CN114907484A

CN114907484A - A strong anti-new crown mutation strain includes four subtypes of bispecific humanized antibody of Ormcken

Info

Publication number: CN114907484A
Application number: CN202210421123.7A
Authority: CN
Inventors: 高福; 仝舟; 仝剑宇; 邢潇; 赵欣; 王奇慧
Original assignee: Shanxi Institute Of Higher Innovation; Institute of Microbiology of CAS
Current assignee: Shanxi Institute Of Higher Innovation; Institute of Microbiology of CAS
Priority date: 2022-04-20
Filing date: 2022-04-20
Publication date: 2022-08-16
Anticipated expiration: 2042-04-20
Also published as: CN114907484B

Abstract

The invention discloses a single chain bispecific antibody capable of resisting new corona and mutant strains thereof, which is formed by combining two new corona human source specific antibodies, and realizes strong protection on all subtypes of an Onckronk mutant strain, namely BA.1 (IC 500.04 mu g/ml), BA.2 (IC 500.05 mu g/ml), BA.3 (IC 500.04 mu g/ml) and BA.1.1 (IC 500.08 mu g/ml). The single-chain bispecific antibody of the invention is used as an effective antibody drug for efficiently resisting all the existing new crown mutation strains and future new crown mutation strains.

Description

A strong anti-new crown mutation strain includes four subtypes of bispecific humanized antibody of Ormcken

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to a strong anti-new crown mutation strain, including four subtypes of double-specific humanized antibody of Ormcken.

Background

With the continuous spread of new coronavirus in people, various variant strains bring great threat to the vaccine immunity defense line constructed globally. The emergence of the variant of Omicron, with its powerful immune escape capacity, has raised an unprecedented concern about the effectiveness of anti-body therapies and vaccines. Therefore, the development of broad-spectrum antibodies against the omitron variants is of crucial importance. However, the rate-limiting step of broad-spectrum antibody screening is a problem to be solved that the immunogenicity of conserved epitopes is poor, resulting in few candidate antibodies. The mainstream human-derived broad-spectrum antibody research idea is to improve the generation probability of the broad-spectrum antibody of a subject by immunogen transformation, sequential immunization and other modes, and simultaneously improve the mining capability of the trace broad-spectrum antibody by using the mode of 'first generation and then mining' of single cells and high-throughput sequencing, for example, the S309 antibody is an excellent case of the idea ^[1] Furthermore, the dorsal epitope of the Receptor Binding Domain (RBD) of the spike protein to which S309 binds, recently proved to be a conserved epitope minimally affected by the Omicron variant ^[2] 。

In addition to mabs, mild symptoms in patients at high risk for COVID-19 would be treated by the combination of two mAb antibodies, however effects similar to this mAb combination could be achieved by a single bispecific antibody (BsAb). It is a specific antibody designed to bind two different antigens simultaneously based on the unique structure of the antibody, and the theory of BsAb antibody was first proposed by Nisonoff and his co-workers in 1960 ^[6] Later, scholars proposed BsAb in 1964, which was produced with antibodies in the eighty-ninety yearsThe continuous development of physics and antibody engineering continuously solves the problems of BsAb molecules in the aspects of adhesion, crosslinking, assembly and the like ^[7] The first bispecific antibody drug for treating malignant ascites disease in the world was approved until 2009 ^[8] In the application of bispecific antibody drugs, 90% of them are used for cancer treatment, and in recent years, the studies of bispecific antibody drugs in infection, inflammation, neurodegenerative diseases, ophthalmology, metabolic diseases, etc. have been increasing, and these studies provide certain basis for developing antibody drugs for treating BsAb of covi-19.

BsAb is mainly classified into two categories according to structure: bispecific antibodies with FC regions (IgG-like bispecific antibodies) and bispecific antibodies without FC regions (non-IgG-like bispecific antibodies). Bispecific antibodies containing Fc regions retain the structure of traditional monoclonal antibodies, having two Fab regions and one Fc region. However, unlike conventional monoclonal antibodies, the two fabs bind different antigens. The antibodies mainly comprise Triomabs, DVD-Ig, 2in 1-IgG, kih IgG, CrossMAb and the like; bispecific antibodies without an Fc region lack an Fc region, consisting of the VH and VL regions of both antibodies or consisting of Fab fragments. Such bispecific antibodies are mainly BiTE, DART, TandAbs, bi-Nanobody, and the like.

