CN116964103A

CN116964103A - Broad-spectrum antibody of SARS-CoV-2 virus and its application

Info

Publication number: CN116964103A
Application number: CN202380009441.5A
Authority: CN
Inventors: 宋德勇; 窦昌林; 董创创; 刘红; 冯健霞; 饶木顶; 邢平平; 于贝贝; 张亚楠; 胡凤娟; 王毅云
Original assignee: Shandong Boan Biotechnology Co Ltd
Current assignee: Shandong Boan Biotechnology Co Ltd
Priority date: 2022-01-28
Filing date: 2023-01-16
Publication date: 2023-10-27
Also published as: WO2023143176A1

Abstract

The invention relates to a broad-spectrum antibody of SARS-CoV-2 virus, which can combine with S protein on SARS-CoV-2 virus to block cytopathy caused by SARS-CoV-2 virus or neutralize SARS-CoV-2 virus. The invention also relates to nucleic acids encoding the broad-spectrum antibodies or antigen-binding fragments thereof; a cell containing the nucleic acid; a pharmaceutical composition comprising said broad-spectrum antibody or antigen-binding fragment thereof, said nucleic acid, said cell; a kit comprising the broad-spectrum antibody or antigen-binding fragment thereof, the nucleic acid, the pharmaceutical composition; and the use of said broad-spectrum antibody or antigen-binding fragment thereof, said nucleic acid, said pharmaceutical composition for preventing, treating, detecting or diagnosing a disease associated with SARS-CoV-2 virus.

Description

Broad-spectrum antibody of SARS-CoV-2 virus and its application

Technical Field

The invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to a broad-spectrum antibody of SARS-CoV-2 virus and application thereof.

Background

The novel coronavirus (2019-nCoV, also called SARS-CoV-2) causes disease COVID-19 (Corona Virus Disease-19) in the global scope, which brings great threat to human life health and immeasurable loss to global economic development.

The S protein (spike protein) on the surface of SARS-CoV-2 virus is combined into a trimer, and the single S protein contains about 1300 amino acids, belongs to a kind of membrane fusion protein, determines the host range and specificity of the virus, and is an important action site of host neutralizing antibody. The S protein contains two subunits (subnits) S1 and S2, S1 mainly comprising a receptor binding domain (receptor binding domain, RBD), responsible for recognizing the receptor of the cell, S2 contains the essential elements required for the membrane fusion process. The current research report shows that the RBD region of S1 subunit of SARS-CoV-2 virus can initiate infection (affinity reaches 15 nM) by combining ACE2 (angiotensin converting enzyme 2) on the surface of human cells, S1 subunit is separated after S1/S2 site is cleaved, S2 cleavage site is exposed and cleavage is carried out, and FP (fusion peptide) is utilized to initiate membrane fusion of virus and cells after a series of conformational changes occur.

Mutations in the novel coronavirus (SARS-CoV-2) are a means of evolution of the virus. Under the precondition of not damaging the key biochemical phenotype of SARS-CoV-2, the antigen epitope drift is induced by the fixed mutation of the antibody target site so as to avoid the recognition of the related antibody. The major variants of SARS-CoV-2 that have been reported include British variant B.1.1.7, south Africa variant B.1.351 and Brazil variant P.1.

The new coronavirus variant of b.1.1.7 was a new coronavirus variant reported to the world guard organization in the united kingdom at 12 months in 2020, which had a transmission capacity about 70% higher than the original strain, and more than 60% of new coronavirus infections in london were derived from the variant virus. The virus strain is characterized by the variation of more than ten key sites, and in the B.1.1.7 mutant strain pedigree, the most notable mutation site is the key amino acid mutation N501Y in RBD (receptor binding region). The structure of the viral protein is changed, and the ACE2 receptor in human bodies is more easily combined, so that the transmission force is stronger.

South Africa variant B.1.351 appeared in month 8 of 2020, and by the end of month 12 of 2020, the infection rate caused by B.1.351 in south Africa was over 80%. Brazil variant P.1 appeared in the brazil agate city at the beginning of month 12 in 2020, and had caused a large outbreak of the entire urban epidemic by the middle of month 1 in 2021. In addition to the presence of D614G, N Y in both south Africa variant B.1.351 and Brazilian variant P.1, E484K variants were found to be present, which have the effect of attenuating anti-viral neutralizing antibodies and may also lead to viral escape from the immune system recognition.

The muir (b.1.621) was first found in columbia to have mutations of T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, D950N, with potential properties for immune escape, and this variant strain exhibited simultaneously stronger infectivity, similar to the two variant strains previously found in uk and south africa, respectively.

Delta variant b.1.617.2 was found in india earlier than 10 in 2020, and had 13 mutation sites. In addition to the D614G mutation as Alpha, beta and Gamma mutants, delta mutants have some unique mutations, especially L4524, which allow the novel coronavirus spinous process proteins to have a stronger affinity for human cell ACE2 receptors; its P681R can then enhance the ability of the mutant to infect cells by promoting cleavage of the pre-spinous process protein into the active S1/S2 configuration.

The Omikovia variant B.1.1.529 was first discovered in 2021 at 11 months, earlier than in south Africa, and had the important amino acid mutation sites of the first 4 VOC variants Alpha, beta, gamma and Delta spike proteins, with triple mutations of "K417N+E484A+N501Y", including mutation sites that enhance cell receptor affinity and viral replication capacity. In addition, there are a number of other mutant epidemiology and laboratory monitoring data for the omnikow variant that may reduce the neutralizing activity of some monoclonal antibodies, showing that cases of the south african infected omnikow variant proliferate and partially replace the Delta variant, the transmission power remains to be further monitored.

Aiming at the novel coronavirus, the development of specific antibodies for blocking the infection of virus S protein to host cells has important significance for preventing and treating the novel coronavirus.

Disclosure of Invention

The invention provides a broad-spectrum antibody or antigen-binding fragment thereof, which can bind to S protein on SARS-CoV-2 virus, block cytopathy caused by SARS-CoV-2 virus or neutralize SARS-CoV-2 virus. The invention also provides multispecific antibodies, bispecific antibodies, antibody combinations derived from the broad spectrum antibodies or antigen-binding fragments thereof, and nucleic acids encoding the broad spectrum antibodies or antigen-binding fragments thereof, the multispecific antibodies, the bispecific antibodies, or nucleic acid combinations; a cell containing said nucleic acid, said combination of nucleic acids; a pharmaceutical composition comprising said broad-spectrum antibody or antigen-binding fragment thereof, said multispecific antibody, said bispecific antibody, said antibody combination, said nucleic acid combination, said cell; a kit comprising the broad-spectrum antibody or antigen-binding fragment thereof, the multispecific antibody, the bispecific antibody, the antibody combination, the nucleic acid combination, the pharmaceutical composition; and the broad-spectrum antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody, the antibody combination, the nucleic acid combination, the pharmaceutical composition for use in preventing, treating, detecting or diagnosing a disease associated with SARS-CoV-2 virus.

One aspect of the present application provides a broad-spectrum antibody or antigen-binding fragment thereof that binds to the S protein on a novel coronavirus (i.e., SARS-CoV-2, also known as 2019-nCoV), blocks cytopathy caused by SARS-CoV-2 virus or neutralizes SARS-CoV-2 virus.

In the scheme of the application, the novel coronavirus, namely SARS-CoV-2 or 2019-nCoV is the collective name of the original strain of the novel coronavirus discovered for the first time in 2019 and the new coronavirus mutant strain appearing subsequently. Further, one or more of the SARS-CoV-2 virus VOCs strain, VOIs strain, VUMs strain, and wild-type strain; more preferably, the VOCs strains include one or more of a b.1.1.7 strain, a b.1.351 strain, a p.1 strain, a b.1.617.2 strain, a ba.1 strain, a ba.1.1 strain, a ba.2 strain, a ba.2.12.1 strain, a ba.2.13 strain, a ba.2.75 strain, a ba.3 strain, a ba.4 strain, and a ba.5 strain; the VOIs strains comprise one or more of a C.37 strain and a B.1.621 strain; the VUMs strains include one or more of a b.1.617.1 strain, a b.1.640 strain, a c.1.2 strain, and a b.1.630 strain; the wild-type strain is a Wuhan-Hu-1 strain.

In aspects of the application, further, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7), beta (B.1.351), gamma (p.1), delta (B.1.617.2), lambda (C.37), mu (B.1.621), omacron, kappa (B.1.617.1), C.1.2, B.1.630, B.1.640, B.1.526, B.1.525, AZ.5, and wild-type strains; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain.

Further, the S protein is S protein (spike protein) on the surface of SARS-CoV-2 virus, and the S protein contains two subunits (subt) S1 and S2. Still further, the antibody can bind to the S protein on the SARS-CoV-2 virus refers to one or more of the S1 and S2 subunits of the S protein, or the RBD protein binding to the S1 subunit.

Furthermore, the antibody or antigen binding fragment of the application can block the pathological changes of ACE 2-expressing cells caused by SARS-CoV-2 virus, or block the infection, invasion and the like of ACE 2-expressing cells by SARS-CoV-2 virus. Still further, the cells include cells that naturally express ACE2 or cells that artificially express ACE 2. Still further, the cell is a mammalian cell. Still further, the mammal includes a human, and a non-human animal such as a mouse or a monkey, and the like.

In a specific embodiment of the application, the antibody or antigen binding fragment thereof blocks cytopathy caused by SARS-CoV-2 virus or neutralizes SARS-CoV-2 virus at a concentration of 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM or less than 0.1 nM.

In one embodiment of the present application, the present application provides a broad-spectrum antibody or antigen-binding fragment thereof comprising 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions capable of binding to the S protein of SARS-CoV-2 virus,

The 3 light chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 38, LCDR2 shown in SEQ ID NO. 39 and LCDR3 shown in SEQ ID NO. 40, and/or the 3 heavy chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 41, HCDR2 shown in SEQ ID NO. 42 and HCDR3 shown in SEQ ID NO. 43;

the 3 light chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or the 3 heavy chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 27, HCDR2 shown in SEQ ID NO. 28 and HCDR3 shown in SEQ ID NO. 29;

the 3 light chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 9, LCDR2 shown in SEQ ID NO. 10 and LCDR3 shown in SEQ ID NO. 11, and/or the 3 heavy chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 12, HCDR2 shown in SEQ ID NO. 13 and HCDR3 shown in SEQ ID NO. 14;

the 3 light chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 15, LCDR2 shown in SEQ ID NO. 16 and LCDR3 shown in SEQ ID NO. 17, and/or the 3 heavy chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 18, HCDR2 shown in SEQ ID NO. 19 and HCDR3 shown in SEQ ID NO. 20; or alternatively

The 3 light chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or the 3 heavy chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 24, HCDR2 shown in SEQ ID NO. 25 and HCDR3 shown in SEQ ID NO. 26.

In the present embodiment, the embodiments of VL (light chain variable region), VH (heavy chain variable region), LCDR (light chain complementarity determining region), HCDR (heavy chain complementarity determining region), LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 may be individually or in any combination.

In one embodiment of the application, the antibody or antigen binding fragment thereof comprises:

(1) A light chain variable region as shown in SEQ ID NO. 36, and/or a heavy chain variable region as shown in SEQ ID NO. 37; the antibody or antigen binding fragment thereof binds to SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain;

(2) A light chain variable region shown in SEQ ID NO. 7, and/or a heavy chain variable region shown in SEQ ID NO. 8; the antibody or antigen binding fragment thereof binds to SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain;

(3) A light chain variable region shown in SEQ ID NO. 1, and/or a heavy chain variable region shown in SEQ ID NO. 2; the antibody or antigen binding fragment thereof binds to SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain;

(4) A light chain variable region shown in SEQ ID NO. 3, and/or a heavy chain variable region shown in SEQ ID NO. 4; the antibody or antigen binding fragment thereof binds to SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain;

(5) A light chain variable region shown in SEQ ID NO. 5, and/or a heavy chain variable region shown in SEQ ID NO. 6; the antibody or antigen binding fragment thereof binds to SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain; or alternatively

(6) A light chain variable region shown in SEQ ID NO. 3, and/or a heavy chain variable region shown in SEQ ID NO. 35; the antibody or antigen binding fragment thereof binds to SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain.

In one embodiment of the invention, the heavy chain constant region of the antibody or antigen binding fragment thereof has the sequence of SEQ ID NO. 30.

Further, the sequence of the light chain constant region of the antibody or antigen binding fragment thereof is SEQ ID NO. 31.

In one embodiment of the present invention, the following antibodies or antigen binding fragments thereof that bind to the SARS-CoV-2 virus S protein are provided:

in embodiments of the invention, the broad-spectrum antibodies or antigen-binding fragments thereof of the invention include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, fab ', F (ab') 2, fv, scFv, or dsFv fragments, and the like.

In a specific embodiment of the invention, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain;

More preferably, the antibody or antigen binding fragment thereof binds to one or more of residues T345, R346, K444, R403, K417, Y453, K458, G476, Y489, F490, Y505, K440, S443, T415, D420, Y421, a475, N487, and R493 of RBD of SARS-CoV-2 virus;

more preferably, the antibody or antigen binding fragment thereof binds to residues T345, R346 and K444 on the Delta RBD; the antibody or antigen binding fragment thereof binds to residues T345, R346, K440, S443 and K444 on the Omicorn ba.1 RBD; the antibody or antigen binding fragment thereof binds to residues R403, K417, Y453, K458, G476, Y489, F490, and Y505 on Delta RBD; or the antibody or antigen binding fragment thereof binds to residues T415, D420, Y421, a475, N487, Y489, and R493 on omacron ba.2 RBD;

more preferably, the antigen binding fragment is a Fab, fab ', F (ab') 2, fv, scFv, or dsFv fragment;

more preferably, BA7208 or antigen binding fragment thereof binds to residues T345, R346 and K444 on the Delta RBD; BA7208 or an antigen binding fragment thereof binds to residues T345, R346, K440, S443 and K444 on the Omicorn ba.1 RBD; BA7125V1 or antigen binding fragment thereof binds to residues R403, K417, Y453, K458, G476, Y489, F490 and Y505 on Delta RBD; or BA7535 or antigen binding fragment thereof binds to residues T415, D420, Y421, a475, N487, Y489 and R493 on omacron ba.2 RBD.

