 6-well pseudovirus control (100 μL/well complete medium and 50 μL/well pseudovirus), place the above 96-well plate in a cell culture incubator (37°C, 5% CO ₂ ) and incubate 1h. When the incubation time is 40 min, take out the Huh-7 cells prepared in the incubator (the confluence rate is 80% to 90%), resuspend the cells in complete medium after digestion, and use a cell counter to count the cells. Dilute cells to ² x 105 cells/mL in complete medium. Incubate for 1 h, add 100 μL of cells to each well of the 96-well plate to make 2×10 ⁴ cells in each well, and make a 6-well cell control (each well contains 150 μL of complete medium and 100 μL of cells). Placed in a cell incubator for 20h at 37°C and 5% _CO2 . After 20 hours, the 96-well plate was taken out of the cell incubator, and 150 μL of supernatant was removed from each loading well with a multi-channel pipette, and then luciferase detection reagent (britelite plus, PerkinElmer) that had been equilibrated at room temperature for 30 min was added. , the addition volume was 100 μL/well, and the reaction was performed at room temperature for 2 min in the dark.

22.3 Experimental Results

After the reaction, the liquid in the reaction wells was repeatedly sucked 6-8 times with a multi-channel pipette to fully lyse the cells, and 100 μL of liquid was aspirated from each well and added to the corresponding 96-well chemiluminescence detection plate (Nunc, 236108). ), placed in a GLOMAX chemiluminescence detector to read the luminescence value. Calculation of neutralization inhibition rate: inhibition rate=[1-(mean luminescence intensity of sample group-mean value of cell control group)/(mean luminescence intensity of virus control group-mean value of cell control group)]×100%. Using Graphpad Prism 7 software, S fitting analysis was performed on the neutralization inhibition rate, and the IC50 of the serum to be tested was calculated.

The experimental results of 2019-nCoV coronavirus pseudovirus are shown in Table 15. The virus neutralization effect of BsAb16, BsAb17 and BsAb18 was significantly better than that of the corresponding mAb molecules.

Table 15 Results of candidate antibodies neutralizing 2019-nCoV coronavirus pseudovirus at the cellular level

抗体名称Antibody name	抗病毒滴度(ng/ml)Antiviral titer (ng/ml)	抗病毒滴度(pM)Antiviral titer (pM)
BsAb16BsAb16	3.763.76	21.1121.11
BsAb17BsAb17	2.092.09	11.7211.72
BsAb18BsAb18	4.014.01	22.5322.53
R15-F7R15-F7	11.0611.06	73.7373.73
P14-F8-43P14-F8-43	12.8112.81	160.13160.13
P14-F8-35P14-F8-35	18.7618.76	234.50234.50
P14-F8-38P14-F8-38	36.9236.92	461.50461.50

Example 23 Functional detection of double antibodies in neutralizing the true virus of 2019-nCoV coronavirus at the cellular level

In this example, the neutralizing effect of candidate double antibodies BsAb16, BsAb17 and corresponding mAbs R15-F7 and P14-F8-35 against 2019-nCoV coronavirus was tested. The binding of the 2019-nCoV coronavirus S protein to the receptor ACE2 on the cell surface is the first step for the virus to infect host cells. The Vero E6 cells used in this example belong to the green monkey kidney cell line and naturally express ACE2. The plaque reduction assay used in the detection method, the plaque reduction neutralization test is the gold standard method for detecting antibodies, and the test takes the antibody concentration that reduces the number of plaques by 90% (PRNT90) or 50% (PRNT50) as its titer.

23.1 Material Preparation

African green monkey kidney cells (Vero) were purchased from the American Standard Biological Collection Center (ATCC) and preserved by the Laboratory of Virology, Institute of Microbial Epidemiology, Academy of Military Medical Sciences. SARS-CoV-2 novel coronavirus Beijing isolates BetaCoV/Beijing/IMEBJ01/2020 and BetaCoV/Beijing/IMEBJ08/2020, whose genome sequence numbers are GWHACAX01000000 and GWHAMKA01000000, respectively, were studied by the Institute of Microbial Epidemiology, Academy of Military Medical Sciences. compartments are separated and stored. The virus stock titers were 5×10 ⁵ pfu/mL and 1×10 ⁷ pfu/mL, respectively.

23.2 Experimental procedure

Vero cell culture: add 12 mL of DMEM complete medium to a 75cm ² culture flask, culture at 37°C in a 5% CO ₂ incubator, and passage once every 3 days. The old medium was removed during passage, the cells were washed once with PBS, and 2 mL of 0.25% trypsin-EDTA was added to digest for 3 min in the incubator. The cells were observed to become rounded under the light microscope, the trypsin was discarded, and then 9 mL of culture medium was added to terminate the digestion. The cells were pipetted into single cells with a pipette. Add new medium to 12mL in a new culture flask, mix well, and continue to culture in a cell incubator at 37°C, 5% CO ₂ .

The antibody was diluted 3-fold with cell maintenance solution, mixed with an equal volume of 2019-nCoV, and incubated at 37°C for 1 h; the virus-antibody mixture (200 μL/well) was added to a 24-well culture containing monolayer dense Vero cells Plate, incubate at 37 °C for 1 h, shaking gently for several times in between; discard the virus antibody mixture, add an appropriate volume of preheated nutrient agar cover to each well, and continue to culture in a 37 °C, 5% CO ₂ incubator. Add an appropriate volume of fixative solution every day, fix at room temperature for 1 h, discard the fixative solution and nutrient agar cap, and wash with fixative solution once; add an appropriate volume of 1.0% crystal violet solution, stain at room temperature for 1 h, discard the crystal violet solution, and wash with fixative solution 1 time, count the number of spots. And the inhibition rate was calculated according to the formula (inhibition rate=(1-antibody group/control group)*100%).

23.3 Experimental Results

The data was organized in Excel. The mean and standard deviation of all experiments were completed in Graphpad Prism 7 software and the relevant statistical charts were drawn. The results are shown in Table 16 and Figures 28A-28B. The results show the virus neutralization effect of BsAb17 Best, showed better virus neutralization effect than monoclonal antibody.

Table 16 Results of neutralization of 2019-nCoV coronavirus by candidate antibodies at the cellular level

Example 24 Activity detection of antibodies of the present disclosure in combination with multiple SARS-CoV-2 mutant Spike recombinant proteins

In this example, the binding activity of double antibody BsAb17, monoclonal antibody R15-F7 and monoclonal antibody P14-F8-35 to Spike mutant recombinant protein in SARS-CoV-2 was detected by ELISA.