Based on this, related studies have reported some BsAbs for treating COVID-19. The first BsAb against SARS-CoV-2 was constructed by linking a non-neutralizing binding agent to a neutralizing binding agent in a bispecific scaffold. In particular to ^[9] The authors first identified fabs that bind to RBDs but not ACE2 by phage display and then assembled them into specific IgG scaffolds with human variable heavy chain (VH) binders that block ACE2, producing VH/Fab bsAb-Bis-4(VH B01, Fab D01), where mab Fab D01 alone had no neutralizing effect on SARS-CoV-2, but Bis-4 was designed and constructed to be 20-25 fold higher in virus neutralizing titer and SARS-CoV-2 could reach 0.107 nm. The second is IgG-like BsAb-CoV-X2 designed by using the knob-into-hole technology, which is designed by combining two monoclonal antibodies C121 and C135 separated from convalescent patients of COVID-19,with the structure domain RBD of S protein of SARS-CoV-2 as target, CoV-X2 molecule can neutralize WT (0.9nm), alpha (0.2nm), beta (12nm), gamma (2.1nm) and other strains ^[10] . The third is DVD-Ig-G BsAb, in which three effective BsAb molecules are screened ^[11] (CV1206_521_ GS, CV503_664_ EL and CV503_521_ GS) wherein GS indicates that two intermolecular light and heavy chains are combined at GGGGSGGGGSGGGG linker, EL indicates that the variable region and the constant region of the light chain are connected by QPKAAPSVTLFPP, the variable region and the constant region of the heavy chain are connected by STKGPSVFPLAP, and mAbs CV503, CV521, CV664 and CV1206 are all isolated from plasma cells and memory cells of patients with COVID-19 and target the RBD domain or NTD domain of the S protein respectively, can neutralize strains such as WT, alpha, beta, gamma, etc., the value of IC50 is less than 0.1ng/mL, and the neutralization is improved by about 10-100 times compared with the mAb used alone. The fourth is bi-Nanobody-BsAb-Nb15-NH-Nb15 ^[12] It is a targetable RBD and human serum albumin screened from the nano-neutralizing antibody database of alpaca, Nb15-NbH-Nb15 shows a neutralizing effect of unit ng/ml on WT and Delta variants of SARS-CoV-2, has a longer half-life in vivo, and in addition Nb15-NbH-Nb15 provides effective protection for prevention and treatment of SARS-CoV-2 infection of transgenic hACE2 mice by intranasal administration. The fifth is also IgG-like BsAb-Clone 2 ^[13] It is a neutralizing antibody obtained by sequencing high-throughput single-cell BCR, and has strong neutralizing effect on WT and B.1.617.2-Delta variant. The sixth is crossMAb-BsAb, targeted at target DDP4 (co-receptor of ACE 2) and RBD, Regdanvimab and Begelomab (mAb) were identified as D614G mutant S proteins blocking the SARSCoV-2 and host DPP4 receptors, respectively, and were modified to prevent heavy and light chain mismatches using KIH (Knobs into holes) and crossMAb techniques ^[14] . The seventh belongs to a nano BsAb which can induce the S protein to form a trimer, prevent the S protein from being combined with a host cell surface receptor ACE2 and neutralize mutant strains of WT, beta, Delta and the like ^[15] . The last BsAb15 was designed based on a pair of noncompetitive human neutralizing antibodies (B38 and H4) that block the binding of SARS-CoV-2 to its receptor ACE2 ^[16] The bsAb15 has higher neutralization efficiency than these mAb antibodies, leading to and retaining the neutralizing effect on most SARS-CoV-2 neutralizing capacity of the relevant variant (stronger neutralizing activity against Delta variants).

Reference to the literature

1.Pinto,D.,et al.,Cross-neutralization of SARS-CoV-2by a human monoclonal SARS-CoV antibody.Nature,2020.583(7815):p.290-295.

2.Zhou,T.,et al.,Structural basis for potent antibody neutralization of SARS-CoV-2variants including B.1.1.529.Science,2022:p.eabn8897.

3.Barnes,C.O.,et al.,SARS-CoV-2neutralizing antibody structures inform therapeutic strategies.Nature,2020.588(7839):p.682-687.