In a second aspect the invention provides a multispecific antibody derived from a broad-spectrum antibody or antigen-binding fragment of the first aspect; preferably, the multispecific antibody comprises a bispecific antibody; more preferably, the multispecific antibody is derived from one or more of BA7054, BA7125, BA7134, BA7208, BA7125V1, BA 7535.

In a third aspect the invention provides a bispecific antibody comprising a first antibody or antigen-binding fragment that binds to an S protein on a SARS-CoV-2 virus, and a second antibody or antigen-binding fragment that binds to an S protein on a SARS-CoV-2 virus, wherein the first antibody or antigen-binding fragment is a broad-spectrum antibody or antigen-binding fragment of the first aspect, and/or the second antibody or antigen-binding fragment is a broad-spectrum antibody or antigen-binding fragment of the first aspect.

Further, the first antibody or antigen-binding fragment is the same as or different from the second antibody or antigen-binding fragment; preferably, the first antibody or antigen-binding fragment binds to the S protein of the same or a different species of SARS-CoV-2 virus than the second antibody or antigen-binding fragment; more preferably, the first antibody or antigen-binding fragment binds to the same or a different epitope on the S protein than the second antibody or antigen-binding fragment.

Preferably, the bispecific antibody binds to one or more of residues T345, R346, K444, R403, K417, Y453, K458, G476, Y489, F490, Y505, K440, S443, T415, D420, Y421, a475, N487 and R493 of RBD of SARS-CoV-2 virus.

Preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain.

Still further, the first antigen-binding fragment is a Fab and the second antigen-binding fragment is a scfv;

more preferably, the bispecific antibody has a knob-hole Fc region;

More preferably, the heavy chain constant region to which the first antigen-binding fragment is attached is a heavy chain constant region having a hole, and the heavy chain constant region to which the second antigen-binding fragment is attached is a heavy chain constant region having a knob;

more preferably, the second antigen-binding fragment is linked to a heavy chain constant region having a knob by VL or VH; more preferably, the VL or VH is linked to a heavy chain constant region having a knob by a linker;

more preferably, the bispecific antibody further has a light chain constant region;

more preferably, the first antibody or antigen binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, the 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or 3 heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO. 27, HCDR2 shown in SEQ ID NO. 28 and HCDR3 shown in SEQ ID NO. 29; the second antibody or antigen binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, the 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 15, LCDR2 shown in SEQ ID NO. 16 and LCDR3 shown in SEQ ID NO. 17, and/or the 3 heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO. 18, HCDR2 shown in SEQ ID NO. 19 and HCDR3 shown in SEQ ID NO. 20;

More preferably, the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO. 7, and/or the heavy chain variable region shown in SEQ ID NO. 8; the second antibody or antigen binding fragment comprises a light chain variable region as set forth in SEQ ID NO. 3 and/or a heavy chain variable region as set forth in SEQ ID NO. 35;

more preferably, the first antibody or antigen binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, the 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or the 3 heavy chain complementarity determining regions of the broad-spectrum antibody or antigen binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 27, HCDR2 shown in SEQ ID NO. 28 and HCDR3 shown in SEQ ID NO. 29; the second antibody or antigen binding fragment comprises 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 38, LCDR2 shown in SEQ ID NO. 39 and LCDR3 shown in SEQ ID NO. 40, and/or the 3 heavy chain complementarity determining regions of the broad-spectrum antibody or antigen binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 41, HCDR2 shown in SEQ ID NO. 42 and HCDR3 shown in SEQ ID NO. 43;

more preferably, the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO. 7, and/or the heavy chain variable region shown in SEQ ID NO. 8; the second antibody or antigen binding fragment comprises a light chain variable region as set forth in SEQ ID NO. 36, and/or a heavy chain variable region as set forth in SEQ ID NO. 37;

More preferably, the first antibody or antigen-binding fragment is BA7208 Fab and the second antibody or antigen-binding fragment is BA7125V1scfv; or the first antibody or antigen-binding fragment is BA7208 Fab and the second antibody or antigen-binding fragment is BA7535scfv.

Still further, the linker between the VL or VH and the heavy chain constant region having knob is a linker polypeptide, preferably, the sequence may be AA. Furthermore, the connecting sequence of VL and VH in scfv can be SEQ ID NO. 32.

Further, in the bispecific antibody, the antibody heavy chain (knob chain) constant region sequence may be SEQ ID NO. 33; the antibody heavy chain (hole chain) constant region sequence may be SEQ ID NO 34.

The third aspect of the present invention also provides an antibody combination comprising a combination of two or more antibodies or antigen-binding fragments that bind to the S protein on SARS-CoV-2 virus;

preferably, the antibody combination is a combination of two antibodies or antigen binding fragments; wherein the first antibody or antigen-binding fragment is a broad-spectrum antibody or antigen-binding fragment of the first aspect and/or the second antibody or antigen-binding fragment is a broad-spectrum antibody or antigen-binding fragment of the first aspect; more preferably, the first antibody or antigen-binding fragment is the same as or different from the second antibody or antigen-binding fragment; more preferably, the first antibody or antigen-binding fragment binds to the S protein of the same or a different species of SARS-CoV-2 virus than the second antibody or antigen-binding fragment; more preferably, the first antibody or antigen-binding fragment binds to the same or a different epitope on the S protein than the second antibody or antigen-binding fragment;

more preferably, the first antibody or antigen-binding fragment is a BA7208 antibody or antigen-binding fragment and the second antibody or antigen-binding fragment is a BA7125V1 antibody or antigen-binding fragment; or the first antibody or antigen-binding fragment is a BA7208 antibody or antigen-binding fragment and the second antibody or antigen-binding fragment is a BA7535 antibody or antigen-binding fragment;

More preferably, the antibody combination binds to one or more of residues T345, R346, K444, R403, K417, Y453, K458, G476, Y489, F490, Y505, K440, S443, T415, D420, Y421, a475, N487 and R493 of RBD of SARS-CoV-2 virus;

preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB.

In aspects of the application, BA7208 or an antigen binding fragment thereof binds to residues T345, R346 and K444 on Delta RBD; BA7208 or an antigen binding fragment thereof binds to residues T345, R346, K440, S443 and K444 on the Omicorn ba.1 RBD; BA7125V1 or antigen binding fragment thereof binds to residues R403, K417, Y453, K458, G476, Y489, F490 and Y505 on Delta RBD; or BA7535 or antigen binding fragment thereof binds to residues T415, D420, Y421, a475, N487, Y489 and R493 on omacron ba.2 RBD. In a version of the application, the epitopes of the antibodies or antigen binding fragments of the application are analyzed by cryoelectron microscopy.

In a fourth aspect the invention provides a nucleic acid encoding said broad spectrum antibody or antigen binding fragment thereof, or said multispecific antibody, said bispecific antibody, or said combination of antibodies. The fourth aspect of the invention also provides a nucleic acid combination comprising a combination of nucleic acids encoding each of the antibodies of the combination of antibodies of the third aspect.

In a fifth aspect the invention provides a vector comprising a nucleic acid encoding said broad spectrum antibody or antigen binding fragment thereof, or said multispecific antibody, or said bispecific antibody; or a combination comprising nucleic acids encoding each of the antibodies in the combination of antibodies. The vector may be used to express the broad-spectrum antibody or antigen-binding fragment thereof, or the multispecific antibody, or the bispecific antibody, or the antibody combination. Preferably, the vector may be a viral vector; preferably, the viral vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, or the like; preferably, the vector may be a non-viral vector; preferably, the vector may be a mammalian cell expression vector; preferably, the expression vector may be a bacterial expression vector; preferably, the expression vector may be a fungal expression vector.

In a sixth aspect the invention provides a cell comprising said nucleic acid, combination of nucleic acids or said vector, said cell being capable of expressing said broad spectrum antibody or antigen binding fragment thereof, or said multispecific antibody, or said bispecific antibody, or said combination of antibodies. Preferably, the cell is a bacterial cell; preferably, the bacterial cells are E.coli cells or the like; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast cells are pichia cells and the like; preferably, the cell is a mammalian cell; preferably, the mammalian cells are chinese hamster ovary Cells (CHO), human embryonic kidney cells (293), B cells, T cells, DC cells, NK cells or the like.

A seventh aspect of the invention provides a pharmaceutical composition comprising the broad spectrum antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody, the antibody combination, nucleic acid combination, vector or cell, preferably the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, preferably the pharmaceutically acceptable carrier comprises one or more of the following: pharmaceutically acceptable solvents, dispersing agents, additives, shaping agents and pharmaceutical excipients.

Further, the pharmaceutical composition is a nasal spray, nasal drop, nebulization or injection formulation; preferably, the injection preparation is an intravenous injection preparation; more preferably, the pharmaceutical composition comprises a therapeutically effective amount, preferably, from about 30mg to about 2400mg, preferably, about 1200mg or about 2400mg of the antibody or antigen-binding fragment, the multispecific antibody, the bispecific antibody, or the antibody combination. In the scheme of the application, the antibody combination comprises a combination of BA7208 and BA7535 monoclonal antibody or a combination of BA7208 and BA7125V1 monoclonal antibody; preferably, in the antibody combination, the molar ratio of BA7208 to BA7535 is 1:1, a step of; preferably, in the antibody combination, the molar ratio of BA7208 to BA7125V1 is 1:1, a step of; more preferably, two antibodies in the antibody combination are administered together after mixing.

Further, the pharmaceutical composition is in a unit formulation which is a nasal spray, nasal drop, nebulization or injection formulation and which comprises a therapeutically effective amount, preferably from 30mg to 2400mg, preferably about 1200mg or about 2400mg, of the antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody or the combination of antibodies; preferably, the pharmaceutical composition contains one selected from the group consisting of the antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody and the antibody combination, and a buffer; more preferably, the buffer comprises one or more of trehalose and polysorbate 80; more preferably, the pharmaceutical composition has a pH of 5.5 to 6.5; more preferably, the buffer further comprises one or more of histidine hydrochloride and histidine; more preferably, the molar ratio of histidine hydrochloride to histidine is 10.5:9.5; more preferably, the pharmaceutical composition comprises 0.04-0.1g/mL trehalose, 0.0001-0.0003g/mL polysorbate 80, and 10-50mg/mL of one selected from the group consisting of the antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody, and the antibody combination, based on the total volume of the pharmaceutical composition; more preferably, the pharmaceutical composition comprises 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL polysorbate 80, and 40+ -4 mg/mL of one selected from the antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody and the antibody combination, based on the total volume of the pharmaceutical composition. More preferably, the pharmaceutical composition comprises 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL polysorbate 80, and 40.+ -.4 mg/mL of the antibody or antigen binding fragment thereof, based on the total volume of the pharmaceutical composition.

Still further, the pharmaceutical composition is administered to a subject for treatment or prophylaxis; the subject being treated includes asymptomatic, light, common, heavy or critical patients infected with SARS-CoV-2 virus; preferably, the subject is asymptomatic, light, common, heavy or critical patients who are infected with SARS-CoV-2 virus in a diagnosis and treatment regimen of coronavirus pneumonia (ninth edition of trial) within 72 hours;

the treatment subjects include asymptomatic, mild, moderate, severe and critically ill patients infected with SARS-CoV-2 virus; preferably, the subject is a patient who is laboratory-checked (e.g., RT-PCR-checked) to confirm infection with SARS-CoV-2 within 72 hours and is asymptomatic, mild, moderate, severe and critical in accordance with NIH guidelines; or alternatively

The preventing subject includes a pre-exposure preventing subject, or a post-exposure preventing subject; preferably, the pre-exposure prophylaxis subjects include a high risk group of exposed to a new coronavirus, healthy subjects, or other subjects unsuitable for vaccination; the post-exposure prophylaxis subjects include post-new coronal pneumonia diagnosed patients and/or intimate contact persons of asymptomatic infected persons.

In an eighth aspect the invention provides a kit comprising a broad-spectrum antibody or antigen-binding fragment thereof, or the multispecific antibody, or the bispecific antibody, or the antibody combination, or a combination comprising nucleic acids encoding a broad-spectrum antibody or antigen-binding fragment thereof, or the multispecific antibody, or the bispecific antibody, or a combination of nucleic acids of each of the antibody combinations, or the cell, or the pharmaceutical composition, of the invention. Further, the kit may further comprise a container or instructions containing suitable buffer reagents.

In a ninth aspect, the invention provides the use of said broad spectrum antibody or antigen binding fragment thereof, said multispecific antibody, said bispecific antibody, said antibody combination, nucleic acid combination, vector or cell in the manufacture of a medicament for the treatment or prophylaxis of a disease.

In a tenth aspect the invention provides the use of said broad spectrum antibody or antigen binding fragment thereof, said multispecific antibody, said bispecific antibody, said antibody combination, nucleic acid, or nucleic acid combination in the preparation of a diagnostic, detection kit.

An eleventh aspect of the invention provides a method of treating or preventing a disease comprising administering to a subject in need thereof the broad spectrum antibody or antigen binding fragment, the multispecific antibody, the bispecific antibody, the antibody combination, nucleic acid combination, vector, cell or pharmaceutical composition of the invention.

Further, the method comprises administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment; preferably, the multispecific antibody, the bispecific antibody or the antibody combination is administered to a subject in need thereof a pharmaceutical composition comprising about 30mg to about 2400mg, preferably about 1200mg or about 2400mg of the antibody or antigen-binding fragment; more preferably, the pharmaceutical composition is a nasal spray, nasal drop, nebulization or injection formulation; preferably, the injection preparation is an intravenous injection preparation; more preferably, the pharmaceutical composition is a unit formulation which is a nasal spray, nasal drop, nebulization or injection formulation and which contains from about 30mg to about 2400mg, preferably about 1200mg or about 2400mg of the antibody or antigen binding fragment, the multispecific antibody, the bispecific antibody or the antibody combination; more preferably, the pharmaceutical composition is a nasal spray, nasal drop, nebulization or injection formulation, preferably, the injection formulation is an intravenous injection formulation.

A twelfth aspect of the invention provides a diagnostic, test method comprising administering the broad-spectrum antibody or antigen-binding fragment, the multispecific antibody, the bispecific antibody, the antibody combination, nucleic acid combination, kit or pharmaceutical composition of the invention to a subject or sample in need thereof.