24.1 Plasmid Construction

The Spike protein sequence (NCBI Protein ID: QHD43416.1) was obtained from NCBI, and the protein sequence was obtained according to the NCBI sequence number. After conversion into the gene sequence, GenScript Biotechnology Co., Ltd. carried out the gene synthesis of the target fragment. According to the reported point mutation information of SARS-CoV-2, the mutations reported in the RBD region on the Spike protein were used to construct Spike-S1 or Spike-RBD protein particles with single point mutation. Each target fragment was amplified by PCR, and then constructed into a eukaryotic expression vector pcDNA3.3 (Invitrogen) by homologous recombination for subsequent expression of recombinant protein. A total of 39 point mutant proteins and 1 wild-type Spike-RBD protein were constructed. For details of the point mutant proteins, see Table 17.

24.2 Plasmid Preparation

24.3 Expression and purification of each protein

Each point mutant recombinant protein was expressed by the Expi293 transient expression system (ThermoFisher, A14635), and the specific method was as follows:

On the day of transfection, confirm that the cell density is about 4.5×10 ⁶ to 5.5×10 ⁶ viable cells per ml, and the cell viability is >95%. At this time, adjust the cells to the final concentration with fresh Expi293 expression medium pre-warmed at 37°C for 3 x 10 ⁶ cells per ml. Dilute the target plasmid with Opti-MEM ^TM pre-cooled at 4°C (add 1 μg plasmid to 1 mL of Opti-MEM ^TM ), and dilute ExpiFectamine ^TM 293 reagent with Opti-MEM ^TM at the same time, then mix the two in equal volumes and mix by gently pipetting. Prepare ExpiFectamine ^TM 293 reagent/plasmid DNA mixture, incubate at room temperature for 10-20min, slowly add it to the prepared cell suspension, shake gently at the same time, and finally place it in a cell culture shaker at 37°C, 8% Cultivated under CO ₂ conditions.

ExpiFectamine ^TM 293 Transfection Enhancer 1 and ExpiFectamine ^TM 293 Transfection Enhancer 2 were added within 18-22 h after transfection, and the flask was placed in a shaker at 32°C and incubated under 5% CO ₂ conditions. After 5-7 days of transfection, the cell expression supernatant was centrifuged at 15000g for 10 min, and the obtained His-tagged protein expression supernatant was subjected to affinity purification with Ni Smart Beads 6FF (Changzhou Tiandiren Biotechnology Co., Ltd., SA036050), and then used The target protein was eluted with a gradient concentration of imidazole. The eluted proteins were respectively replaced into PBS buffer by ultrafiltration concentration tubes (Millipore, UFC901096). After identification by SDS-PAGE and activity identification, they were frozen at -80°C until use.

24.4 Detection of recombinant mutant protein binding activity by ELISA

On a 96-well ELISA plate, the recombinant mutant proteins were coated, respectively, with a coating concentration and a coating volume of 2 μg/mL and 30 μL/well, overnight at 4°C. The next day, the plate was washed 3 times with PBST and then blocked with 5% skim milk for 2 h. After washing the plate 3 times with PBST, the candidate antibody BsAb17, R15-F7, P14-F8-35 or negative control antibody IPI was added in gradient dilution. (purchased from BMS company), and incubated for 1 h. After that, after washing 3 times with PBST, 100 μL/well of HRP-labeled anti-human Fc secondary antibody (Abeam, ab98624 (goat anti-human IgG Fc (HRP) pre-adsorbed antibody) diluted at 1:5000) was added and incubated for 1 h. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). The results are summarized in Table 17, and the corresponding original ELISA data are shown in Figure 29A-F, Figure 30A -F and in Figures 31A-F.

The results of Table 17, Figures 29A-F, Figures 30A-F and Figures 31A-F show that, except for the Spike protein E484K mutation, the binding activity of mAb R15-F7 is lost, and the binding activity of P14-F8-35 is slightly decreased. The point mutation on Spike protein RBD did not affect the binding activity of double antibody BsAb17 and monoclonal antibody R15-F7 and P14-F8-35. At the same time, the results in Table 17, Figures 29A-F, Figures 30A-F and Figures 31A-F also show that in each component of the double antibody, if a certain antigen (or epitope) binding part occurs, the virus mutation escapes phenomenon (such as R15-F7 escape phenomenon in the case of E484K point mutation), because the probability of another antigen (or epitope) binding part also occurs at the same time mutation escape phenomenon is very low, so for many mutant strains ( Such as amino acid positions 323, 330, 339, 341, 344, 367, 384, 408, 414, 417, 435, 439, 444, 445, 446, 450, 452, 453, 455, 458, 475, 476, 477, 483 , 484, 485, 486, 490, 493, 501, 518, 519, 614, 681), the double antibody binding to different epitopes of the present disclosure can still play a role in neutralizing the virus. The E484K point mutation was found for the first time in the B.1.351 mutant strain isolated from the South African epidemic strain. Therefore, in Example 25, the focus of the BsAb17 bispecific antibody on the South African epidemic strain (“NF” containing N501Y, E484K, K417N mutations was determined ) neutralization activity.

Table 17 Statistical table of binding activity of candidate antibodies to Spike protein RBD/S1-recombinant mutant protein

Remarks: "***" means strong binding activity, "**" means second binding activity, and "inactive" means that the mutant antigen has no binding activity to the corresponding antibody.

Example 25 Detection of neutralizing activity of antibodies of the present disclosure against wild strains of SARS-CoV-2 and mutant strains (B.1.351 mutant strains) appearing in South Africa

By detecting the binding activity of candidate antibodies BsAb17, R15-F7 and P14-F8-35 to recombinant mutant proteins, it was found that the antibodies have the possibility of reduced or absent neutralization effect on SARS-CoV-2 mutant strains with E484K mutation. Therefore, in this example, a strain (No.: NPRC 2.062100001, purchased from the National Collection of Pathogenic Microorganisms) with the same mutation site (that is, including N501Y, E484K, K417N mutation sites) as the B.1.351 mutant was used for Virus neutralization activity detection, the strain was isolated and obtained by Guangzhou CDC, and its genome sequence number is PRJNA694014. SARS-CoV-2 novel coronavirus Beijing isolates wild strains BetaCoV/Beijing/IMEBJ01/2020 and BetaCoV/Beijing/IMEBJ08/2020, whose genome sequence numbers are GWHACAX01000000 and GWHAMKA01000000, respectively. Separated and preserved in the research laboratory.

25.1 Material Preparation

Configure cell culture medium: in MEM medium (Invitrogen), add glutamine (final concentration 2mM), penicillin (final concentration 100U/mL), streptomycin (final concentration 100μg/mL), inactivated FBS (final concentration) 10%); configure virus medium: in MEM medium (Invitrogen), add glutamine (final concentration 2mM), penicillin (final concentration 100U/mL), streptomycin (final concentration 100μg/mL), inactivation FBS (final concentration 5%). Prepare antibody dilutions: Dilute the test antibody and negative isotype control (Isotype IgG) in serum-free medium, take 50 μL of each antibody concentration into a 96-well cell culture plate, and set up 3 replicate wells.