4.Hansen,J.,et al.,Studies in humanized mice and convalescent humans yield a SARS-CoV-2antibody cocktail.Science,2020.369(6506):p.1010-1014.

5.Liu,H.,et al.,Cross-Neutralization of a SARS-CoV-2Antibody to a Functionally Conserved Site Is Mediated by Avidity.Immunity,2020.53(6):p.1272-1280.e5.

6.Yuan,M.,et al.,A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2and SARS-CoV.Science,2020.368(6491):p.630-633.

7.Kipriyanov,S.M.,et al.,Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics.J Mol Biol,1999.293(1):p.41-56.

6.Nisonoff,A.,F.C.Wissler,and L.N.Lipman,Properties of the major component of a peptic digest of rabbit antibody.Science,1960.132(3441):p.1770-1.

7.Labrijn,A.F.,et al.,Bispecific antibodies:a mechanistic review of the pipeline.Nat Rev Drug Discov,2019.18(8):p.585-608.

8.Heiss,M.M.,et al.,The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelial cancer:Results of a prospective randomized phase II/III trial.Int J Cancer,2010.127(9):p.2209-21.

9.Lim,S.A.,et al.,Bispecific VH/Fab antibodies targeting neutralizing and non-neutralizing Spike epitopes demonstrate enhanced potency against SARS-CoV-2.MAbs,2021.13(1):p.1893426.

10.De Gasparo,R.,et al.,Bispecific IgG neutralizes SARS-CoV-2variants and prevents escape in mice.Nature,2021.593(7859):p.424-428.

11.Cho et al.,eabj5413(2021)Bispecific antibodies targeting distinct regions of the spike protein potently neutralize SARS-CoV-2variants of concern.Sci.Transl.Med.,2021.10.5413.

12.Wu,X.,et al.,A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2infection via intranasal administration.Cell Rep,2021.37(3):p.109869.

13.Peng,L.,et al.,Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617.bioRxiv,2021.

14.Ojha,R.,et al.,Designing of a bispecific antibody against SARS-CoV-2spike glycoprotein targeting human entry receptors DPP4 and ACE2.Hum Immunol,2022.

15.Hanke,L.,et al.,A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2in vivo.Nat Commun,2022.13(1):p.155.

16.Li,Z.,et al.,An engineered bispecific human monoclonal antibody against SARS-CoV-2.Nat Immunol,2022.23(3):p.423-430。

Disclosure of Invention

The bispecific antibody is successfully constructed by utilizing the characteristics of different epitopes of the IMCAS-123 antibody and the IMCAS-364 antibody and based on the characteristic of a long Linker efficiently producing Monomer form in a reference document. The affinity of the mutant strain is improved by 5-10 times compared with the original IMCAS-123 and IMCAS-364 for the receptor binding domains of different variants. Thus, the present invention has been completed.

The invention provides a bispecific humanized antibody of a potent anti-new crown mutation strain, which is formed by connecting a first antibody heavy chain, a second antibody light chain and a first antibody heavy chain by a connecting peptide linker. Wherein the content of the first and second substances,

the heavy chain variable region of the first antibody (IMCAS-364) comprises the CDR1 amino acid sequence: SEQ ID NO. 1: GFTFSRYG; the CDR2 amino acid sequence is SEQ ID NO. 2: IWYDGSNK; the CDR3 amino acid sequence is SEQ ID No. 3: AKQEGTYCSGGSCYSGLDY, respectively;

the variable region of the light chain of the first antibody (IMCAS-364) comprises the CDR1 amino acid sequence of SEQ ID No. 4: QSISSY; the CDR2 amino acid sequence is SEQ ID No. 5: AAS; the CDR3 amino acid sequence is SEQ ID NO. 6: QQSYSTPLT are provided.