In a thirteenth aspect the invention provides the use of said broad spectrum antibody or antigen binding fragment thereof, said multispecific antibody, said bispecific antibody, said combination of antibodies, nucleic acid combination, vector, cell or pharmaceutical composition for the treatment, prophylaxis of a disease.

In a fourteenth aspect the invention provides the use of said broad spectrum antibody or antigen binding fragment thereof, said multispecific antibody, said bispecific antibody, said combination of antibodies, nucleic acid, combination of nucleic acids, kit, or pharmaceutical composition for detection, diagnosis.

In a fifteenth aspect the present invention provides the use of said broad spectrum antibody or antigen binding fragment thereof, said multispecific antibody, said bispecific antibody, said combination of antibodies, said nucleic acid, said combination of nucleic acids, or said pharmaceutical composition for the prevention, treatment, detection or diagnosis of a disease associated with SARS-CoV-2 virus.

In the present embodiment, the disease is COVID-19 pneumonia and other related complications. Further, the broad-spectrum antibody or antigen-binding fragment, the multispecific antibody, the bispecific antibody, the combination of antibodies, the ability to block infection, invasion, etc. of cells by SARS-CoV-2 virus or its pseudovirus, or to neutralize SARS-CoV-2 virus or its pseudovirus. Still further, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omicron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain.

The invention also provides the use of BA7054, BA7125, BA7134, BA7208, BA7125V1, BA7535, BA7208-7125V 1-linker 4, BA7208-7125V 1-linker 6 antibody, BA 7208-7535-linker 4, BA7208 in combination with BA7125V1 antibody, BA7535 in combination with BA7208 antibody for the prevention, treatment, detection or diagnosis of a disease associated with SARS-CoV-2 virus; still further, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omicron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain.

Table 1 shows the names, the places of occurrence and the major mutations of the major strains of SARS-CoV-2 virus.

The broad-spectrum antibodies or antigen-binding fragments thereof provided herein have one or more of the following advantages:

1. has affinity to S protein or S1 protein or S2 protein of SARS-CoV-2 virus;

2. the binding of Spike RBD protein of SARS-CoV-2 virus and ACE2 is blocked;

3. blocking infection of cells by pseudoviruses of SARS-CoV-2 virus;

4. blocking infection of cells by a real virus of SARS-CoV-2 virus;

5. has good metabolic stability, small side effect and toxicity and high safety;

6. the in vivo experiments of mice show that the broad-spectrum antibody has good prevention or treatment effect.

Drawings

FIG. 1 shows S protein immunized mice serum titers;

FIG. 2 shows ELISA detection candidates BA7054, BA7125, BA7134, BA7208 blocking binding of RBD protein of wild type, B.1.351 and B.1.617.2 strains to hACE 2;

FIGS. 3A-3C show RBD of antibodies BA7054, BA7134, BA7208, BA7125V1 blocking B.1.351, B.1.617.2 and B.1.1.529.1 strains, and binding of 3 control antibodies to hACE 2; FIG. 3D shows that antibody BA7535 blocks binding of 11 strain proteins to hACE 2; FIGS. 3E-3M show that antibody BA7535 blocks the binding of 9 strain proteins to hACE 2.

FIGS. 4A-4I are graphs showing the binding kinetics of the dual antibody BA7208/7125V1 and its parent mab to RBDs of the B.1.617.2, B.1.529.1 and B.1.621 variants. FIGS. 4J-4K are graphs showing the binding kinetics of BA7535, BA7208 to BA.1 (B.1.1.529.1) RBD.

FIGS. 5A-5H show pseudovirus neutralization curves of BA7208, BA7125V1, BA7054, BA7134, BA7208/BA7125V1 antibodies and 3 control antibodies in Table 13 for various SARS-CoV-2 variants, data collection from two biological replicates expressed as mean+ -SD. FIG. 5I shows the pseudovirus neutralization profile of BA7535 for 2 SARS-CoV-2 variants.

FIG. 6 shows the neutralization activity of BA7208 antibodies against a number of pseudoviruses.

Fig. 7 shows that BA7208 has a broad spectrum and excellent neutralizing activity against various mutants of omacron.

Fig. 8 shows that BA7535 has a broad spectrum and excellent neutralizing activity.

FIGS. 9A-9D show the neutralizing activity of antibodies BA7535, BA7208, the combination of BA7535+BA7208, and LY-COV1404 (Gift's marketed antibodies) on more than 30 previous and emerging variants in a pseudoviral system.

FIGS. 10A-10G show the live virus neutralization of SARS-CoV-2 variants by anti-SARS-CoV-2 antibodies in FRNT assays.

FIGS. 11A-11B show the neutralization activity of BA7208 and BA7208/7125V1 on SARS-CoV-2 variant Omicron BA.1 and BA.2. FIGS. 11C-11E show the real virus neutralization curves of the Omicron BA.1, BA.2, BA.5 variant by BA7535, three biological replicates were performed.

FIG. 12A shows the pharmacokinetic profile of Balb/c mice single intravenous antibody drugs BA7208, BA7125V1, BA7054, BA7134, BA7208/7125V1 double anti-injection; FIG. 12B shows a pharmacokinetic profile of BA7208 administered multiple times to rats; fig. 12C shows a single intravenous pharmacokinetic profile of BA 7535. Figures 12D-12F show the results of single dosing toxicity test for BA7208 mice. FIG. 12G shows the mouse PK data-neutralization curve for double anti-BA 7208/7535-linker 4 (BA 7208 Fab,7535 scfv).

FIGS. 12H-12L show the results of BA7208 mediated ADCC, ADCP experiments, where FIG. 12H shows the ADCC activity of BA7208 with CHO-K1 cells expressing SARS-CoV-2 Spike (wild type) as target cells. FIG. 12I shows ADCP activity of BA7208 on CHO-K1 cells expressing SARS-CoV-2 Spike (wild type) as target cells. FIG. 12J shows ADCP activity of BA7208 using HEK293T cells expressing SARS-CoV-2 BA.1 Spike as target cells. FIG. 12K shows the ADCC activity of BA7208 using HEK293T cells expressing SARS-CoV-2 BA.1 Spike as target cells. The experiments were performed in duplicate using unrelated mabs with the same constant regions as isotype controls (not shown). Data are expressed as mean ± SD.

FIG. 13 shows a schematic of the structure of an anti-2019-nCoV (or SARS-CoV-2) bispecific antibody.

FIG. 14A shows ELISA detection of binding of the RBD protein to hACE2 by the dual anti-BA 7208-7125V 1-linker 4 and BA7208-7125V 1-linker 6 blocking B.1.1.529 (also known as Omicoron strain).

FIG. 14B shows ELISA detection of binding of the double anti-BA 7208-7125V 1-linker 4 and BA7208-7125V 1-linker 6 to the RBD protein of B.1.621 (also known as the muu (Mu) strain) to hACE 2.

FIG. 15 shows the neutralization profile of pseudoviruses of double anti-BA 7208/7535-linker 4 (BA 7208 Fab,7535 scfv).

FIG. 16 shows a cryo-electron microscopy image of Delta Spike Trimer with BA7208-Fab/BA7125V1-Fab (BA 7125-Fab on the figure is the Fab of BA7125V1 described in the examples).

FIG. 17 shows a cryo-electron microscopy image of Omicron Spike Trimer with BA 7208-Fab.

FIG. 18 shows the interaction of the Omicorn RBD mutation site with the BA7208 Fab heavy chain.

FIGS. 19A-19H show structural overlay analysis of BA7208, BA7125V1, BA7254 (BA 7125 on FIGS. 19A-19H is BA7125V1 described in the examples).

Fig. 20 shows competitive binding results of the Biological Layer Interferometer (BLI) based competitive binding assay compared to the BA7535, BA7208 antibodies.

Figures 21A-21B show pulmonary live virus titers in prophylaxis and treatment groups following BA7208 injection administration to mice. Figures 21C-21D show pulmonary live virus titers in prophylaxis and treatment groups following nasal and aerosol administration of BA7208 to mice.

FIG. 22A shows a prophylactic and therapeutic trial route for BA7535 and BA7535/BA7208 combinations in hACE2 transgenic mice. Fig. 22B shows monitoring body weight changes (n=4), and symbols represent mean ± SEM. FIG. 22C shows the Viral load (visual burden) of the lung and brain analyzed by lesion formation (FFA) at 2 and 4dpi, with the dashed line representing the limit of detection (LOD). Fig. 22D shows pathological changes in H & E stained lung sections collected at 4dpi in prophylaxis and treatment groups. No significant lung lesions were observed in the prophylaxis group as well as in the high and low dose treatment groups compared to the PBS control group. The pictures show low magnification (up; scale bar, 500 μm) and high magnification (down; scale bar, 100 μm). Representative images of each group n=4.

FIG. 23 shows the mutation sites on RBD of various SARS-CoV-2 variants.

FIG. 24A shows the crystal structure of the BA.2 Spike trimer and BA7535-Fab complex. FIG. 24B shows the crystal structure of the BA.2 RBD and BA7535-Fab complexes.

Figure 25 shows that BA7535 partially coincides with the binding epitope of RBD and the binding epitope of ACE 2.

FIG. 26 shows a comparison of the crystal structures of RBD/BA7535-Fab and REGN10987 (PDB ID:6 XDG).

Detailed Description

The invention will be further illustrated with reference to specific examples. The described embodiments are some, but not all, embodiments of the invention. It is to be understood that the following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compositions of the present invention may be utilized and are not intended to limit the scope of what the present invention may be used. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 production of anti-2019-nCoV (or SARS-CoV-2) monoclonal antibody

1.1 proteins and mice used are shown in Table 2.

TABLE 2 sources of S proteins and mice used

The immunization of the transgenic mice is shown in Table 3.

TABLE 3 immunization of transgenic mice

The Spike RBD antigen proteins of the total 5 mutants, B.1.617.1 RBD, B.1.617.2 RBD, B.1.351 RBD and p.1 RBD proteins, B.1.1.529.1 RBD, and the Spike proteins of the 2 mutants, B.1.617.2 Spike and B.1.351 Spike proteins were immunized separately in the manner shown in Table 3. Immunization by abdominal subcutaneous injection, the immunization dose of the Spike RBD antigen proteins (i.e., B.1.617.1 RBD, B.1.617.2 RBD, B.1.351 RBD, p.1 RBD protein and B.1.1.529.1 RBD) was 40 μg/dose, and the immunization dose of the Spike proteins (i.e., B.1.617.2 Spike and B.1.351 Spike proteins) of the 2 mutants was 35 μg/dose. First, freund's complete adjuvant is used for emulsifying the antigen, and second, third and fourth, freund's incomplete adjuvant is used for emulsifying the antigen. After immunization of mice with the above proteins according to Table 3, the results of the serum titers of the mice are shown in FIGS. 1, 2500X,12500X, and 62500X, which represent the serum dilution ratio.

1.2 establishment of phage library

Mice were sacrificed, spleens were dissected and removed, spleens were crushed by syringe plugs and filtered with a filter screen, filtered spleen cells were frozen and prepared, RNA was extracted to obtain cDNA, and phage library was established according to the usual method. The library capacity data of the constructed library are shown in table 4.

TABLE 4 construction of phage library storage capacities for immunized mice with each strain

1.3 screening was performed in two ways

Plate screening with b.1.617.2 Spike RBD protein (i.e., b.1.617.2 RBD) (sense upin 40592-V08H 90) or b.1.351 Spike RBD protein (i.e., b.1.351 RBD) (sense upin 40592-V08H 85) or b.1.617.1 Spike RBD protein (i.e., b.1.617.1 RBD) (sense upin 40592-V08H 88) or p.1 Spike RBD protein (i.e., p.1 RBD) (sense upin 40592-V08H 86) or b.1.617.2 Spike protein (b.1.617.2 Spike) (sense upin 40589-V08H 10) or b.1.351 Spike protein (i.1.351 Spike) (sense upin 40589-V08H 13) was carried out by the biological sciences of b.1.1.529. The following day phage library was added for 2h, washed 4-10 times and the specifically bound phage was eluted with elution buffer.

1.3.2 magnetic bead screening, biotinylating the Spike RBD protein of B.1.617.2 or B.1.351 or B.1.617.1 or p.1 or the Spike RBD protein of B.1.529.1RBD protein according to the steps of the kit, combining with Thermo magnetic beads, blocking by BSA, incubating with a phage library for 2h, washing for 4-10 times, and eluting specifically combined phage by using an Elutation Buffer. The antibody clones obtained by screening and the sources are shown in Table 5.

Table 5 screening strategy and antibody Source

EXAMPLE 2 molecular construction and production of intact antibodies

177 positive clones IgG1 were constructed and sequenced, with 4 lead antibody variable region amino acid sequences as shown in table 6 below: (CDR regions are underlined, the analysis system is the IMGT system), BA7125V1 is the antibody obtained by point mutation of the heavy chain variable region of BA 7125.

TABLE 6 variable region sequences of active cloned amino acid sequences

The antibody variable region genes were amplified by conventional molecular biology PCR (2X Phanta Max Master Mix manufacturer: vazyme cat# P515-P1-AA lot # 7E351H 9), and the antibody heavy chain variable region genes were ligated into vector pCDNA3.4 (Life Technology) having the nucleic acid sequences of the antibody heavy chain constant region sequences, and the antibody light chain variable region genes were ligated into vector pCDNA3.4 having the nucleic acid sequences of the antibody light chain constant region sequences, respectively, by homologous recombination.

The variable region sequences of each antibody in the examples of the present application are shown in Table 7, and the heavy and light chain constant region sequences are shown in Table 7.

TABLE 7

The positive clone after sequencing is subjected to plasmid extraction and then is co-transfected into HEK293 cells at 37 ℃ and 8 percent CO ₂ Culturing in a shaking table at 125rpm, transiently expressing for 7 days, purifying the supernatant by Protein A affinity chromatography to obtain the antibody, and determining the concentration of the antibody by combining a UV280 with a theoretical extinction coefficient.