25.2 Virus and Cell Incubation

Virus and antibody mixed incubation: add an equal volume of 50 μL of virus containing 100 TCID50 (diluted in serum-free MEM), mix well, and incubate at room temperature for 60 min; virus and antibody mixture infect cells: collect freshly cultured Vero cells, use virus The culture medium (5% FBS-MEM) was prepared with a cell concentration of 2 × 10 ⁵ cells/mL, and 100 μL was added to the above-mentioned culture plate containing the virus-antibody mixture and mixed, and placed at 35°C, 5% CO ₂ cell culture in the box.

25.3 Cytopathic observation and statistics

Observation of cytopathic changes and calculation of neutralizing effect of antibodies: cell growth was observed under a microscope on the 2nd day after inoculation, and cytopathic changes (CPE) were observed and recorded on the 4th day. The final judgment result on the 6th day. The judgment results must meet: the antibody itself has no obvious cytotoxicity; the normal cell control is established; the virus control CPE reaches ++++. The neutralization end point was calculated by Karber's method (the antibody dilution was converted to logarithm), that is, the highest dilution of the antibody that could protect 50% of cells from infection with 100 TCID50 challenge virus fluid was the titer of the antibody.

The results are shown in Figures 32A-32C. Figure 32A shows the neutralization activity detection results of BsAb17 on SARS-CoV-2 wild strain and B.1.351 mutant strain, and Figure 32B shows P14-F8-35 on SARS-CoV-2 wild strain and B.1.351 mutant strain Figure 32C shows the neutralization activity detection results of R15-F7 on SARS-CoV-2 wild strain and B.1.351 mutant strain. The results showed that the bispecific antibody BsAb17 and the nanobody P14-F8-35 had good neutralizing activity against SARS-CoV-2 wild strain and B.1.351 mutant strain. In the neutralization activity test results of the B.1.351 mutant strain, the neutralization activities (IC50 values) of BsAb17 and P14-F8-35 only decreased by 2-3 times. The LY-CoV555 and CB6 monoclonal antibodies developed by Eli Lilly and Shanghai Junshi Biomedical Technology Co., Ltd. (hereinafter referred to as "Junshi") have lost their neutralizing activity against the B.1.351 mutant strain. The monoclonal antibodies developed by Regeneron and Brii Biosciences The neutralizing activity of anti-cocktail therapy against the B.1.351 mutant strain also decreased by 5-10 times; the neutralizing activity of the vaccine developed by Moderna and Pfizer after immunization against the B.1.351 mutant strain decreased by more than 10 times. (Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization, bioRxiv 2021 Jan 26; 2021.01.25.428137.doi:10.1101/2021.01.25.428137.). Therefore, in terms of the neutralizing activity with the B.1.351 mutant strain, compared with other clinically under development mAbs and sera after vaccine immunization reported in the aforementioned literature, the results of this example show great advantages.

Example 26 Detection of the effect of the antibodies of the present disclosure on the prevention and treatment of SARS-CoV-2 infected mice

In this example, the preventive treatment effect of bispecific antibody BsAb17 on SARS-CoV-2 infected mice was detected by means of prophylactic administration. Infection and death of experimental mice caused by SARS-CoV-2 mutant viruses were prevented by pre-administrating sufficient doses of dual neutralizing antibodies before mice were exposed to the SARS-CoV-2 mutant virus environment. The SARS-CoV-2 strain used was MASCp6 (preserved by the Laboratory of Virology, Institute of Microbial Epidemiology, Academy of Military Medical Sciences), which was derived from the SARS-CoV-2 wild-type strain in BALB/c ACE2 wild After multiple passages in vivo, MASCp6 can infect BALB/c mice in which ACE2 is non-humanized. Compared with the wild-type SARS-CoV-2 strain, there are a total of five mutations in the MASCp6 strain, four of which lead to changes in amino acid residues and are located in the open reading frame 1ab (ORF1ab), S and nucleocapsid (N). ) gene, the N501Y mutation is included in the four mutations, which is a mutation of the popular mutant strains in the UK, South Africa and Brazil (including mutant strains B.1.1.7, B.1.351 and P.1). Point (Rapid adaptation of SARS-CoV-2 in BALB/c mice: Novel mouse model for vaccine efficacy, May 2020, DOI: 10.1101/2020.05.02.073411).

The experimental operation is as follows. The experiment was divided into 2 groups. The animals were BALB/c mice, the control group was injected with PBS as a negative control, and the experimental group was injected with 20 mg/kg of BsAb17 antibody for one time. 12h after the first administration, BALB/c mice were intravenously injected with 50 μL of the SARS-CoV-2 strain MASCp6, and the model diagram of the administration of prophylactic treatment is shown in Figure 33, to inject the mice with the virus strain MASCp6 The time of pre-administration of BsAb17 antibody was -1 day. Next, the survival curve of mice infected with SARS-CoV-2 was counted, and the cadavers of the mice were dissected to detect the copy number of the virus infected in the lungs and bronchus. The results of the survival curve infected with SARS-CoV-2 are shown in the figure 34, copy number analysis of pulmonary and bronchial infection viruses is shown in Figures 35A-B.

Figure 34 shows that all the mice in the double-antibody BsAb17 prophylactic treatment group survived, while the mice in the control group died within 1 week due to virus infection. At the same time, Figures 35A and 35B show that the number of virus copies in the lungs and bronchi of the mice in the prophylactic treatment group is significantly lower than that in the mice in the control group, wherein the number of virus copies in the lungs of the mice in the prophylactic treatment group and the control group is per gram of tissue, respectively. 1×10 ^6.7 and 1×10 ^11.7 ; while the virus copy numbers in the bronchi of the prophylactic treatment group and the control group were 1×10 ^8.1 and 1×10 ^10.8 per gram of tissue, respectively. -2 virus infection has excellent preventive and therapeutic effects. Compared with the wild-type SARS-CoV-2 strain, there are five mutations in the MASCp6 strain, one of which is the N501Y mutation of the Spike protein, and the N501 residue is the five keys for SARS-COV-2 receptor recognition. One of the residues, this mutation is the common mutation site of mutant strains B.1.1.7, B.1.351 and P.1. Therefore, the double antibody of the present disclosure exhibits obvious advantages in the prevention and treatment of new coronary pneumonia caused by mutant strains B.1.1.7, B.1.351 and P.1 containing the N501Y mutation site.

Example 27 Antibody Neutralization Experiment of New Coronavirus Wild Strain and Mutant Strain at the Cell Level in Vitro

In this example, the anti-CD20 antibody Rituxmab (Rituxmab) was used as a negative isotype control (Isotype IgG). neutralization effect. The Vero E6 cells used belong to the green monkey kidney cell line and naturally express ACE2. Assays Vero E6 cells were used for viral infection assays. The test is carried out according to the following steps.

27.1 Material Preparation

Prepare cell culture medium: in MEM medium (GIBCO, Life, USA), add 1% final concentration of 100IU/mL PS (Penicillin Streptomycin, GIBCO, Life, USA), 1M HEPES (GIBCO, Life, USA), inactivated FBS (GIBCO, Life, USA, final concentration 10%).

Prepare virus medium: in MEM medium (GIBCO, Life, USA), add 1% PS (Penicillin Streptomycin, Penicillin Streptomycin, GIBCO, Life, USA), 1% 1M HEPES (GIBCO, Life, USA), inactivated FBS (GIBCO, Life, USA, final concentration 2%).

Prepare antibody dilutions: use serum-free medium to dilute the test antibody and negative isotype control (Isotype IgG) with an initial concentration of 100nM, 2- or 4-fold serial dilution, and a total of 12 dilutions (the configuration table is as follows, the first 3 The well and the last well are 4-fold dilution, the rest are 2-fold dilution, and the prepared antibody concentrations are 100, 25, 6.25, 3.125, 1.5625, 0.7813, 0.3906, 0.1953, 0.0977, 0.0488, 0.0244, 0.0061 nM, respectively. Take 50 μL of each antibody concentration into a 96-well cell culture plate, and set up 3-4 replicate wells.

27.2 Test Procedure

Mixed incubation of virus and antibody: Obtained SARS-CoV-2 virus (wild strain, mutant strain B.1.617.2 and B.1.351, IDs are GDPCC-nCOV10, GDPCC 2.00096 and GDPCC-nCOV84, respectively (Guangdong CDC, China) .Compared to the wild strain, the S protein of the mutant strain B.1.617.2 contains the mutation sites T19R, R158G, L452R, T478K, D614G, P681R, D950N, deletions of amino acids 156 and 157 (Δ156/157); and optionally The presence of mutation site G142D. Compared with the wild strain, the S protein of the mutant strain B.1.351 contains mutation sites D80A, D215G, K417N, E484K, N501Y, D614G and A701V; and deletions of amino acids 241, 242 and 243 (Δ241 /242/243). Each SARS-CoV-2 strain was diluted in serum-free MEM and used to infect Vero E6 cells. After 6 days of infection, the 50% cell culture infectious dose (TCID50) was calculated using the Karber method. An equal volume of 50 μL of the SARS-CoV-2 virus containing 100 TCID50 was added to the antibody diluent, mixed well, and incubated at room temperature for 120 min.

Infected cells with virus and antibody mixture: Collect freshly cultured Vero E6 cells, prepare a cell concentration of 2 x 105 cells/mL with virus medium ( ⁵ % FBS-MEM), and add 100 μl to the culture of the above virus-antibody mixture Plate and mix well, place in a 37°C, 5% _CO2 cell incubator.

Observation and statistics of cytopathic changes: Observation of cytopathic changes and calculation of neutralizing effect of antibodies: the growth of cells was observed under a microscope on the 2nd day after inoculation, and the condition of cytopathic changes (CPE) was observed and recorded on the 4th day after inoculation. The final judgment result on the 6th day. The judgment results must meet: the antibody itself has no obvious cytotoxicity; the normal cell control is established; the virus control CPE reaches ++++. CPE grading standard: "+" means less than 25% of cells show CPE; "++" means more than 25%, less than 50% of cells show CPE; "+++" means 50% to 70% of cells show CPE; "++++" means that more than 75% of cells have CPE. The neutralization end point was calculated by Karber's method (the antibody dilution was converted to logarithm), that is, the highest dilution of the antibody that could protect 50% of cells from infection with 100 TCID50 challenge virus fluid was the titer of the antibody.

27.3 Experimental Results

The results are shown in Table 18. The results showed that the bispecific antibody BsAb17 could significantly inhibit the infection of Vero E6 cells by the wild strain and mutant strain B.1.617.2 of SARS-CoV-2 virus, and its antibody titers to protect 50% of the cells from infection with 100TCID50 virus fluid were respectively were 0.78 nM and 0.52 nM, whereas the neutralizing antibody titer of the bispecific antibody BsAb17 against mutant B.1.351 was 25 nM. At the same time, the titers of the corresponding mAb P14-F8-35 were 0.39nM, 1.04nM and 0.26nM, and the virus neutralizing activity was stronger. Although the monoclonal antibody R15-F7 has strong neutralizing activity on the wild strain with a titer of 1.56nM, the neutralizing activity of the mutant strains B.1.617.2 and B.1.351 is significantly weakened, and the titer is higher than 100nM.

Table 18 Cell-level neutralization activity detection of antibodies against SARS-CoV-2 wild-type strains, mutant strains B.1.351 and B.1.617.2 (unit: nM)

	BsAb17BsAb17	R15-F7R15-F7	P14-F8-35P14-F8-35	同种型对照isotype control
SARS-CoV-2野生株SARS-CoV-2 wild strain	0.780.78	1.561.56	0.390.39	>100>100
SARS-CoV-2 B.1.617.2SARS-CoV-2 B.1.617.2	0.520.52	>100>100	1.041.04	>100>100
SARS-CoV-2 B.1.351SARS-CoV-2 B.1.351	25.0025.00	>100>100	0.260.26	>100>100

From the above experimental results, it can be seen that the double antibody of the present disclosure can show obvious advantages in the prevention and treatment of new coronary pneumonia caused by B.1.617.2 and B.1.351 mutant strains.

Example 28 In vitro neutralization effect test of antibody on new coronavirus wild strain and mutant strain B.1.617.2

In this example, the anti-CD20 antibody Rituxmab (Rituxmab) was used as a negative isotype control (Isotype IgG). Virus neutralizing effect. Binding of the SARS-CoV-2 coronavirus S protein to the receptor ACE2 on the cell surface is the first step for the virus to infect host cells. The human alveolar epithelial cell Calu-3 used in this experiment naturally expresses ACE2. Studies have shown that it is an important target cell infected by the new coronavirus SARS-CoV-2, so it can be used as a cell model for in vitro efficacy evaluation of anti-coronavirus drug candidates. The test is carried out according to the following steps.

28.1 Material Preparation

Preparation of cell culture medium: in MEM medium (Invitrogen, 41500-034), add glutamine (final concentration 2 mM), penicillin (final concentration 100 U/mL), streptomycin (final concentration 100 μg/mL), inactivation FBS (final concentration 10%).