Example 3 characterization of anti-2019-nCoV monoclonal antibody molecules

3.1 ELISA detection antibodies blocked binding of RBD proteins of wild-type, beta (B.1.351) and Delta (B.1.617.2) strains to hACE2

3 Spike RBD proteins were diluted to 0.125. Mu.g/mL (wild-type RBD, WT RBD), 0.25. Mu.g/mL (B.1.35 1 RBD) and 0.125. Mu.g/mL (B.1.617.2 RBD) with pH 9.6 CBS, respectively, coated with an ELISA plate, 100. Mu.L/well, and incubated overnight at 4 ℃; after washing the plates, they were blocked with skimmed milk powder. After washing the plates, 50. Mu.L of PBST diluted antibody was added to each well, and the final concentration of the antibody was 4. Mu.g/mL, 1. Mu.g/mL, 0.25. Mu.g/mL. Biotin-labeled ACE2 protein (Novoprotein, C05Y) was then added, 50. Mu.L/well and incubated at 37℃for 1h; after washing the plates, PBST diluted STREP/HRP, 100. Mu.L/well was added and incubated for 1h at 37 ℃. After washing the plate, 100. Mu.L TMB was added to each well for color development, and after 10min, 50. Mu.L 2M H was added to each well ₂ SO ₄ The color development was terminated, and the OD450 was read by using a microplate reader, and the values in tables 8 to 10 except for the antibody concentration represent the OD450 values. The results in fig. 2 and tables 8-10 show that each of BA7054, BA7125, BA7134, BA7208 blocks binding of 3 Spike RBD proteins to ACE 2.

TABLE 8 protein binding sensitivity of detection antibodies to blocking wild-type Spike RBD and hACE2

TABLE 9 detection of antibody drug blocking protein binding of B.1.351 (also known as Beta (Beta) strain) Spike RBD to hACE2 Sensitivity of combination

Table 10 detects protein binding sensitivity of antibody drug blocking B.1.617.2 (also known as Delta strain) Spike RBD to hACE2

3.2 ELISA detection antibody blocks binding of each strain protein to hACE2

3.2.1 antibodies BA7054, BA7134, BA7208, BA7125V1 block binding of RBD of B.1.351 (also known as Beta (Beta) strain), B.1.617.2 (also known as Delta (Delta) strain) and B.1.1.529.1 (also known as Omicron (Omicron) BA.1 strain) to hACE2

3 Spike RBD proteins (i.e., RBD proteins of strains B.1.351, B.1.617.2 and B.1.1.529.1) were diluted to 0.125. Mu.g/mL with pH 9.6 CBS, respectively, and the ELISA plates were coated, 100. Mu.L/well, and incubated overnight at 4 ℃; after washing the plates, they were blocked with skimmed milk powder. After washing the plate, 50. Mu.L of PBST diluted antibody was added to each well, and the final concentration of the antibody was (4. Mu.g/mL, 1. Mu.g/mL, 0.25. Mu.g/mL, 0.0625. Mu.g/mL, 0.015625. Mu.g/mL, 0.00390625. Mu.g/mL, 0.0009765625. Mu.g/mL, 0.000244140625. Mu.g/mL). Biotin-labeled ACE2 protein (Novoprotein, C05Y) was then added, 50. Mu.L/well and incubated at 37℃for 1h; after washing the plates, PBST diluted STREP/HRP, 100. Mu.L/well was added and incubated for 1h at 37 ℃. After washing the plate, 100. Mu.L TMB was added to each well for color development, and after 10min, 50. Mu.L 2M H was added to each well ₂ SO ₄ The color development was terminated, OD450 was read with a microplate reader, and the inhibition ratio was calculated as%inhibition= (OD 450 of PBST-OD 450 of antibody)/OD 450 of PBST ×100% according to the following formula. The results are shown in FIGS. 3A-3C, which are graphs showing the blocking activity of four candidate antibodies and three control antibodies on RBDs that bind to hACE2 by ELISA for B.1.351, B.1.617.2 and B.1.1.529.1. Experiments were performed 3 times in parallel, numerical = mean ± SD. As can be seen from FIGS. 3A-3C, each antibody blocked the binding of the B.1.351 (also known as Beta strain) RBD protein and the B.1.617.2 (also known as Delta strain) RBD protein to ACE2, but for the B.1.1.529.1 (also known as Omicker) BA.1 strain) RBD protein only BA7208 And BA7134 blocks its binding to ACE2, other antibodies have no blocking activity.

3.2.2 antibody BA7535 blocks binding of 11 Strain proteins to hACE2

The detection method is the same as 3.2.1, and is different in that the Spike RBD protein comprises Gamma (p.1) strain, delta (B.1.617.2) strain, omicron B.1.1.529.1 (namely BA.1 strain), lambda (C.37) strain, mu (B.1.621), kappa (B.1.617.1) strain, C.12 strain, B.1.630 strain, B.1.640 strain, wild-type strain (namely Wuhan-Hu-1 strain or Original WT), RBD proteins of Beta (B.1.351) are purchased from Beijing Qiazhou, and the detection result is shown in FIG. 3D, and as can be seen from FIG. 3D, the antibody BA7535 can block the binding of each strain protein to hACE 2.

The IC50 (. Mu.g/mL) of each mutant RBD blocked ACE2 binding by BA7535 is shown in Table 10-1.

TABLE 10-1

3.2.3 ELISA detection antibody BA7535 blocks the binding of RBD proteins of P.1, B.1.617.2, C.37, B.1.621, B.1.617.1, C.1.2, B.1.630, B.1.640, BA.1 strains to hACE2

The detection method is the same as 3.2.1, except that the Spike RBD proteins comprise the RBD proteins of P.1, B.1.617.2, C.37, B.1.621, B.1.617.1, C.1.2, B.1.630, B.1.640, and BA.1 strains, each of which is purchased from Beijing Yizhushen, and the detection results are shown in FIG. 3E-3M, and FIG. 3E-3M, wherein antibody BA7535 shows broad blocking activity against the RBDs of 9 SARS-CoV-2 variants (P.1, B.1.617.2, C.37, B.1.621, B.1.617.1, C.1.2, B.1.630, B.1.640, and BA.1).

3.3 detection of the affinity of antibodies to RBD proteins of the respective mutant strains

3.3.1 binding kinetics of antibodies BA7054, BA7134, BA7208, BA7125V1, double antibodies BA7208-7125V1 to respective mutant Spike RBD proteins

Measured using a BIAcore 8K instrument based on surface plasmon resonance (surface plasmon resonance, SRP) technology.

For kinetic measurements, each strain SpiHBS-EP for ke RBD protein ⁺ 1× (cytova, BR-1006-69) buffer was serially diluted 2-fold, starting at 50nM, diluted 2-fold for 4 concentration gradients, and set at 0 concentration. Startup 3 times. Antibody: 2. Mu.g/ml, sample injection time 100s, flow rate 10. Mu.L/min, capture with ProA chip (cytova, 29127556); antigen protein: combining 120s, and dissociating 600s at the flow rate of 30 mu L/min; regeneration: with MgCl ₂ The buffer was regenerated for 30s at a flow rate of 30. Mu.L/min. Binding constants (ka) and dissociation constants (KD) were calculated using a 1:1binding model (BIAcore Evaluation Software version 3.2), with equilibrium dissociation constants (KD) calculated as a ratio KD/ka. The affinity data are shown in Table 11, table 12, respectively.

TABLE 11 Biacore 8K detection antibody affinity

As can be seen from table 11, although each antibody has a difference in RBD affinity from the different mutants, overall our antibody binding is relatively broad spectrum; however, BA7125V1 and BA7054 did not bind to Omicron strain BA.1 (B.1.1.529.1), BA7208 bound best, relatively broad spectrum, and had higher affinity, binding only slightly weaker to Mu (B.1.621), and most affinity to critical BA.1 (B.1.1.529.1).

TABLE 12 Biacore 8K detection antibody affinity

Table 12 shows the binding kinetics of the RBDs of the mab BA7208/7125V1 and its parent mab BA7208, BA7125V1 to the B.1.617.2, B.1.529.1 and B.1.621 variants, for comparison of mab to the diabody affinities, see examples 4-5 below for sequence and structural information, see FIGS. 4A-4I. From table 12 and fig. 4A-4I, it can be seen that the affinity of diabodies is better than 2 maternal monoclonal antibodies, and the advantages of both maternal monoclonal antibodies are combined, possessing the best broad spectrum.

3.3.2 binding kinetics of antibodies BA7535, BA7208 to RBD protein of mutant BA.1 (B.1.1.529.1)

The detection method is the same as 3.3.1, and the results are shown in FIG. 4J-4K, and it can be seen that the affinity of BA7535 to BA.1 RBD is higher than that of BA7208, and the measured equilibrium constants (KD) are 0.10+ -0.02 nM and 1.81+ -0.26 nM, respectively.

3.4 neutralizing Activity of antibodies on pseudoviruses

3.4.1 neutralizing Activity of antibodies BA7208, BA7125V1, BA7054 and BA7134 against pseudoviruses

The pseudo-viruses of each strain S protein were packaged with VSV (vesicular stomatitis virus) vector, and the reagent consumables are shown in Table 12. mu.L of pseudovirus was incubated with 100. Mu.L of antibody to be tested at 37℃for 1h and then infected with 100. Mu.L of 293T-ACE2 cells, number of cells per well: 4E5 cells/hole, incubating for 20-28h at 37 ℃, detecting a luminescence value RLU of the Luciferase by adopting a chemiluminescence method, and calculating the pseudo-virus inhibition rate of the antibody to be detected according to the RLU reading value. The buffer system of the antibodies was pH7.4,0.01M PBS buffer, the reagent consumables are shown in Table 13, and the neutralization activity detection results of each antibody pseudovirus are shown in Table 14.

TABLE 13

TABLE 14 neutralizing Activity of antibodies BA7208, BA7125V1, BA7054 and BA7134 against pseudoviruses

Note that: the lower the IC50 value, the higher the neutralization activity is represented

As can be seen from Table 14, and FIGS. 5A-5H, BA7208, BA7125V1, BA7054, and BA7134 have broad pseudovirus neutralization activities. BA7208 and BA7125V1 have broad spectrum and complementary neutralizing activity. In addition to B.1.640 and Mu, BA7208 could neutralize 11 of 13 mutants with IC50 between 2.81ng/mL and 6.22ng/mL, and with the best neutralization activity for BA.1 and BA.2, IC50 was 3.52ng/mL and 3.43ng/mL, respectively. BA7125V1 can neutralize 11 mutants other than 2 Omicorn mutants with IC50 between 11.36ng/mL and 42.31 ng/mL. BA7054 can neutralize 9 mutants other than Mu, omicorn and B.1.640, with IC50 between 4.47ng/mL and 10.11 ng/mL. BA7134 can neutralize 11 mutants other than B.1.640 and Mu with IC50 between 3.81ng/mL and 147.60 ng/mL. BA7208, BA7125V1 and BA7054 have a significantly higher neutralizing activity than VIR-7381, REGN10933 and REGN 10987.

It can be further seen that the broad spectrum of the double antibody BA7208-7125V1 can neutralize all 13 mutants, and the IC50 is between 2.33ng/mL and 116.10 ng/mL; the neutralizing activity of the double antibody BA7208-7125V1 is slightly lower than that of the best monoclonal antibody in BA7208 and BA7125V1 in most strains (the activity of 10.8ng/mL is also very good); double antibody BA7208-7125V1 was 3-fold less active than monoclonal antibody BA7208 on the key strain Omcicron. The combination of BA7125V1 and BA7208 is similar to that of the double antibody BA7208-7125V1, and has the widest spectrum neutralization activity, so that the respective disadvantages of 2 monoclonal antibodies can be overcome.

3.4.2 antibody BA7535 neutralizing Activity against SARS-Cov-2 pseudovirus of BA.1.1 and BA.2

FIG. 5I shows the neutralizing activity of antibody BA7535 against SARS-Cov-2 pseudoviruses of BA.1.1 and BA.2, and it can be seen that BA7535 has good neutralizing activity against both BA.1.1 and BA.2 SARS-Cov-2 pseudoviruses.

Table 15 shows the SARS-Cov-2 pseudovirus IC50 (μg/mL) of antibody BA7535 to BA.1.1 and BA.2

3.4.3 Pseudovirus neutralization activity data of BA7208 antibody supplement-1

Pseudo virus neutralization activity experimental procedure: mu.L of each novel coronavirus (Beijing three drug) was mixed with 100. Mu.L of the gradient diluted BA7208 antibody and incubated at 37℃for 1h, 50. Mu.L+100. Mu.L of DMEM as negative control and 150. Mu.L of DMEM as blank, and then 100. Mu.L of 293T cell suspension (4X 10) overexpressing human ACE2 was added ⁵ cells/mL), after incubation at 37 ℃ for 20-28h, 150 μl of supernatant was aspirated, and 100 μl of briitelite plus (PerkinElmer) was added as substrate, and the microplate reader read fluorescence values, and the inhibition ratio = 1- (sample mean RLU value-blank mean RLU value)/(negative control mean RLU value-blank mean RLU value) ×100%) was calculated. The IC50 was calculated by fitting GraphPad Prism software, plotted as sample concentration and inhibition. Table 16 shows the pseudovirus neutralization activity data of the BA7208 antibody.

Table 16

As can be seen from fig. 6 and table 16, BA7208 has a broad spectrum and excellent neutralizing activity with IC50<10ng/mL for 15 of the tested strains.

Data on pseudovirus neutralization activity of 3.4.4BA7208 antibody supplement-2

Pseudovirus neutralization activity experimental procedure see 3.4.2, each new coronavirus was purchased from beijing three drugs, the results are shown in fig. 7 and table 17.

TABLE 17

As can be seen from FIG. 7 and Table 17, BA7208 had broad spectrum and excellent neutralization activity against Omicron mutants, and the neutralization activity against BA.2.75 in the tested strains had an IC50<10ng/mL.