Preparation of virus medium: in MEM medium (Invitrogen, 41500-034), add glutamine (final concentration 2mM), penicillin (final concentration 100U/ml), streptomycin (final concentration 100μg/mL), inactivation FBS (final concentration 5%).

Prepare antibody dilutions: Dilute the test antibody and negative isotype control (Isotype IgG) in MEM medium containing 5% FBS, with an initial concentration of 100 nM, 2-fold serial dilution, and a total of 12 dilutions. The prepared antibody concentrations were 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.098 nM, respectively). Take 50 μL of each antibody concentration into a 96-well cell culture plate, and set up 4 replicate wells.

28.2 Experimental Procedure

Mixed incubation of virus and antibody: SARS-CoV-2 virus (wild strain and mutant strain B.1.617.2) was obtained, diluted in serum-free MEM, and infected Calu-3 cells. The 50% tissue culture (cells in this example) infectious dose (TCID50) was calculated 6 days after infection using the Karber method. An equal volume of 50 μL of the SARS-CoV-2 virus containing 200 TCID50 was added to each antibody dilution, mixed well and incubated at 35 degrees for 120 min. At this time, the final concentrations of the test antibodies were: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.098, 0.049 nM.

Infected cells with virus and antibody mixture: Collect freshly cultured Calu-3 cells, prepare a cell concentration of 2 × 10 ⁵ cells/mL with virus medium (5% FBS-MEM), and add 100 μl to the above virus-antibody mixture. The plates were mixed and placed in a 35°C, 5% CO2 cell incubator.

Cytopathic observation and statistics: cell growth was observed under a microscope on the 2nd day after inoculation, and cytopathic effect (CPE) was observed and recorded on the 4th day. The final judgment result on the 6th day. Determination of CPE in test wells: greater than or equal to 10% of cells showed CPE as +, and there was no protective effect. Less than 10% of cells exhibited CPE as +/-. Determination criteria for control wells: the antibody group itself has no obvious cytotoxicity, the normal cell control group with only medium is shown as cell growth CPE-, and the virus control group with only virus added is shown as CPE+. The neutralization end point of the antibody to the virus was calculated by Karber method (the antibody dilution was converted into logarithm), that is, the highest dilution concentration of the antibody that could protect 90% of cells from infection by 100 TCID50 challenge virus solution was the titer of the antibody.

28.3 Experimental Results

The experimental results are shown in Table 19. The test results showed that the bispecific antibody BsAb17 could significantly inhibit the infection of Calu-3 cells by the wild strain and mutant strain B.1.617.2 of SARS-CoV-2 virus, and it protected 90% of the cells from antibody droplets infected with 100TCID50 virus fluid The specific IC90 were 0.049Nm (wild strain) and 0.195nM (mutant strain B.1.617.2), respectively. At the same time, the corresponding IC90 of mAb R15-F7 were 0.098nM (wild strain) and 6.25nM (mutant strain B.1.617.2); the IC90 of mAb P14-F8-35 against both viruses were 0.39nM.

Table 19. Detection of antibody neutralization activity at the cellular level against SARS-CoV-2 wild-type strain and B.1.617.2 mutant strain (unit nM)

	BsAb17BsAb17	R15-F7R15-F7	P14-F8-35P14-F8-35	同种型对照isotype control
SARS-CoV-2野生株SARS-CoV-2 wild strain	0.0490.049	0.0980.098	0.390.39	>100>100
SARS-CoV-2 B.1.617.2SARS-CoV-2 B.1.617.2	0.1950.195	6.256.25	0.390.39	>100>100

From the above experimental results, it can be seen that the double antibody of the present disclosure can show obvious advantages in the prevention and treatment of new coronary pneumonia caused by wild strains and B.1.617.2 mutant strains.

Example 29 In vivo efficacy test of antibodies against novel coronavirus

This example adopts the new coronavirus mouse adaptation strain model (Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy. Science, 2020, eabc4730.doi:10.1126/science.abc4730.) and SARS -CoV-2 B.1.351 infected Balb/C mouse model (Informa database, https://pharma.id.informa.com.) to test the in vivo efficacy of the bispecific antibody BsAb17.

29.1 Experimental Materials

8-9 month old female BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. All animals were kept in SPF environment, and all animal experiments were approved by the Experimental Animal Ethics Committee of the Academy of Military Medical Sciences of the Chinese People's Liberation Army Academy of Military Sciences.

The new coronavirus mouse-adapted strain BetaCoV/Beijing/IMEBJ05-P15/2020 (No.: GWHACFH01000000) was isolated and preserved by the Virology Laboratory of the Institute of Microbial Epidemiology, Academy of Military Medical Sciences, and the new coronavirus B.1.351 mutant strain (No. NPRC2. 062100001) were purchased from the National Collection of Pathogenic Microorganisms, and the strain was isolated from the Guangzhou Center for Disease Control and Prevention. The original titers of the mouse-adapted strain and the B.1.351 mutant strain were 4×10 ⁵ PFU/ml and 4×10 ⁵ PFU/ml, respectively.

29.2 Experimental Procedure

Balb/C mice aged 8-9 months were randomly divided into three groups: antibody prevention + treatment group (mice were given antibody 12 hours before virus infection, 50mg/kg), antibody treatment group (mice after virus infection) Antibodies were administered 2 hours at 50 mg/kg) and controls (mice were given PBS 12 hours before infection and 2 hours after infection). Mice were anesthetized before infection, and mice were inoculated with 4×10 ⁵ PFU/ml of SARS-CoV-2 B.1.351 or 4×10 ⁴ PFU/ml of new coronavirus mouse-adapted strain by intranasal method (30 μl/mice). ), and injected BsAb17 bispecific antibody 50 mg/kg by intraperitoneal route before or after virus infection, respectively.

The state of the mice was observed every day, and on the 5th day after infection, the mice were dissected, lung tissue and trachea were taken and placed in a grinding tube, 1 ml of cell maintenance solution was added, and the mice were ground with a tissue grinder until uniform, and centrifuged at 8000 rpm/min for 10 min. Take 100 μl of supernatant, extract viral nucleic acid according to QIAGEN's QIAamp Viral RNA Mini Kit instructions, and use Takara's One Step RT-PCR kit (RR064A) for real-time quantitative RT-PCR detection of viral nucleic acid. Another part of the lung tissue was fixed with 4% PFA, and the sections were stained with HE and RNAscope to observe the histopathological changes. The test results are shown in Figure 36-38.