Data supplementation of pseudovirus neutralization activity of 3.4.5BA7535 antibody

Pseudo virus neutralization activity experimental procedure: mu.L of each novel coronavirus (purchased from Beijing three) was mixed with 100. Mu.L of a gradient diluted BA7208 antibody and incubated at 37℃for 1h, 50. Mu.L+100. Mu.L of DMEM as a negative control and 150. Mu.L of DMEM as a blank, and 100. Mu.L of 293T cell suspension (4X 10) overexpressing human ACE2 was added ⁵ cells/mL), after incubation at 37 ℃ for 20-28h, 150 μl of supernatant was aspirated, and 100 μl of briitelite plus (PerkinElmer) was added as substrate, and the microplate reader read fluorescence values, and the inhibition ratio = 1- (sample mean RLU value-blank mean RLU value)/(negative control mean RLU value-blank mean RLU value) ×100%) was calculated. The IC50 was calculated by fitting GraphPad Prism software, plotted as sample concentration and inhibition. The pseudovirus neutralization activity data for the BA7535 antibodies are shown in Table 18, where BA.4/5 represents BA.4 and BA.4, and RBD mutations of BA.4 and BA.5 are identical, and thus the pseudoviruses of BA.4 and BA.5 are identical.

TABLE 18

As can be seen from fig. 8 and table 18, BA7535 has a broad spectrum and excellent neutralizing activity with IC50<10ng/mL for 19 of the tested strains.

3.4.6 neutralizing Activity of antibodies BA7535, BA7208 and combinations thereof, LY-COV1404 (antibodies marketed by Gift) on more than 30 previous and emerging variants in pseudoviral systems

Pseudovirus neutralization activity experimental procedure see 3.4.3, each pseudovirus purchased from beijing three drugs, experimental results see fig. 9A-9D, and table 19.

TABLE 19

From FIGS. 9A-9D and Table 19, it can be seen that BA7535 alone can neutralize all of the previous and emerging SARS-CoV-2 variants tested; d614G, alpha, bq.1.1 and XBB only impair the efficacy of BA7535, but none of the variants can escape neutralization. The combination of BA7535 and BA7208 (in the antibody combination, the molar ratio of BA7208 to BA7535 is 1:1; the two antibodies in the antibody combination are administered together after mixing) provides a broader neutralizing efficacy and a higher resistance; LY-Cov1404 was still effective against the previously circulating Omacron variants, but was escaped by BA.2.38.1 (K444N), BQ.1 (K444T), BQ.1.1 (K444T) and XBB (V445 P+G446S).

3.5 neutralizing Activity of antibodies on real Virus

3.5.1 Neutralization Activity of BA7208, BA7125V1, BA7054, BA7208/7125V1 on real Virus

The 50. Mu.L of antibody was mixed with 50. Mu.L of SARS-CoV-2 virus (180 FFU, lesion formation unit) in 96-well plates, incubated at 37℃for 1h, transferred to 96-well plates plated with Vero E6 cells (ATCC), and incubated at 37℃for 1h to infect the virus. The medium was removed, the plates were incubated at 37℃for 24H with 100. Mu.L MEM containing 1.2% carboxymethylcellulose, the cover removed, the cells were incubated for 30 min after 4% paraformaldehyde was added to the cells, the cells were permeabilized with 0.2% Triton X-100, cross-reacted with rabbit anti-SARS-CoV-N IgG (Cat 40143-R001) antibody for 1H at room temperature, and then HRP-labeled goat anti-rabbit IgG (H+L) antibody (1:4000 dilution) was added (Jackson ImmunoResearch) and incubated at room temperature. TrueBlue peroxidase substrate (KPL) was added to the reaction, and SARS-CoV-2 spot was counted by means of an EliSpot reader (Cellular Technology Ltd). Each true virus was from university of guangzhou medical science.

The results are shown in Table 20, and FIGS. 10A-10G (true virus neutralization curves based on FRNT method).

TABLE 20 neutralization assay IC50 for true viruses

From Table 20, and FIGS. 10A-10G, it can be seen that: our 4 antibodies had better neutralizing activity against both wild type virus and b.1.351 real virus, with BA7208 having the best neutralizing activity against ba.1.1 real virus.

3.5.2 BA7208 has excellent effect of neutralizing real virus of Omicron BA.1 and BA.2 variants

See 3.5.1 for experimental procedure for the true viruses, each from university of medical science, guangzhou, and FIGS. 11A-11B and Table 21-1 for experimental results.

TABLE 21-1

FIGS. 11A-11B show that BA7208 and double antibody BA7208/7125V1 have strong neutralization effect on SARS-CoV-2 variant Omicron BA.1 and BA.2, and IC50 value is between 0.0329 and 0.316. Mu.g/ml. As can be seen from Table 21-1, the IC50 values of the BA7208 antibody for neutralization of Omciron BA.1 and BA.2 variants were: 53.2, 18.17ng/mL.

3.5.3 BA7535 has excellent real virus neutralization activity to Omicron BA.1, BA.2, BA.5 variant

For the course of the virus experiments, see 3.5.1, each true virus was from university of Guangzhou medical science, and the results of the experiments are shown in FIGS. 11C-11E and Table 21-2. As can be seen, BA7535 effectively neutralized BA.1, BA.2 and BA.5 live viruses with IC 50's of 0.38ng/mL, 1.1ng/mL and 2.2ng/mL, respectively (FIGS. 11C-11E).

TABLE 21-2

3.6 pharmacokinetics

3.6.1 Single intravenous injection pharmacokinetics of the dual antibodies BA7208, BA7125V1, BA7054, BA7134 and BA7208/7125V1

Blood samples were collected at 10mg/kg for each of 5 antibodies (BA 7208, BA7125V1, BA7054, BA7134 and BA7208/7125V1 diabodies) by single intravenous injection using Balb/c mice (body weight: 25.+ -. 3 g), and at 5 minutes, 30 minutes, 1 hour, 4 hours, 8 hours, 1 (24 hours), 3 (72 hours), 5 (120 hours), 7 (168 hours), 10 (240 hours) and 14 (336 hours) days after the end of the administration, the serum drug concentration was measured by ELISA method, and the pharmacokinetic parameters were calculated at Phoenix WinNonlin 6.4.6.4. The pharmacokinetic parameters of each antibody are shown in Table 22 and the pharmacokinetic profile is shown in FIG. 12A. The results showed that each antibody had good metabolic stability in mice, except for BA 7054.

Table 22 Balb/c Single intravenous antibody drug injection pharmacokinetic parameters in mice

3.6.2 Pharmacokinetics of multiple dosing in BA7208 rats

Pharmacokinetic experimental procedure the experimental parameters are shown in table 23 below:

table 23BA7208 pharmacokinetic experimental procedure experimental parameters

Fig. 12B shows the pharmacokinetic test results of multiple BA7208 administrations in rats. Mice according to LY-CovMab had a T1/2 of 9.5 days and humans had a T1/2 of 28 days; the T1/2 of the rat of BA7208 is 8.4-11.2 days, and the T1/2 of the human body can be predicted to be 28 days. Since the IC50 of BA7208 against BA.2 real virus is 18.17ng/mL (effective concentration), the blood concentration is predicted to be higher than the IC50 after half a year of preventive administration according to the T1/2 of human body, and the protective effect of single administration on human body can be predicted to be maintained for more than half a year.

3.6.3BA7535 Single intravenous injection pharmacokinetics

Blood samples were collected from Balb/c mice (body weight 25.+ -.3 g) and each of the antibodies BA7535 was administered intravenously at 10mg/kg for 5 minutes, 1 hour, 6 hours, 1 (24 hours), 3 (72 hours), 5 (120 hours), 7 (168 hours), 10 (240 hours) and 14 (336 hours) days after the completion of the administration, and the concentration of the drug in serum was measured by ELISA, and pharmacokinetic parameters were calculated by Phoenix Winnlin 6.4.

The pharmacokinetic profile is shown in fig. 12C, and it can be seen that the BA7535 antibody has good metabolic stability in mice.

3.6.4 PK parameters of Balb/c mice given BA7535 intravenously in a single pass

BA7535 showed satisfactory half-life and AUC (0-t), terminal half-life (t _1/2 λz) was about 95 hours, and AUC (0-t) was about 12785 hours. The results are shown in Table 24-1 below.

TABLE 24-1

3.7 Toxicity test of single administration of BA7208 mice

The results of the toxicity test of the single administration of BA7208 are shown in figures 12D-12F, and the survival rate, the mental state, the weight and the feeding amount of animals are not obviously abnormal in 14 days, viscera are not abnormal in dissection, and the whole animal is relatively tolerant.

3.8 mouse PK experiments with double anti-BA 7208/7535-linker 4 (BA 7208 Fab,7535 scfv)

The murine PK experiment procedure for the dual antibody BA 7208/7535-linker 4 was carried out with the murine PK of 3.6.3BA7535 mab and the results were seen in FIG. 12G, and it can be seen that the dual antibody was relatively stable in mice with a terminal half-life (t 1/2, λz) of about 92.3 hours and an AUC (0-t) of about 12108 hours. Mu.g/mL, similar to that of mab.

The PK parameters for the dual anti-BA 7208/7535-linker 4 are shown in Table 24-2 below:

TABLE 24-2

3.9 BA7208 mediated ADCC, ADCP capacity

ADCC experimental procedure:

ADCC reporter bioassays were performed with CHO-K1-Spike cells (Genscript) as target cells and Jurkat cells (G7011, promega) as effector cells. Target cells, effector cells and serial dilutions of antibody were mixed in a white 96-well plate and in a cell incubator (37 ℃,5% co ₂ ) Is cultured for 6 hours. The Bio-Lite chromogenic solution was then added and incubated for 15 minutes at Room Temperature (RT). Plates were read with a Tecan microplate reader. Experiments were performed in duplicate, value = mean ± SD.

ADCP Experimental procedure:

for ADCP, CHO-K1-Spike cells (Genscript) were used as target cells and Jurkat-FcgammaRIIA-H131 cells (Vazyme) were effector cells. Target cells, effector cells and serial dilutions of antibodies were mixed in a white 96-well plate and incubated in a cell incubator (37 ℃,5% co) ₂ ) Is cultured for 6 hours. The Bio-Lite chromogenic solution is then added and incubated at RT for 2-5 minutes. Plates were read with a Tecan microplate reader. Experiments were performed in duplicate, value = mean ± SD.

The experimental results are shown in fig. 12H-12L, and it can be seen that BA7208 shows strong ability to mediate ADCC, ADCP.

Example 4 construction of anti-2019-nCoV (or SARS-CoV-2) double antibody molecule

The structural schematic of the anti 2019-nCoV (or SARS-CoV-2) diabody of the present application is shown in FIG. 13, and FIG. 13 is merely used as an example and should not be construed as limiting the present application. Wherein the structure of the right half part is A-linker (linker) 1-B-linker (linker) 2-CH2-CH3. The double antibody is divided into two structures of a connector 4 and a connector 6.

The structure and sequence of linker 4 and linker 6 are shown in Table 25 below:

table 25

Construction of heavy chain 1 (scFv-knob chain): the heavy chain variable region gene and the light chain variable region gene of an antibody (e.g., 7125V1 antibody) were amplified by conventional molecular biology PCR (2X Phanta Max Master Mix manufacturer: vazyme, cat. No.: P515-P1-AA) respectively, then joined together by linker (e.g., SEQ ID NO: 32) by overlay PCR Technology, and then joined together by homologous recombination (ClonExpress II rapid cloning kit (Vazyme, cat. No.: C113-02-AB) with the amino acid sequence of the heavy chain variable region of the antibody (e.g., 7125V1 antibody) of SEQ ID NO:35 and the amino acid sequence of the light chain variable region of the antibody (e.g., 7125V1 antibody) of SEQ ID NO:3, see Table 6, and finally joined into the vector pCDNA3.4 (Life Technology) of the light chain variable region of the antibody of which the amino acid sequence is SEQ ID NO:35, and the antibody heavy chain variable region of which antibody (kg. B) is shown in Table 26. SEQ ID NO: 33.

Construction of heavy chain 2 (hole chain): amplifying the heavy chain variable region gene of an antibody (such as BA 7208) by conventional molecular biology Technology PCR, connecting the heavy chain variable region gene with the heavy chain (hole chain) constant region sequence of the antibody by homologous recombination, and finally connecting the heavy chain variable region gene into a vector pCDNA3.4 (Life Technology); construction of a light chain: the antibody (e.g., BA 7208) light chain variable region gene is ligated into vector pcdna3.4 with the nucleic acid sequence of the antibody light chain constant region sequence. The amino acid sequence of the heavy chain variable region of the BA7208 antibody is SEQ ID NO. 8, and the amino acid sequence of the light chain variable region of the BA7208 antibody is SEQ ID NO. 7, see Table 6. The constant region sequence of the heavy chain (hole chain) of the antibody is SEQ ID NO. 34 as shown in Table 26; the antibody light chain constant region sequence is SEQ ID NO. 31 as shown in Table 7.

Heavy chain 1 and heavy chain 2 were integrated into heterodimers by the Knob intohole technique.

The constant region sequences of the anti-2019-nCoV (or SARS-CoV-2) diabody molecules are shown in Table 26.

TABLE 26 anti 2019-nCoV (orConstant region sequence of SARS-CoV-2) diabody

Example 5 characterization of anti-2019-nCoV (or SARS-CoV-2) diabody molecules or antibody combinations

5.1 ELISA detection of binding of RBD protein of double anti-BA 7208-7125V 1-linker 4 and BA7208-7125V 1-linker 6 to hACE2 by B.1.1.529 (also known as Omicron) and B.1.621 (also known as muir) strains

2 Spike RBD proteins were diluted to 0.125. Mu.g/mL each with pH 9.6 CBS and coated on the ELISA plate, 100. Mu.L/well, incubated overnight at 4 ℃; after washing the plates, they were blocked with skimmed milk powder. After washing the plate, 50. Mu.L of PBST diluted antibody was added to each well, and the final concentration of the antibody was about 125KD, which was 4. Mu.g/mL, 1. Mu.g/mL, 0.25. Mu.g/mL, 0.0625. Mu.g/mL, 0.015625. Mu.g/mL, 0.00390625. Mu.g/mL, 0.0009765625. Mu.g/mL, 0.000244140625. Mu.g/mL. Biotin-labeled ACE2 protein (Novoprotein, C05Y) was then added, 50. Mu.L/well and incubated at 37℃for 1h; after washing the plates, PBST diluted STREP/HRP, 100. Mu.L/well was added and incubated for 1h at 37 ℃. After washing the plate, 100. Mu.L TMB was added to each well for color development, and after 10min, 50. Mu.L 2M H was added to each well ₂ SO ₄ The color development was terminated and OD450 was read with a microplate reader. FIGS. 14A and 14B show that BA7208-7125V 1-linker 4 and BA7208-7125V 1-linker 6, respectively, are capable of blocking the binding of 2 Spike RBD proteins (B.1.1.529 RBD (Omikovine) protein, B.1.621 (muir) RBD protein) to ACE2, and IC 50's are shown in Table 27, respectively, below. The structure of BA7208-7125V 1-linker 4 and BA7208-7125V 1-linker 6 is described in example 4.

Table 27 BA7208-7125V 1-linker 4 and BA7208-7125V 1-linker 6 blocked binding of 2 Spike RBD proteins to ACE2

5.2 detection of the affinity of the double anti-BA 7208-7125V 1-linker 6 for the RBD proteins of the B.1.1.529 (Omikovia), B.1.621 (muir) and B.1.617.2 (delta) strains

The binding kinetics of the antibodies to each mutant Spike RBD protein was measured using a BIAcore 8K instrument based on the surface plasmon resonance (surface plasmon resonance, SRP) technique.

For kinetic measurements, each strain Spike RBD protein was subjected to HBS-EP ⁺ 1× (cytova, BR-1006-69) buffer was serially diluted 2-fold, starting at 50nM, diluted 2-fold for 4 concentration gradients, and set at 0 concentration. Startup 3 times. Antibody: 2. Mu.g/ml, sample injection time 100s, flow rate 10. Mu.L/min, capture with ProA chip (cytova, 29127556); antigen protein: combining 120s, and dissociating 600s at the flow rate of 30 mu L/min; regeneration: with MgCl ₂ The buffer was regenerated for 30s at a flow rate of 30. Mu.L/min. Binding constants (ka) and dissociation constants (KD) were calculated using a 1:1binding model (BIAcore Evaluation Software version 3.2), with equilibrium dissociation constants (KD) calculated as a ratio KD/ka. The results show that each antibody drug can bind to the corresponding antigen and the affinity data are shown in table 28.

Table 28 affinity of the diabodies for the antigens

5.3 neutralizing Activity of double antibodies on pseudoviruses

5.3.1 neutralizing Activity of the double anti-BA 7208-7125V 1-linker 4 on pseudoviruses.

The pseudo-viruses of each strain S protein were packaged with VSV (vesicular stomatitis virus) vector, and the reagent consumables are shown in Table 13. mu.L of pseudovirus was incubated with 100. Mu.L of antibody to be tested at 37℃for 1h and then infected with 100. Mu.L of 293T-ACE2 cells, number of cells per well: 4E5 cells/well, after incubation for 20-28h at 37 ℃, luminescence value RLU of Luciferase is detected by adopting a chemiluminescence method, pseudo-virus inhibition rate of the antibody to be detected is calculated according to the RLU reading value, and the result of detecting neutralizing activity of the pseudo-virus of the antibody is shown in Table 18. The double anti-BA 7208-7125V 1-linker 4 has wide pseudo-virus neutralization activity, can well neutralize B.1.617.2 (delta) strain, B.1.1.529 (Omikovia) strain and B.1.351 (Beta) strain, and has IC50 of 0.012nM, 0.017nM and 0.010nM respectively, see Table 29.

Table 29 double antibody BA7208-7125V 1-ligationNeutralization Activity of son 4 on pseudoviruses

5.3.2 The neutralizing activity of the mab combination of BA7535 and BA7208 on pseudoviruses is seen in 3.4.6.

5.3.3 neutralization Activity of the double anti-BA 7208/7535-linker 4 (BA 7208 Fab,7535 scfv) pseudoviruses

The experimental procedure of pseudoviruses is shown in 3.4.3, and each pseudovirus is purchased from Beijing three drugs, and the experimental results are shown in FIG. 15 and Table 29-1.

Table 29-1 neutralizing Activity of double anti-BA 7208/7535-linker 4 on pseudoviruses

Supplementing: double anti BA 7208/7535-connector 4 pseudovirus data-IC ₅₀

From the above results, it can be seen that the diabodies can combine the advantages of 2 kinds of monoclonal antibodies into a whole, and exhibit broad spectrum and excellent neutralizing activity.

5.5 neutralizing Activity of the double anti-BA 7208/7125V1 on the true virus see 3.5.1.

Example 6 antibody epitope analysis

6.1. Epitope analysis of antibodies or antigen binding fragments of the application by cryoelectron microscopy

The experimental method is as follows: omicron S protein (i.e., omicron Spike Trimer) (40589-V08H 26, yinqiao) or Delta S protein (i.e., delta Spike Trimer) (40589-V08H 10, yinqiao) was mixed with antibody Fab, incubated on ice for 40min, centrifuged at 4℃for 5min, and then molecular sieve purified using GE micro Akta and Superose 6 columns to collect unimodal samples. 4. Mu.L of the sample was placed on a 300 mesh glow discharge grid (Quantifoil, R1.2/1.3) supported by a thin layer of graphene oxide, the water was blotted with filter paper, flash frozen with liquid ethane via Thermo scientific Vitrobot Mark IV instrument, and frozen electron microscopy imaged with Thermo Fisher Titan Krios G i electron microscope equipped with K3 camera and BioQuantum imaging filter. And carrying out multi-round 2D and 3D classification and refinement on the imaged particle data, and further carrying out data processing and 3D modeling to obtain crystal structure data. The experimental results are shown in FIG. 16 and tables 30-31, and in FIG. 17 and table 32.

FIG. 16 shows a cryo-electron microscopy image of Delta Spike Trimer with BA7208-Fab, BA7125V1-Fab, and the BA7125-Fab on FIG. 16 is the Fab of BA7125V1 described in the examples above. FIG. 16a. Complexes of three BA7208-Fab (green) and three BA7125V1-Fab (yellow) with Delta Spike Trimer (blue (dodger blue), dark purple (plus), and brown (rosy brown)). FIG. 16 b.complex of BA7208-Fab (light green, fab light chain; dark green, fab heavy chain) with Delta Spike RBD (blue). FIG. 16 c.complex of BA7125V1-Fab (pale yellow, fab light chain; dark yellow, fab heavy chain) with Delta Spike RBD (blue). FIG. 16d is an enlarged view of the binding site of Fabs on Delta RBD showing the interactions of the side chains of the residues forming hydrogen bonds, the salt bridge and one cation.

Table 30. Protein interaction sites of Delta RBD with BA7208 Fab.

Italic residues are located in the BA7208Fab light chain and underlined residues are located in the BA7208Fab heavy chain

TABLE 31 protein interaction site between Delta RBD and BA7125V1Fab

Italic residues are located in the BA7125V1Fab light chain and underlined residues are located in the BA7125V1Fab heavy chain

FIG. 17 shows a cryo-electron microscopy image of Omicron Spike Trimer with BA 7208-Fab. Fig. 17a, complexes of three BA7208Fab (green) with Omicron Spike Trimer (blue (dodger blue), dark purple (plus), and brown (rosy brown)). 17 b.complex of BA7208-Fab (light green, fab light chain; dark green, fab heavy chain) with Omicron Spike RBD in downward conformation (dark purple). Complexes of BA7208-Fab with Omicron Spike RBD in the upward conformation (blue). FIG. 17d.7208-Fab is an enlarged view of the binding site on Omicon RBD showing the interaction of the side chains of the residues forming hydrogen bonds, the salt bridge and one cation.

Table 32.Omicorn BA.1 RBD protein interaction site between BA7208 Fab

Italic residues are located in the BA7208 Fab light chain and underlined residues are located in the BA7208 Fab heavy chain

As can be seen from the results of fig. 16 and tables 30-31, and fig. 17 and table 32, BA7208 mab binds to residues T345, R346, K444 on Delta RBD; the BA7125V1 mab binds to residues R403, K417, Y453, K458, G476, Y489, F490, Y505 on Delta RBD; BA7208 mab binds to residues T345, R346, K440, S443, K444 on the Omicorn ba.1 RBD.

Further structural analysis found that 14 of the 15 mutation sites of Omicorn ba.1 RBD did not bind directly to BA7208-Fab, only N440K formed hydrogen bonds with W34 and D56 of BA7208 heavy chain Fab, and the other 3 RBD mutation sites of Omicorn ba.2 (T376A, D405N and R408S) were not located at the binding site of BA7208 (see fig. 18). FIG. 18 shows that BA7208 and Omicon RBD binding sites avoided 17 out of the 18 mutation sites of BA.1 and BA.2.

FIGS. 19A-H show a BA7208/BA7125V1 structural overlay analysis, with BA7125 on FIG. 19 being BA7125V1 described in the examples above. In fig. 19A, the complex of RBD (cyan) and BA7208-Fab (green) is superimposed with the complex of RBD (cyan) and ACE2 (blue), and it can be seen that BA7208 does not overlap with the ACE2 binding site. In fig. 19B, the complex of RBD (cyan) and BA7125V1-Fab (yellow) is superimposed with the complex of RBD (cyan) and ACE2 (blue). In fig. 19C, the complex of RBD (cyan) and BA7054-Fab (pink) is superimposed with the complex of RBD (cyan) and ACE2 (blue). The complexes of RBD (cyan) and BA7208 Fab (green) in FIGS. 19D-G are superimposed with the complexes of RBD (cyan) and antibody (blue) REGN10987, VIR-7381, LY-Cov1404 and A23-58.1, respectively. FIGS. 19D-F show that BA7208-Fab has a similar binding pattern to REGN10987, the broad-spectrum antibodies VIR-7381 and LY-CoV 1404. FIG. 19H complex of RBD (cyan) and BA7125V1-Fab (yellow) is overlaid with complex of RBD (cyan) and A23-58.1 antibody (blue).

As shown in fig. 19A-19C, BA7208-Fab and BA7054-Fab did not directly prevent ACE2 binding to Spike-RBD by epitope overlap, whereas BA7125V1-Fab directly competed with ACE 2. This result of BA7208-Fab and BA7054-Fab is not consistent with their significant blocking activity as IgG forms, suggesting that the bivalent binding of one intact IgG molecule to 2 RBDs of one Spike or the intact antibody molecule binding to RBDs coated in a plate may sterically interfere with ACE2 binding to RBDs.

As shown in FIGS. 19D-19H, the binding model of BA7208-Fab and REGN10987, VIR 7381 and LY-COV1404 was similar to that of A23-58.1, and BA7125V1 was similar to that of A23-58.1.

Taken together, virus variation on SPIKE-RBD is more prone to occur in the ACE2 binding region, and BA7208 epitopes outside of the ACE2 binding region are predictive of reduced risk of future exposure to variation.

6.2. We compared their primary binding epitope to Delta RBD to Vir-7381, REGN10933 and REGN10987 using a competitive binding assay based on Biological Layer Interferometers (BLI).

The data in Table 33 shows that BA7054, BA7208, vir-7381 and REGN10987 compete with each other, indicating that their epitopes are close to each other. Both BA7054 and BA7208 compete with Vir-7381 and REGN10987, suggesting that BA7054 and BA7208 may also bind an epitope at the edge of the ACE2 binding region as Vir-7381 and REGN10987. BA7208 and BA7125V1 did not compete with each other, indicating that their epitopes did not overlap.

Table 33

6.3 competitive binding assay based on Biological Layer Interferometry (BLI) compared to competitive binding of BA7535, BA7208 antibodies

Competitive binding assay procedure based on Biological Layer Interferometry (BLI):

competitive binding of antibodies was performed on the ForteBio Octet Red 96 system (Pall Forte BioCorporation, menlo Park, CA) using tandem format sorting. The RBD protein of the biotinylated Omicron BA.1 variant (Sino Biological, cat.40592-V08H 121) was loaded onto the SA sensor (Fortebio, cat.18-5019). The sensor is then exposed to 30 μg/mL of a primary antibody (e.g., antibody A) or PBST for 100 seconds, and then to 30 μg/mL of a secondary antibody (e.g., antibody B) for 100 seconds. Data were processed using ForteBio's data analysis software 9.0.

BLI results referring to FIG. 20, FIG. 20 (mock is buffer without antibody, i.e., PBST) 4 columns each represent the binding signal intensity of antibody A and antibody B after the chip binds omicron RBD. From left to right, the first column represents RBD and reacts with buffer solution (buffer) and then is combined with BA7208, the signal intensity can reach about 1.2, the second column represents RBD and is combined with BA7208 to be saturated, then is combined with BA7208 again, the combined signal can be about 0.15, namely, the combined BA7208 occupies RBD standard position and is combined with BA7208 basically, the third column represents buffer solution (buffer) and is combined with BA7535 again, the combined signal can reach about 1.2, the fourth column represents RBD and is combined with BA7208 to be saturated and then is combined with BA7535 again, the combined signal still can reach about 1, and the combined epitope of BA7208 and BA7535 is different, and BA7208 does not interfere with the combination of RBD and BA7535, namely, BA7535 and BA7208 can not compete mutually, and the combined application potential of COVID-19 can be realized.

EXAMPLE 7 preparation of the pharmaceutical composition (in the form of an injectable solution) of the application

The BA7208 antibody solution after virus removal filtration is concentrated to 30-70g/L through a 30KD ultrafiltration membrane bag, then buffer solution is replaced by dialysis buffer solution (9.5 mM histidine, 10.5mM histidine hydrochloride, pH is 5.5-6.5), dialysis volume is 6-8 times, TMP is controlled to be less than or equal to 1.5bar in the whole process, and then the BA7208 antibody solution is washed out of an ultrafiltration system after being concentrated to 70-100g/L, so that the concentration of BA7208 antibody protein is ensured to be more than 55 g/L. Then adding auxiliary material mother liquor (9.5 mM histidine, 10.5mM histidine hydrochloride, 32% trehalose, 0.08% polysorbate 80 (II), pH 5.5-6.5) into the BA7208 antibody protein solution, then diluting the antibody protein solution to 40.0+/-4.0 mg/mL by using dialysis buffer (9.5 mM histidine, 10.5mM histidine hydrochloride, pH 5.5-6.5), sterilizing and filtering to obtain the pharmaceutical composition (9.5 mM histidine, 10.5mM histidine hydrochloride, 8% trehalose, 0.02% polysorbate 80 (II) and 40.0+/-4.0 mg/mL of the BA7208 antibody.