29.3 Experimental Results

The results in Figure 36 show that in the SARS-CoV-2B.1.351 infection of the Balb/C mouse model, the use of BsAb17 antibody at a dose of 50 mg/kg can reduce the virus titer in lung tissue under both the prevention + treatment and treatment models. Among them, the viral loads in the lung tissue of the mice in the control group were 10 ^10.84 RNA copies/g on the 5th day after infection, respectively. Compared with the mice in the control group, the viral loads in the lung tissue of the mice in the prevention + treatment group and the treatment group on the 5th day after infection were 10 ^7.30 RNA copies/g and 10 ^7.38 RNA copies/g, respectively, and decreased by 10 after administration. ^3.54 versus 10 ^3.46 times, with a significant difference (**p<0.005) (Figure 36A). Similarly, in a murine-adapted strain infection model, the use of BsAb17 antibody at a dose of 50 mg/kg reduced viral titers in lung and tracheal tissue under both prophylactic + treatment and therapeutic models. Among them, the viral loads in the lung tissue of the mice in the control group were 10 ^10.95 RNA copies/g on the 5th day after infection, respectively. The viral loads in the lung tissue of the mice in the prevention + treatment group and the treatment group were 10 ^5.58 RNA copies/g and 10 ^6.68 RNA copies/g on the 5th day after infection, respectively, which were decreased by 10 ^5.37 and 10 ^4.27 times after administration (Figure 36B). ).

The results of H&E staining showed that B.1.351 mutant strain and mouse-adapted strain control mice developed moderate or severe interstitial pneumonia in lung tissue on the 5th day after infection, including local alveolar thickening and inflammatory cell infiltration, alveolar There is some exudate in the cavity, hemorrhage. Compared with the control group mice, the lung tissue lesions of the treatment group mice were reduced, with mild or moderate interstitial pneumonia, alveolar septum thickening, inflammatory cell infiltration and hemorrhage area significantly reduced. In the lung tissue of mice in the group, only mild interstitial pneumonia was seen, and inflammatory cell infiltration and hemorrhage were significantly reduced (Figure 37). The results of RNAscope assay showed that SARSCoV-2-specific RNA could be detected in the lung tissue of mice in the control group, a small amount of viral RNA could be observed in the treatment group, and almost no viral RNA could be observed in the prevention + treatment group (Figure 38).

Example 30 Antibody sequence analysis

Antibodies R15-F7, P14-F8, and modified P14-F8-hVH8, P14-F8-35, P14-F8-38, P14-F8-43 and diabodies BsAb16, BsAb17 and BsAb18 were selected based on the above examples , they were analyzed and sequenced. The variable regions of human antibody sequences were defined based on the IMGT database (http://www.imgt.org/), and the sequences (SEQ ID NOs: 1-7) of the light and heavy chain variable regions of the antibodies of the present invention were determined; The variable region sequence was analyzed, and the CDRs were defined by AbM, and the complementarity determining region sequences (SEQ ID NO: 8-21) of the antibody heavy chain and light chain were determined, and the full-length antibody sequence (SEQ ID NO: 22- 41). For specific sequence information, please refer to Sequence Listing 20, Table 21, Table 22 and Table 23.

Those skilled in the art will further realize that the present disclosure may be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it is to be understood that other variations are considered to be within the scope of the present disclosure. Therefore, the present disclosure is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims to indicate the scope and content of the invention.

Sequence Listing Overview

Table 20 Antibody variable region amino acid sequences

Table 21 Antibody Complementarity Determining Region (CDR) Amino Acid Sequences

Table 22 Full-length antibody amino acid sequence

Table 23 Full-length antibody nucleotide sequences

Claims

A polypeptide complex that specifically binds to the coronavirus S protein, the polypeptide complex comprising:

(a) a first epitope binding portion comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region together form a specific an antigen-binding domain that binds the first epitope of the coronavirus S protein, and

(b) a second epitope binding portion comprising a single domain antibody or a VHH fragment thereof that specifically binds to the second epitope of the coronavirus S protein,

wherein the first epitope binding moiety and the second epitope binding moiety are fused to each other, and

wherein the first epitope is different from the second epitope.
The polypeptide complex of claim 1, wherein the first epitope binding moiety and the second epitope binding moiety do not compete for epitopes with each other.
The polypeptide complex of claim 1, wherein the first epitope binding moiety comprises a human antibody, a humanized antibody, or a chimeric antibody or an antigen-binding fragment thereof.
The polypeptide complex of claim 2, wherein the first epitope binding portion comprises a human antibody, a humanized antibody, or a chimeric antibody or an antigen-binding fragment thereof.
The polypeptide complex of any one of claims 1-4, wherein

The heavy chain variable region comprises:

The first heavy chain CDR1 in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes,

The first heavy chain CDR2 in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes, and

The first heavy chain CDR3 in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 or a variant thereof with no more than 2 amino acid changes; and/or

The light chain variable region comprises:

The light chain CDR1 in the light chain variable region amino acid sequence shown in SEQ ID NO:2 or a variant thereof with no more than 2 amino acid changes,

The light chain CDR2 in the light chain variable region amino acid sequence shown in SEQ ID NO: 2 or a variant thereof with no more than 2 amino acid changes, and

The light chain CDR3 in the light chain variable region amino acid sequence shown in SEQ ID NO: 2 or a variant thereof with no more than 2 amino acid changes.
The polypeptide complex according to claim 5, wherein the first heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:8; the first heavy chain CDR2 comprises or is represented by SEQ ID NO:9 Said first heavy chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; said light chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; said The light chain CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12; and/or the light chain CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13.
The polypeptide complex of any one of claims 1-4 and 6, wherein:

The heavy chain variable region comprises or consists of the following sequence: the sequence shown in SEQ ID NO: 1 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% thereof , 98% or 99% identical sequences, and/or

The light chain variable region comprises or consists of the following sequence: the sequence shown in SEQ ID NO: 2 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% thereof , 98% or 99% identical sequences.
The polypeptide complex of claim 5, wherein:

The heavy chain variable region comprises or consists of the following sequence: the sequence shown in SEQ ID NO: 1 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% thereof , 98% or 99% identical sequences, and/or

The light chain variable region comprises or consists of the following sequence: the sequence shown in SEQ ID NO: 2 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% thereof , 98% or 99% identical sequences.
The polypeptide complex of any one of claims 1-4, 6 and 8, wherein the first epitope binding moiety comprises a heavy chain comprising the heavy chain variable region and a variable comprising the light chain The light chain of the region, and wherein the heavy chain comprises or consists of the following sequence: the sequence shown in SEQ ID NO: 22 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% therewith A sequence of %, 97%, 98% or 99% identity; and/or the light chain comprises or consists of the sequence shown in SEQ ID NO: 23 or has at least 90%, 91%, 92% therewith , 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences.
The polypeptide complex of any one of claims 1-4, 6 and 8, wherein the single domain antibody comprises or consists of a VHH fragment.
The polypeptide complex of any one of claims 1-4, 6 and 8, wherein the single domain antibody comprises:

The second heavy chain CDR1 in the VHH amino acid sequence shown in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes;

The second heavy chain CDR2 in the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes; and

The second heavy chain CDR3 in the VHH amino acid sequence set forth in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes.
The polypeptide complex of claim 11, wherein the second heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, 18 or 20; the second heavy chain CDR2 comprises or consists of SEQ ID NO: 14, 18 or 20; and/or the second heavy chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, 17 or 19.
The polypeptide complex of claim 12, wherein:

The second heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14; the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; and the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; The chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

The second heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; and the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; The chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; or

The second heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20; the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; and the second heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; The chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:16.
The polypeptide complex of any one of claims 1-4, 6, 8, 12 and 13, wherein the single domain antibody or VHH fragment thereof comprises or consists of the following sequence: SEQ ID NO: 5, 6 or The VHH amino acid sequence shown in 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
The polypeptide complex of any one of claims 1-4, 6, 8, 12 and 13, wherein the N-terminus of the second epitope binding moiety is at least one of the first epitope binding moiety C-terminal fusion of the heavy chain.
The polypeptide complex of claim 14, wherein the N-terminus of the second epitope binding moiety is fused to the C-terminus of at least one heavy chain of the first epitope binding moiety.
The polypeptide complex of any one of claims 1-4, 6, 8, 12 and 13, wherein the N-terminus of the second epitope binding moiety is at least one of the first epitope binding moiety C-terminal fusion of the light chain.
The polypeptide complex of claim 14, wherein the N-terminus of the second epitope binding moiety is fused to the C-terminus of at least one light chain of the first epitope binding moiety.
The polypeptide complex of any one of claims 1-4, 6, 8, 12 and 13, wherein the C-terminus of the second epitope binding moiety and at least one of the first epitope binding moiety N-terminal fusion of the heavy chain.
The polypeptide complex of claim 14, wherein the C-terminus of the second epitope binding moiety is fused to the N-terminus of at least one heavy chain of the first epitope binding moiety.
The polypeptide complex of any one of claims 1-4, 6, 8, 12 and 13, wherein the C-terminus of the second epitope binding moiety and at least one of the first epitope binding moiety N-terminal fusion of the light chain.
The polypeptide complex of claim 14, wherein the C-terminus of the second epitope binding moiety is fused to the N-terminus of at least one light chain of the first epitope binding moiety.
The polypeptide complex of any one of claims 1-4, 6, 8, 12 and 13, wherein the second epitope binding moiety comprises at least 2 identical or different VHH fragments starting with The first epitope binding moiety is fused in tandem or separately to the first epitope binding moiety.
The polypeptide complex of claim 14, wherein the second epitope binding moiety comprises at least 2 identical or different VHH fragments fused in tandem to the first epitope binding moiety or respectively fused to the first epitope binding moiety.
The polypeptide complex of any one of claims 16, 18, 20 and 22, wherein the second epitope binding moiety comprises at least 2 identical or different VHH fragments connected in tandem to The first epitope binding moiety is fused or separately fused to the first epitope binding moiety.
The polypeptide complex of any one of claims 1-4, 6, 8, 12, 13, 16, 18, 20, 22, and 24, wherein the first epitope binding portion comprises an Fc region.
The polypeptide complex of claim 26, wherein the Fc region is an IgG1 Fc or an IgG4 Fc.
The polypeptide complex of claim 26, wherein the Fc region is an IgG1 Fc with L234A and L235A or an IgG4 Fc with the S228P mutation.
The polypeptide complex of any one of claims 1-4, 6, 8, 12, 13, 16, 18, 20, 22, 24, 27, and 28, wherein the first epitope binding moiety and the The second epitope binding moieties are fused to each other via peptide bonds or peptide linkers.
The polypeptide complex of claim 29, wherein the peptide linker has a length of no more than about 30 amino acids.
The polypeptide complex of claim 30, wherein the peptide linker comprises a peptide selected from the group consisting of (G)n, (GS)n, (GSGGS)n, (GGGS)n, (GGGGS)n and SEQ ID NO: 42- The group consisting of amino acid sequences shown in 45, wherein n is an integer of at least 1.
The polypeptide complex of any one of claims 1-4, 6, 8, 12, 13, 16, 18, 20, 22, 24, 27, 28, 30 and 31, wherein the single domain antibody is Camelid single domain antibody or humanized single domain antibody.
The polypeptide complex of claim 32, wherein the polypeptide complex comprises or has at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence set forth in SEQ ID NO: 29, 30 or 31 , 95%, 96%, 97%, 98% or 99% identical sequences.
The polypeptide complex of any one of claims 1-4, 6, 8, 12, 13, 16, 18, 20, 22, 27, 28, 30, 31 and 33, wherein the polypeptide complex is A bispecific antibody complex consisting of 2 heavy chains and 2 light chains, the heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 29, 30 or 31, and Described light chain is made up of amino acid sequence shown in SEQ ID NO:23.
An isolated polynucleotide encoding the polypeptide complex of any one of claims 1-34.
The polynucleotide of claim 35, comprising the sequence shown in SEQ ID NO:33 and the sequence shown in SEQ ID NO:39, 40 or 41.
An isolated vector comprising the polynucleotide of claim 35 or 36.
A host cell comprising the polynucleotide of claim 35 or 36 or the vector of claim 37.
A method of expressing the polypeptide complex according to any one of claims 1 to 34, the method comprising culturing the host cell according to claim 38 under conditions suitable for expressing the polypeptide complex, and The polypeptide complex of any one of claims 1 to 34 is optionally recovered from the host cell or from the culture medium.
A pharmaceutical composition comprising the polypeptide complex according to any one of claims 1 to 34 and a pharmaceutically acceptable carrier.
A detection kit comprising the polypeptide complex according to any one of claims 1 to 34.
Use of the polypeptide complex according to any one of claims 1 to 34 in the preparation of a medicament for the treatment and/or prevention of coronavirus infection in a subject.
The use of claim 42, wherein the coronavirus is the SARS-CoV-2 virus and the coronavirus infection is COVID-19.
Use of the polypeptide complex according to any one of claims 1 to 34 in the preparation of a diagnostic agent or kit for detecting coronavirus or diagnosing coronavirus infection.
The use of claim 44, wherein the coronavirus is the SARS-CoV-2 virus and the coronavirus infection is COVID-19.
A method for detecting coronavirus contamination in an environment in vitro, comprising: providing an environmental sample; making the environmental sample and the polypeptide complex according to any one of claims 1-34 or the detection reagent according to claim 41 contacting the cassette; and detecting the formation of the complex between the polypeptide complex of any one of claims 1-34 and the coronavirus S protein.
The method of claim 46, wherein the coronavirus is the SARS-CoV-2 virus.
An antibody or antigen-binding fragment thereof that specifically binds to the coronavirus S protein, comprising:

(a) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: the first heavy chain CDR1 in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 or any other region thereof The variant with more than 2 amino acid changes, the first heavy chain CDR2 in the heavy chain variable region amino acid sequence shown in SEQ ID NO:1 or a variant with no more than 2 amino acid changes, and the variant shown in SEQ ID NO:1 The first heavy chain CDR3 or a variant thereof with no more than 2 amino acid changes in the heavy chain variable region amino acid sequence shown; and wherein the light chain variable region comprises: the light chain variable region shown in SEQ ID NO:2 The light chain CDR1 in the amino acid sequence of the variable region or a variant thereof with no more than 2 amino acid changes, the light chain CDR2 in the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2 or a variant thereof with no more than 2 amino acid changes A variant, and the light chain CDR3 in the light chain variable region amino acid sequence shown in SEQ ID NO:2 or a variant thereof with no more than 2 amino acid changes; or

(b) a VHH fragment comprising: the second heavy chain CDR1 in the VHH amino acid sequence shown in SEQ ID NO: 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes; SEQ ID NO: The second heavy chain CDR2 in the VHH amino acid sequence shown in 5, 6 or 7 or a variant thereof with no more than 2 amino acid changes; and the second heavy chain CDR2 in the VHH amino acid sequence shown in SEQ ID NO: 5, 6 or 7 Heavy chain CDR3 or a variant thereof with no more than 2 amino acid changes.
The antibody or antigen-binding fragment thereof of claim 48,

wherein the first heavy chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; the first heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9; the first heavy chain CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9; The chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; the light chain CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; the light chain CDR2 comprises or consists of SEQ ID NO: 12 and/or the light chain CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; or wherein the VHH fragment comprises: the first amino acid sequence shown in the formula GFRFGSYX 1 MS; Double chain CDR1, wherein X 1 is Y, T or V; the second heavy chain CDR2 of the amino acid sequence shown in the formula DINTRGX 2 X 3 TR, wherein X 2 is E or I, and X 3 is T or V; and the second heavy chain CDR3 of the amino acid sequence shown in the formula AASX 4 X 5 TFX 6 GRSDPDY, wherein X 4 is G or P, X 5 is D or A, and X 6 is E or F.
The antibody or antigen-binding fragment thereof according to claim 48 or 49, wherein the antibody is a monoclonal antibody, the variable region of its heavy chain is composed of the amino acid sequence shown in SEQ ID NO: 1, and the variable region of the light chain is composed of the amino acid sequence shown in SEQ ID NO: 1. The region consists of the amino acid sequence shown in SEQ ID NO:2.
The antibody or antigen-binding fragment thereof of claim 48 or 49, wherein the antibody is a Nanobody, and the VHH fragment consists of the VHH amino acid sequence shown in SEQ ID NO:5.
Use of an antibody or antigen-binding fragment thereof according to any one of claims 48 to 51 in the manufacture of a medicament for the treatment and/or prevention (such as prophylactic treatment) of a coronavirus infection in a subject.
The use of claim 52, wherein the coronavirus is the SARS-CoV-2 virus and the coronavirus infection is COVID-19.
The use of claim 45 or 53, wherein the SARS-CoV-2 virus is a SARS-CoV-2 wild-type or a SARS-CoV-2 mutant.
The use according to claim 54, wherein compared with the SARS-CoV-2 wild type, the S protein of the SARS-CoV-2 mutant has a mutation site in the S1 subunit and/or the S2 subunit and / or missing.
The use according to claim 55, wherein the S protein of the SARS-CoV-2 mutant has a mutation site in the receptor binding domain of the S1 subunit compared to the wild type of SARS-CoV-2.
The use according to any one of claims 54-56, wherein the wild-type S protein of SARS-CoV-2 is the S protein of GenBank accession number QHD43416.1.
The use of claim 56 or 57, wherein the mutation site comprises or is selected from one or more of the following: amino acid positions 323, 330, 339, 341, 344, 367, 384, 408, 414, 417 , 435, 439, 444, 445, 446, 450, 452, 453, 455, 458, 475, 476, 477, 478, 483, 484, 485, 486, 490, 493, 501, 518, 519.
The use of claim 55 or 57, wherein the mutation site comprises or is selected from one or more of the following: amino acid positions 19, 80, 142, 158, 215, 323, 330, 339, 341, 344 , 367, 384, 408, 414, 417, 435, 439, 444, 445, 446, 450, 452, 453, 455, 458, 475, 476, 477, 478, 483, 484, 485, 486, 490, 493 , 501, 518, 519, 614, 681, 701 and 950.
The use according to claim 55 or 57, wherein the S protein of the SARS-CoV-2 mutant is present at amino acid positions 14-318 and/or 533-685 compared to the SARS-CoV-2 wild type Mutation site.
The use of claim 60, wherein the mutation site comprises or is selected from one or more of the following: amino acid positions 19, 80, 142, 158, 215, 614, 681.
The use of claim 55 or 57, wherein the mutation sites comprise or are selected from one or more of those shown in Table 17.
The use according to claim 62, wherein the S protein of the SARS-CoV-2 mutant comprises a mutation site N501Y.
The use according to claim 63, wherein the S protein of the SARS-CoV-2 mutant comprises mutation sites N501Y, E484K and K417N.
The use of claim 55 or 57, wherein the S protein of the SARS-CoV-2 mutant has a deletion at one or more of the following amino acid positions compared to the SARS-CoV-2 wild type: amino acid position 69, 70, 156, 157, 241, 242 and 243;

Preferably, the S protein of the SARS-CoV-2 mutant is deleted at the following amino acid positions:

(1) amino acid positions 69 and/or 70;

(2) amino acid positions 156 and 157; or

(3) Amino acid positions 241, 242 and 243.
The use according to claim 55 or 57, wherein the S protein of the SARS-CoV-2 mutant comprises mutation sites L452R, T478K and P681R compared to the SARS-CoV-2 wild type.
The use according to claim 54, wherein the SARS-CoV-2 mutant is SARS-CoV-2 mutant strain B.1.1.7.
The use according to claim 54, wherein the SARS-CoV-2 mutant is SARS-CoV-2 mutant strain B.1.351.
The use according to claim 54, wherein the SARS-CoV-2 mutant is SARS-CoV-2 mutant strain B.1.617.2.