The prescription composition of the pharmaceutical composition of the application (containing 40.0.+ -. 4.0mg/mL of antibody) is shown in Table 34:

watch 34

pH 6.0(5.5-6.5)

EXAMPLE 8 prophylactic and therapeutic efficacy of antibodies in vivo

8.1 BA7208 is administered by injection to effectively prevent and treat Omicron infection

Omicron BA.1 and BA.2 live viruses infect Balb/Cwt mice or new coronal pneumonia hACE2 transgenic mice K18 model, the lungs of the mice are taken the next day after the challenge, the live virus load of the lungs is detected by adopting an FFA method, and the protective effect of the antibody on treatment or prevention is evaluated.

The route of injection and grouping of mice are shown in table 35 below:

table 35

As can be seen from fig. 21A-21B, both the prophylaxis and treatment groups showed significantly reduced pulmonary live virus titers, and the basic clearance of pulmonary virus, BA7208, was excellent in protection against mice.

8.2 BA7208 is effective in preventing and treating Omicron infection by both nasal drip and aerosol inhalation

The K18hACE2 transgenic mice were purchased from Jiangsu Jixiaokang, omicron variant BA.2, isolated and stored by Guangzhou customs technical center biosafety tertiary laboratory. True virus-related assays were conducted by Guangzhou respiratory health institute at BSL-3 grade laboratories.

The BA7208 sample formula is: 9.5mM histidine, 10.5mM histidine hydrochloride, 8% trehalose, 0.02% polysorbate 80 (PS 80), antibody BA7208 concentration 38.905mg/mL.

The Omacron BA.2 live virus was used to infect K18hACE2 transgenic mice by nasal drip, by 50. Mu.L nasal drip (1 mg/kg dose, PBS as dilution buffer) or by aerosol inhalation (3 mg/kg dose, PBS as dilution buffer), and the mice were sacrificed at 4h post-infection (treatment group) or 24h pre-infection (prophylaxis group) after 24h challenge (1 dpi).

Dissecting the mice, putting the lungs of the mice into 1mL PBS buffer solution for tissue grinding, centrifuging the ground tissue solution, and taking the supernatant for detecting the titer of the novel coronavirus, wherein the detection method is an FFA method.

Nasal route of administration and grouping of mice are shown in table 36 below:

table 36

The route of administration and grouping of mice by nebulization are shown in table 37 below:

table 37

As can be seen from fig. 21C-21D, after the administration of BA7208 by nasal drip and atomization, the titer of live lung virus in both prophylaxis and treatment groups was significantly reduced, down to the limit of detection, indicating that the lung virus was substantially cleared, and BA7208 was excellent in protection against mice.

8.3 Neutralization potency of BA7535, BA7208 and combinations thereof in vivo

We assessed the prophylactic and therapeutic activity of BA7535 alone or in combination with BA7208 on Omacron BA.5 variant infection in K18-hACE2 transgenic mouse models.

The experimental process comprises the following steps: the in vivo prophylactic and therapeutic efficacy of BA7535 and BA7535/BA7208 cocktails (i.e., the BA7535 and BA7208 mab combination) against SARS-CoV-2 Omicron ba.5 was evaluated in K18-hACE 2-transgenic mice (collectable drug rehabilitation). At 1X 10 infection ⁵ FFU SARS-CoV-2 Omicron BA.5 was intraperitoneally injected with 2mg/kg or 10mg/kg of BA7535 or BA7535+BA7208 mab combination 24 hours before or 8 hours after six to eight weeks of mice. Mice injected with Phosphate Buffered Saline (PBS) were infected with the same dose of SARS-CoV-2 as a control. To investigate the presence of SARS-CoV-2 in the lungs and brain, virus titers in the lungs and brain were collected by Focal Formation (FFA) 2 days and 4 days after infection. At the same time, lung tissues were collected and stained for histopathological analysis to monitor body weight changes. SARS-CoV-2 Omicron BA.5 strain was supplied by disease prevention control center in Guangdong province of China. Experiments relating to the true SARS-CoV-2 virus were performed at the Guangzhou customs technical center ABSL-3 laboratory.

Analysis of experimental results: lungs and brains were collected 2 days and 4 days after infection and replication of the virus was quantified, respectively. The amounts of 2 and 10mg/kg of BA7535 reduced the pulmonary viral titer by approximately 2.5 orders of magnitude compared to the control group. Complete elimination of viral replication was observed in both the lung and brain (figures 22A and 22C). Likewise, the use of BA7535 in combination with BA7208 (1 mg/kg or 5mg/kg of each mAb) completely abrogated (abolished) replication of the BA.5 virus in the lungs and brain, animals receiving the mAb (BA 7535/BA 7208) cocktail appeared to benefit from the additional contribution of the BA7208mAb, as mAb (BA 7535/BA 7208) cocktail treated animals maintained relatively stable body weight, while those of the control animals had drastically reduced body weight (FIG. 22B). The K18-hACE2 transgenic mice infected with SARS-CoV-2 were subjected to hematoxylin and eosin staining for lung sections (FIG. 22D), and the control group (PBS) showed lung lesions, increased inflammatory cells around blood vessels and branches, and prominent inflammatory cell infiltration. No significant lung lesions were observed in the prophylaxis group, as well as in the high dose BA7535 treated group (BA 7535-T-10 mg/kg) and the low dose BA7535 treated group (BA 7535-T-2 mg/kg) compared to the control group (PBS). In summary, BA7535, either alone or in combination with BA7208, protected K18-hACE2 transgenic mice from SARS-CoV-2 BA.5 infection.

EXAMPLE 9 BA7208 is insensitive to 30 more mutation sites on each variant RBD except R346K

The key mutation sites for RBD in the 19 SARS-CoV-2 virus variants are shown in FIG. 23. With reference to antibodies Vir-7381, REGN10933 and REGN10987, it was evaluated whether the four antibodies BA7208, BA7125V1, BA7054 and BA7134 were effective in inhibiting these variants. As shown in FIG. 23, light gray represents the sites where our antibodies had neutralizing activity and black represents where our antibodies had no neutralizing activity. It can be seen that BA7208 is insensitive to other mutation sites on RBD, except R346, and is relatively broad spectrum.

EXAMPLE 10 clinical study of BA7208 nasal spray formulation II T

1. Study purposes: investigation of the effect of BA7208 nasal spray on the prevention of infection in New crown susceptible people

2. Application crowd and scene: centralizing isolation points, business trip personnel in high risk areas, medical staff, close contact of new patients, and the like.

3. Study design:

exploratory clinical trial designed as single arm, open label, dose escalation aimed at evaluating the pharmacokinetics, primary safety of BA7208 nasal spray formulation as a prophylactic in susceptible populations of new crown infections

And preliminary efficacy studies.

100-300 new crown-infected persons scheduled to be in the group 18-65 years old, and subjected to BA7208 nasal spray preparation administration after necessary health screening.

The study was performed in two phases:

IIT clinical a

SAD (1), 4 dose groups of 10 persons each, dosed with different concentrations of BA7208 nasal spray formulation; and evaluating pharmacokinetics and preliminary safety, and selecting a candidate dose to carry out multiple administration dose tests.

(2) MAD,3 dose groups of 20 persons each, dosed with different concentrations of BA7208 nasal spray; and evaluating pharmacokinetics and preliminary safety, and selecting candidate doses to perform a dose expansion test.

IIT clinical b

Sample expansion stage: and (5) expanding the sample size according to the candidate dose determined in the first stage, and performing preliminary pharmacodynamic study. The test was divided into two groups, a dosing group and a placebo group, and the difference in positive rate of new crown infection was observed between the dosing group and the placebo group.

EXAMPLE 11 BA7535 antibody epitope Studies

Omicron BA.2 Spike protein was mixed with antibody Fab, incubated on ice for 20min and after molecular sieve purification unimodal samples were collected. 3.5. Mu.L of the sample was placed on a glow discharge grid supported by a thin layer of graphene oxide, the water was blotted with filter paper, rapidly frozen with liquid ethane via Thermo scientific Vitrobot Mark IV instrument, and frozen electron microscopy imaged with Thermo Fisher Titan Krios G i electron microscope. And carrying out multi-round 2D and 3D classification and refinement on the imaged particle data, and further carrying out data processing and 3D modeling to obtain crystal structure data.

The crystal structure of the ba.2 Spike trimer and BA7535-Fab complex is shown in fig. 24a, with 3 RBDs of the Spike trimer in an "up" conformation, 1 Spike protein can bind 3 BA 7535-fabs, and the binding region is located on top of the RBDs. As can be seen in the crystal structure of the BA.2 RBD and BA7535-Fab complexes (FIG. 24B), the RBD binding epitope of BA.2 includes 7 residues in total of T415, D420, Y421, A475, N487, Y489 and R493 (Table 38), forming 6 hydrogen bonds and 1 salt bridge, T415-Y106, D420-Y106, Y421-L103, A475-T28, N487-R98 and Y489-R98 and R493-E50 (with RBD amino acids in front, BA7535 amino acids in front), respectively. The binding epitope of BA7535 can avoid most RBD mutations, mutations at F486 only, and N417T and R493Q mutations may affect binding of BA7535 to RBD.

After superimposing the crystal structure of the BA.2 RBD/BA7535-Fab complex with the Spike-RBD/ACE2 complex crystal structure (PDB ID:6VW 1), as shown in FIG. 25, the binding epitope of BA7535 to RBD and the binding epitope of ACE2 partially overlap, indicating that the neutralization mechanism of BA7535 occupies the binding site of ACE2 by binding to RBD, thus directly blocking the binding of RBD to ACE 2.

The crystal structures of RBD/BA7535-Fab and REGN10987 (PDB ID:6 XDG) were compared, and as shown in FIG. 26, the binding epitopes of the two antibodies did not coincide and the binding patterns were also different. Although BA7535 binds to the top of RBD, most mutation sites on RBD are avoided, and the broad spectrum of BA7535 can be greatly improved. The binding regions of both antibodies to RBD were analyzed by PISA with BA7535 having a larger binding region than REGN10987 and BA7535 having a binding region area of And REGN10987 isOnly 4 hydrogen bonds and 1 salt bridge were formed in the REGN10987 epitope compared to 6 hydrogen bonds and 1 salt bridge in the BA7535 epitope, indicating that BA7535 has a higher affinity for RBD than REGN 10987. The solvation free energy Δg= -6.3kcal/mol of the heavy chain of BA7535 bound to RBD, which is lower than Δg= -3.8kcal/mol of REGN10987, also indicates that BA7535 has a higher affinity for RBD than REGN 10987.

Table 38

Claims

An antibody or antigen-binding fragment thereof that binds to an S protein on SARS-CoV-2 virus, wherein the antibody or antigen-binding fragment thereof comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, wherein

The 3 light chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 38, LCDR2 shown in SEQ ID NO. 39 and LCDR3 shown in SEQ ID NO. 40, and/or the 3 heavy chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 41, HCDR2 shown in SEQ ID NO. 42 and HCDR3 shown in SEQ ID NO. 43;

the 3 light chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or the 3 heavy chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 27, HCDR2 shown in SEQ ID NO. 28 and HCDR3 shown in SEQ ID NO. 29;

The 3 light chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 9, LCDR2 shown in SEQ ID NO. 10 and LCDR3 shown in SEQ ID NO. 11, and/or the 3 heavy chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 12, HCDR2 shown in SEQ ID NO. 13 and HCDR3 shown in SEQ ID NO. 14;

the 3 light chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 15, LCDR2 shown in SEQ ID NO. 16 and LCDR3 shown in SEQ ID NO. 17, and/or the 3 heavy chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 18, HCDR2 shown in SEQ ID NO. 19 and HCDR3 shown in SEQ ID NO. 20; or alternatively

The 3 light chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or the 3 heavy chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise HCDR1 shown in SEQ ID NO. 24, HCDR2 shown in SEQ ID NO. 25 and HCDR3 shown in SEQ ID NO. 26.
The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region, and/or a heavy chain variable region;

The light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 36 and/or the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 37;

the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 7 and/or the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 8;

the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID No. 1 and/or the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID No. 2;

the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 3 and/or the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 4;

The light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID No. 5 and/or the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID No. 6; or alternatively

The light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 3 and/or the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 35.
The antibody or antigen binding fragment thereof of claim 1, wherein the SARS-CoV-2 virus comprises one or more of Alpha (b.1.1.7), beta (b.1.351), gamma (p.1), delta (b.1.617.2), lambda (c.37), mu (b.1.621), omacron, kappa (b.1.617.1), c.1.2, b.1.630, b.1.640, b.1.526, b.1.525, az.5, and wild-type strains;

preferably, the wild-type strain is a Wuhan-Hu-1 strain;

Preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain;

more preferably, the antibody or antigen binding fragment thereof binds to one or more of residues T345, R346, K444, R403, K417, Y453, K458, G476, Y489, F490, Y505, K440, S443, T415, D420, Y421, a475, N487, and R493 of RBD of SARS-CoV-2 virus;

more preferably, the antibody or antigen binding fragment thereof binds to residues T345, R346 and K444 on the Delta RBD; the antibody or antigen binding fragment thereof binds to residues T345, R346, K440, S443 and K444 on the Omicorn ba.1 RBD; the antibody or antigen binding fragment thereof binds to residues R403, K417, Y453, K458, G476, Y489, F490, and Y505 on Delta RBD; or the antibody or antigen binding fragment thereof binds to residues T415, D420, Y421, a475, N487, Y489, and R493 on omacron ba.2 RBD;

more preferably, the antigen binding fragment is a Fab, fab ', F (ab') 2, fv, scFv, or dsFv fragment;

more preferably, BA7208 or antigen binding fragment thereof binds to residues T345, R346 and K444 on the Delta RBD; BA7208 or an antigen binding fragment thereof binds to residues T345, R346, K440, S443 and K444 on the Omicorn ba.1 RBD; BA7125V1 or antigen binding fragment thereof binds to residues R403, K417, Y453, K458, G476, Y489, F490 and Y505 on Delta RBD; or BA7535 or antigen binding fragment thereof binds to residues T415, D420, Y421, a475, N487, Y489 and R493 on omacron ba.2 RBD.
An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody comprises the heavy chain constant region shown in SEQ ID No. 30 or comprises the light chain constant region shown in SEQ ID No. 31.
A multispecific antibody derived from the antibody or antigen-binding fragment thereof of any one of claims 1-4; preferably, the multispecific antibody comprises a bispecific antibody.
A bispecific antibody comprising a first antibody or antigen-binding fragment that binds to an S protein on a SARS-CoV-2 virus, and a second antibody or antigen-binding fragment that binds to an S protein on a SARS-CoV-2 virus, wherein the first antibody or antigen-binding fragment is an antibody or antigen-binding fragment according to any one of claims 1-4, and/or the second antibody or antigen-binding fragment is an antibody or antigen-binding fragment according to any one of claims 1-4;

preferably, the first antibody or antigen-binding fragment is the same as or different from the second antibody or antigen-binding fragment;

preferably, the first antibody or antigen-binding fragment binds to the S protein of the same or a different species of SARS-CoV-2 virus than the second antibody or antigen-binding fragment; more preferably, the first antibody or antigen-binding fragment binds to the same or a different epitope on the S protein than the second antibody or antigen-binding fragment;

More preferably, the bispecific antibody binds to one or more of residues T345, R346, K444, R403, K417, Y453, K458, G476, Y489, F490, Y505, K440, S443, T415, D420, Y421, a475, N487 and R493 of RBD of SARS-CoV-2 virus;

preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB.
The bispecific antibody of claim 6, wherein the first antigen-binding fragment is Fab and the second antigen-binding fragment is scfv;

more preferably, the bispecific antibody has a knob-hole Fc region;

More preferably, the heavy chain constant region to which the first antigen-binding fragment is attached is a heavy chain constant region having a hole, and the heavy chain constant region to which the second antigen-binding fragment is attached is a heavy chain constant region having a knob;

more preferably, the second antigen-binding fragment is linked to a heavy chain constant region having a knob by VL or VH; more preferably, the VL or VH is linked to a heavy chain constant region having a knob by a linker;

more preferably, the bispecific antibody further has a light chain constant region;

more preferably, the first antibody or antigen binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, the 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or 3 heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO. 27, HCDR2 shown in SEQ ID NO. 28 and HCDR3 shown in SEQ ID NO. 29; the second antibody or antigen binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, the 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 15, LCDR2 shown in SEQ ID NO. 16 and LCDR3 shown in SEQ ID NO. 17, and/or the 3 heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO. 18, HCDR2 shown in SEQ ID NO. 19 and HCDR3 shown in SEQ ID NO. 20;

More preferably, the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO. 7, and/or the heavy chain variable region shown in SEQ ID NO. 8; the second antibody or antigen binding fragment comprises a light chain variable region as set forth in SEQ ID NO. 3 and/or a heavy chain variable region as set forth in SEQ ID NO. 35;

more preferably, the first antibody or antigen binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, the 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or the 3 heavy chain complementarity determining regions of the antibody or antigen binding fragment thereof comprising HCDR1 shown in SEQ ID NO. 27, HCDR2 shown in SEQ ID NO. 28 and HCDR3 shown in SEQ ID NO. 29; the second antibody or antigen binding fragment comprises 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 38, LCDR2 shown in SEQ ID NO. 39 and LCDR3 shown in SEQ ID NO. 40, and/or the antibody or antigen binding fragment comprises 3 heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO. 41, HCDR2 shown in SEQ ID NO. 42 and HCDR3 shown in SEQ ID NO. 43;

more preferably, the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO. 7, and/or the heavy chain variable region shown in SEQ ID NO. 8; the second antibody or antigen binding fragment comprises a light chain variable region as set forth in SEQ ID NO. 36, and/or a heavy chain variable region as set forth in SEQ ID NO. 37;

More preferably, the first antibody or antigen-binding fragment is BA7208 Fab and the second antibody or antigen-binding fragment is BA7125V1scfv; or the first antibody or antigen-binding fragment is BA7208 Fab and the second antibody or antigen-binding fragment is BA7535 scfv.
An antibody combination comprising a combination of two or more antibodies or antigen-binding fragments that bind to the S protein on SARS-CoV-2 virus;

preferably, the antibody combination is a combination of two antibodies or antigen-binding fragments, wherein a first antibody or antigen-binding fragment is an antibody or antigen-binding fragment according to any one of claims 1-4, and/or a second antibody or antigen-binding fragment is an antibody or antigen-binding fragment according to any one of claims 1-4;

more preferably, the first antibody or antigen-binding fragment is the same as or different from the second antibody or antigen-binding fragment; more preferably, the first antibody or antigen-binding fragment binds to the S protein of the same or a different species of SARS-CoV-2 virus than the second antibody or antigen-binding fragment; more preferably, the first antibody or antigen-binding fragment binds to the same or a different epitope on the S protein than the second antibody or antigen-binding fragment;

More preferably, the first antibody or antigen binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, the 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or 3 heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO. 27, HCDR2 shown in SEQ ID NO. 28 and HCDR3 shown in SEQ ID NO. 29; the second antibody or antigen binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, the 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 15, LCDR2 shown in SEQ ID NO. 16 and LCDR3 shown in SEQ ID NO. 17, and/or the 3 heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO. 18, HCDR2 shown in SEQ ID NO. 19 and HCDR3 shown in SEQ ID NO. 20;

more preferably, the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO. 7, and/or the heavy chain variable region shown in SEQ ID NO. 8; the second antibody or antigen binding fragment comprises a light chain variable region as set forth in SEQ ID NO. 3 and/or a heavy chain variable region as set forth in SEQ ID NO. 35;

More preferably, the first antibody or antigen binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, the 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 22 and LCDR3 shown in SEQ ID NO. 23, and/or the 3 heavy chain complementarity determining regions of the antibody or antigen binding fragment thereof comprising HCDR1 shown in SEQ ID NO. 27, HCDR2 shown in SEQ ID NO. 28 and HCDR3 shown in SEQ ID NO. 29; the second antibody or antigen binding fragment comprises 3 light chain complementarity determining regions comprising LCDR1 shown in SEQ ID NO. 38, LCDR2 shown in SEQ ID NO. 39 and LCDR3 shown in SEQ ID NO. 40, and/or the antibody or antigen binding fragment comprises 3 heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO. 41, HCDR2 shown in SEQ ID NO. 42 and HCDR3 shown in SEQ ID NO. 43;

more preferably, the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO. 7, and/or the heavy chain variable region shown in SEQ ID NO. 8; the second antibody or antigen binding fragment comprises a light chain variable region as set forth in SEQ ID NO. 36, and/or a heavy chain variable region as set forth in SEQ ID NO. 37;

more preferably, the first antibody or antigen-binding fragment is a BA7208 antibody or antigen-binding fragment and the second antibody or antigen-binding fragment is a BA7125V1 antibody or antigen-binding fragment; or the first antibody or antigen-binding fragment is a BA7208 antibody or antigen-binding fragment and the second antibody or antigen-binding fragment is a BA7535 antibody or antigen-binding fragment;

More preferably, the antibody combination binds to one or more of residues T345, R346, K444, R403, K417, Y453, K458, G476, Y489, F490, Y505, K440, S443, T415, D420, Y421, a475, N487 and R493 of RBD of SARS-CoV-2 virus;

preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, and wild-type strain; preferably, the wild-type strain is a Wuhan-Hu-1 strain; preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB.
A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the multispecific antibody of claim 5, or the bispecific antibody of any one of claims 6-7.
A combination of nucleic acids comprising a combination of nucleic acids encoding each of the antibodies of the combination of antibodies of claim 8.
A cell comprising the nucleic acid of claim 9 or the nucleic acid combination of claim 10.
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the multispecific antibody of claim 5, or the bispecific antibody of any one of claims 6-7, or the antibody combination of claim 8, or the nucleic acid of claim 9, the nucleic acid combination of claim 10, or the cell of claim 11.
The pharmaceutical composition of claim 12, which is a nasal spray, nasal drop, nebulization or injection formulation; preferably, the injection preparation is an intravenous injection preparation; more preferably, the pharmaceutical composition comprises a therapeutically effective amount, preferably, about 30mg to about 2400mg, preferably, about 1200mg or about 2400mg of the antibody or antigen-binding fragment of any one of claims 1 to 4, or the multispecific antibody of claim 5, or the bispecific antibody of any one of claims 6 to 7, or the antibody combination of claim 8;

More preferably, the pharmaceutical composition is a unit formulation which is a nasal spray, nasal drop, nebulization or injection formulation and which contains a therapeutically effective amount, preferably from 30 mg to 2400mg, preferably about 1200mg or about 2400mg of the antibody or antigen binding fragment thereof according to any one of claims 1-4, or the multispecific antibody according to claim 5, or the bispecific antibody according to any one of claims 6-7, or the antibody combination according to claim 8; preferably, the pharmaceutical composition contains one selected from the group consisting of the antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody and the antibody combination, and a buffer; more preferably, the buffer comprises one or more of trehalose and polysorbate 80; more preferably, the pharmaceutical composition has a pH of 5.5 to 6.5; more preferably, the buffer further comprises one or more of histidine hydrochloride and histidine; more preferably, the molar ratio of histidine hydrochloride to histidine is 10.5:9.5; more preferably, the pharmaceutical composition comprises 0.04-0.1g/mL trehalose, 0.0001-0.0003g/mL polysorbate 80, and 10-50mg/mL of one selected from the group consisting of the antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody, and the antibody combination, based on the total volume of the pharmaceutical composition; more preferably, the pharmaceutical composition comprises 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL polysorbate 80, and 40+ -4 mg/mL of one selected from the antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody and the antibody combination, based on the total volume of the pharmaceutical composition.
A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the multispecific antibody of claim 5, or the bispecific antibody of any one of claims 6-7, or the antibody combination of claim 8, or the nucleic acid of claim 9, or the nucleic acid combination of claim 10, or the cell of claim 11, or the pharmaceutical composition of any one of claims 12-13.
Use of the antibody or antigen binding fragment thereof of any one of claims 1-4, the multispecific antibody of claim 5, the bispecific antibody of any one of claims 6-7, the antibody combination of claim 8, the nucleic acid of claim 9, or the nucleic acid combination of claim 10, the cell of claim 11, or the kit of claim 14 in the manufacture of a medicament for preventing, treating, detecting, or diagnosing a disease associated with SARS-CoV-2 virus;

preferably, the SARS-CoV-2 virus comprises one or more of Alpha (B.1.1.7) strain, beta (B.1.351) strain, gamma (p.1) strain, delta (B.1.617.2) strain, lambda (C.37) strain, mu (B.1.621) strain, omacron strain, kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain;

Preferably, the wild-type strain is a Wuhan-Hu-1 strain;

preferably, the omacron strain comprises one or more of ba.1 (b.1.1.529.1), ba.1.1, ba.2, ba.2.12.1, ba.2.13, ba.2.38, ba.2.38.1, ba.2.74, ba.2.75, ba.2.76, ba.2.77, ba.2.79, ba.2.80, ba.3, ba.4, ba.5, ba.4.6, ba.4.7, ba.5.5.1, bq.1, bq.1.1, XBB strain.
The use according to claim 15, wherein the medicament is a nasal spray, nasal drop, nebulization or injection formulation; preferably, the injection preparation is an intravenous injection preparation; more preferably, the medicament comprises a therapeutically effective amount, preferably from about 30mg to about 2400mg, preferably from about 1200mg or about 2400mg, of the antibody or antigen-binding fragment of any one of claims 1 to 4, or the multispecific antibody of claim 5, or the bispecific antibody of any one of claims 6 to 7, or the antibody combination of claim 8;

preferably, the medicament is a unit formulation which is a nasal spray, nasal drop, nebulization or injection formulation and which contains a therapeutically effective amount, preferably from 30mg to 2400mg, preferably about 1200mg or about 2400mg of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, or the multispecific antibody according to claim 5, or the bispecific antibody according to any one of claims 6-7, or the antibody combination according to claim 8; preferably, the medicament contains a member selected from the group consisting of the antibody or antigen-binding fragment thereof, the multispecific antibody, one of the bispecific antibody and the antibody combination, and a buffer; more preferably, the buffer comprises one or more of trehalose and polysorbate 80; more preferably, the drug pH is from 5.5 to 6.5; more preferably, the buffer further comprises one or more of histidine hydrochloride and histidine; more preferably, the molar ratio of histidine hydrochloride to histidine is 10.5:9.5; more preferably, the medicament comprises 0.04-0.1g/mL trehalose, 0.0001-0.0003g/mL polysorbate 80, and 10-50mg/mL of one selected from the antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody and the antibody combination, based on the total volume of the medicament; more preferably, the medicament comprises 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL polysorbate 80, and 40+ -4 mg/mL of one selected from the antibody or antigen binding fragment thereof, the multispecific antibody, the bispecific antibody and the antibody combination, based on the total volume of the medicament.
A method of treating or preventing a disease caused by SARS-CoV-2 virus, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof of any one of claims 1-4, the multispecific antibody of claim 5, the bispecific antibody of any one of claims 6-7, the antibody combination of claim 8, the pharmaceutical composition of any one of claims 12-13;

the subject in need thereof includes a treated subject or a prevented subject;

the subject being treated includes asymptomatic, light, common, heavy or critical patients infected with SARS-CoV-2 virus; preferably, the subject is asymptomatic, light, common, heavy or critical patients who are infected with SARS-CoV-2 virus in a diagnosis and treatment regimen of coronavirus pneumonia (ninth edition of trial) within 72 hours;

the treatment subjects include asymptomatic, mild, moderate, severe and critically ill patients infected with SARS-CoV-2 virus; preferably, the subject is a patient who is laboratory-checked (e.g., RT-PCR-checked) to confirm infection with SARS-CoV-2 within 72 hours and is asymptomatic, mild, moderate, severe and critical in accordance with NIH guidelines; or alternatively

The preventing subject includes a pre-exposure preventing subject, or a post-exposure preventing subject; preferably, the pre-exposure prophylaxis subjects include a high risk group of exposed to a new coronavirus, healthy subjects, or other subjects unsuitable for vaccination; the post-exposure prophylaxis subjects include post-new coronal pneumonia diagnosed patients and/or intimate contact persons of asymptomatic infected persons.