The variable region of the heavy chain of the second antibody (IMCAS-123) comprises the CDR1 amino acid sequence: SEQ ID NO. 7: GFTFSSYA; the CDR2 amino acid sequence is SEQ ID NO. 8: ISGSGGST; the CDR3 amino acid sequence is SEQ ID NO. 9: AKDHLITMVQPEYFHHW, respectively; further, the CDR3 amino acid sequence is mutated, namely AKDHLSTMVQPEYFHHW、AKDHLMTMVQPEYFHHW、AKDHLITLVQPEYFHHW、AKDHFITMVQPEYFHHW、AKDHFITMVQPEYFYHW。

The variable region of the light chain of the second antibody (IMCAS-123) comprises the CDR1 amino acid sequence of SEQ ID No. 10: QGISRW; the CDR2 amino acid sequence is SEQ ID NO. 11: AAG; the CDR3 amino acid sequence is SEQ ID No. 12: CQADSSF;

more preferably, the variable region amino acid sequence of the heavy chain of said first antibody (IMCAS-364) is SEQ ID NO. 13: QVQLQESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKQEGTYCSGGSCYSGLDYWGQGTLVTVSSAS, respectively; the variable region amino acid sequence of the light chain of the second antibody (IMCAS-364) is SEQ ID No. 14: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKG are provided.

The variable region amino acid sequence of the heavy chain of the second antibody (IMCAS-123) is SEQ ID NO. 15: QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHLITMVQPEYFHHWGQGTLVTVSSAS, respectively; the variable region amino acid sequence of the light chain of the second antibody (IMCAS-123) is SEQ ID NO. 16: DIQMTQSPSSVSASVGDSVTITCRASQGISRWLAWYQQRPGKAPKLLIYAAGNLETGVPSRFSGSGSGTDFTLTISDLQAEDFATYYCQQADSFPLTFGGGTKVDIKG are provided.

Specifically, the first antibody heavy chain, the linker1, the second antibody light chain, the linker2, the second antibody heavy chain, the linker1 and the first antibody light chain are connected in sequence in the above order, and more preferably, the connection mode is that the first antibody heavy chain, the linker1, the second antibody light chain, the linker2 and the second antibody heavy chain are connected in sequence.

Preferably, the amino acid sequence of Linker1 is SEQ ID NO. 17: RADAAAAGGPGS, respectively; the amino acid sequence of Linker2 is SEQ ID NO. 18: RADAAAAGGGGSGGGGSGGGGSGGGGS are provided.

Preferably, the amino acid sequence of the bispecific humanized antibody of the potent anti-new crown mutation strain is SEQ ID NO. 19:

QVQLQESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKQEGTYCSGGSCYSGLDYWGQGTLVTVSSASRADAAAAGGPGSDIQMTQSPSSVSASVGDSVTITCRASQGISRWLAWYQQRPGKAPKLLIYAAGNLETGVPSRFSGSGSGTDFTLTISDLQAEDFATYYCQQADSFPLTFGGGTKVDIKGRADAAAAGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHLITMVQPEYFHHWGQGTLVTVSSASRADAAAAGGPGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKGTKGPSVTSGQAGQ

further preferably, the bispecific humanized antibody against the new crown mutation strain further comprises a signal peptide, and therefore the connection sequence is formed by connecting the signal peptide, a first antibody heavy chain, a linker1, a second antibody light chain, a linker2, a second antibody heavy chain, a linker1 and the first antibody light chain in sequence. In an embodiment the amino acid sequence of the signal peptide is SEQ ID NO. 20: METDTLLLWVLLLWVPGSTGD is added. Further, it comprises a tag sequence for isolation and purification, such as 6 × His tag.

Most preferably, the amino acid sequence of the bispecific humanized antibody against the novel crown mutation strain is SEQ ID No. 21:

METDTLLLWVLLLWVPGSTGDQVQLQESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKQEGTYCSGGSCYSGLDYWGQGTLVTVSSASRADAAAAGGPGSDIQMTQSPSSVSASVGDSVTITCRASQGISRWLAWYQQRPGKAPKLLIYAAGNLETGVPSRFSGSGSGTDFTLTISDLQAEDFATYYCQQADSFPLTFGGGTKVDIKGRADAAAAGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHLITMVQPEYFHHWGQGTLVTVSSASRADAAAAGGPGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKGTKGPSVTSGQAGQHHHHHH*

the invention also provides the coding nucleic acid of the bispecific humanized antibody of the high-efficiency anti-new crown mutation strain. Preferably, the nucleotide sequence thereof is SEQ ID No. 22:

ATGCAGGTGCAGCTGCAAGAGAGCGGCGGCGGTGTCGTGCAGCCTGGCCGGAGCCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTTTCTAGATACGGCATGCACTGGGTGAGACAGGCTCCTGGCAAGGGGCTGGAATGGGTGGCCGTGATCTGGTACGACGGCTCCAACAAGTACTACGCCGATAGCGTGAAGGGCAGATTCACCATCTCCCGGGACAACAGCAAAAATACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGACGACACGGCCGTGTACTACTGCGCCAAACAGGAGGGCACATATTGCAGCGGCGGAAGCTGCTACAGCGGACTGGACTACTGGGGACAGGGCACACTGGTGACCGTGAGCAGCGCTTCTCGGGCCGATGCCGCTGCTGCCGGCGGCCCCGGCTCTGATATCCAGATGACCCAGTCCCCAAGCTCTGTGAGCGCCTCTGTTGGCGACAGCGTGACAATCACATGCCGCGCCAGCCAGGGCATCAGCAGATGGCTGGCCTGGTATCAGCAAAGACCTGGCAAGGCCCCTAAACTGCTGATCTACGCCGCTGGGAACCTGGAAACCGGCGTGCCTAGCAGGTTCAGCGGCTCCGGTAGCGGCACCGACTTTACACTTACCATCAGCGACCTGCAAGCAGAGGACTTCGCCACATACTATTGTCAGCAGGCCGACTCATTCCCCCTGACCTTCGGCGGCGGCACAAAGGTGGACATTAAGGGCAGAGCCGACGCCGCGGCCGCCGGCGGAGGAGGCTCGGGCGGCGGAGGTAGCGGCGGCGGAGGTTCTGGAGGCGGCGGCTCACAGGTGCAGCTGGTCGAGAGCGGCGGCGGCCTGGTGCAGCCTGGCGGCAGCCTCAGACTGAGCTGTGCCGCCAGCGGATTCACCTTTAGTAGCTACGCCATGAGCTGGGTTAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGTCCGCCATCAGCGGCTCCGGAGGCAGCACCTACTACGCCGACAGCGTCAAGGGCAGATTTACCATCAGCAGAGATAATAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTCCGGGCCGAAGATACCGCTGTGTACTACTGTGCCAAGGACCACCTGATCACCATGGTGCAGCCCGAGTACTTCCACCACTGGGGCCAAGGAACCCTGGTGACAGTGTCTTCCGCCAGCAGAGCCGATGCCGCCGCCGCCGGCGGACCCGGCGACATTCAGATGACACAGAGCCCTTCTTCTCTGTCTGCTTCTGTCGGCGATCGGGTGACCATCACCTGCAGAGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAAAAGCCCGGCAAGGCCCCAAAGCTGCTGATCTACGCGGCCTCTAGCCTGCAGAGCGGAGTGCCCAGCCGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCCTGCAACCTGAGGACTTCGCCACATACTACTGCCAGCAGAGTTATAGCACACCTCTGACCTTCGGCGGTGGAACAAAGGTGGACATCAAGGGTACCAAGGGCCCTAGCGTGACCAGCGGCCAGGCCGGC。

or SEQ ID NO. 23:

ATGGAAACTGACACCCTGCTGCTCTGGGTGCTGCTGCTGTGGGTGCCAGGCTCTACCGGAGATCAGGTGCAGCTGCAAGAGAGCGGCGGCGGTGTCGTGCAGCCTGGCCGGAGCCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTTTCTAGATACGGCATGCACTGGGTGAGACAGGCTCCTGGCAAGGGGCTGGAATGGGTGGCCGTGATCTGGTACGACGGCTCCAACAAGTACTACGCCGATAGCGTGAAGGGCAGATTCACCATCTCCCGGGACAACAGCAAAAATACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGACGACACGGCCGTGTACTACTGCGCCAAACAGGAGGGCACATATTGCAGCGGCGGAAGCTGCTACAGCGGACTGGACTACTGGGGACAGGGCACACTGGTGACCGTGAGCAGCGCTTCTCGGGCCGATGCCGCTGCTGCCGGCGGCCCCGGCTCTGATATCCAGATGACCCAGTCCCCAAGCTCTGTGAGCGCCTCTGTTGGCGACAGCGTGACAATCACATGCCGCGCCAGCCAGGGCATCAGCAGATGGCTGGCCTGGTATCAGCAAAGACCTGGCAAGGCCCCTAAACTGCTGATCTACGCCGCTGGGAACCTGGAAACCGGCGTGCCTAGCAGGTTCAGCGGCTCCGGTAGCGGCACCGACTTTACACTTACCATCAGCGACCTGCAAGCAGAGGACTTCGCCACATACTATTGTCAGCAGGCCGACTCATTCCCCCTGACCTTCGGCGGCGGCACAAAGGTGGACATTAAGGGCAGAGCCGACGCCGCGGCCGCCGGCGGAGGAGGCTCGGGCGGCGGAGGTAGCGGCGGCGGAGGTTCTGGAGGCGGCGGCTCACAGGTGCAGCTGGTCGAGAGCGGCGGCGGCCTGGTGCAGCCTGGCGGCAGCCTCAGACTGAGCTGTGCCGCCAGCGGATTCACCTTTAGTAGCTACGCCATGAGCTGGGTTAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGTCCGCCATCAGCGGCTCCGGAGGCAGCACCTACTACGCCGACAGCGTCAAGGGCAGATTTACCATCAGCAGAGATAATAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTCCGGGCCGAAGATACCGCTGTGTACTACTGTGCCAAGGACCACCTGATCACCATGGTGCAGCCCGAGTACTTCCACCACTGGGGCCAAGGAACCCTGGTGACAGTGTCTTCCGCCAGCAGAGCCGATGCCGCCGCCGCCGGCGGACCCGGCGACATTCAGATGACACAGAGCCCTTCTTCTCTGTCTGCTTCTGTCGGCGATCGGGTGACCATCACCTGCAGAGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAAAAGCCCGGCAAGGCCCCAAAGCTGCTGATCTACGCGGCCTCTAGCCTGCAGAGCGGAGTGCCCAGCCGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCCTGCAACCTGAGGACTTCGCCACATACTACTGCCAGCAGAGTTATAGCACACCTCTGACCTTCGGCGGTGGAACAAAGGTGGACATCAAGGGTACCAAGGGCCCTAGCGTGACCAGCGGCCAGGCCGGCCAGCACCATCACCACCACCACTGA。

the invention also provides an expression vector and a recombinant cell of the nucleic acid.

The invention also provides the application of the bispecific humanized antibody of the strong anti-new crown mutation strain in the preparation of drugs for treating diseases caused by new crown viruses, wherein the new crown viruses are selected from SARS-CoV-2 and SARS strains. More preferably, the SARS-CoV-2 is selected from the group consisting of a prototype strain, an alpha variant, a beta variant, a delta variant, a gamma variant, and an Onck Ron variant (more specifically, subtypes BA.1, BA.2, BA.3, and BA.1.1).

Accordingly, the present invention provides a pharmaceutical composition for the above use, wherein the bispecific humanized antibody against the novel crown mutation strain is a potent antibody against the novel crown mutation strain as an active ingredient. Preferably, a pharmaceutically acceptable carrier is also included, as well as in combination with other drugs.

The invention successfully constructs the bispecific antibody by utilizing the characteristics of different epitopes of the IMCAS-123 antibody and the IMCAS-364 antibody. The neutralization effect is greatly improved for different subtypes of Omicron. The invention has realized that the strong protection to all subtypes of the Ormckrozen mutant strain comprises BA.1(0.04 mu g/ml), BA.2(0.05 mu g/ml), BA.3(0.04 mu g/ml) and BA.1.1(0.08 mu g/ml), is an effective antibody drug for efficiently resisting all existing and future new crown mutant strains, and thus has completed the invention.

Drawings

FIG. 1 shows the purification results of the dual specific antibody Tand ab123-64 protein of the present invention.

FIG. 2 the affinity of the bispecific antibody Tandab123-64 to the novel coronavirus receptor binding domain protein.

FIG. 3 shows the neutralization experiment of the bispecific antibody Tand ab123-64 and SARS-CoV-2 pseudovirus.

Detailed Description

In order to break through the traditional mode of 'generation before screening' and quickly focus candidate antibodies on conserved epitopes, the invention utilizes antigen subtraction screening of a spike protein receptor binding domain of a prototype strain and a spike protein receptor binding domain of a Beta variant strain to quickly focus antibodies of conserved target positions of ClassIII and ClassVI in epitope classification on a platform for screening weak immunogenic antibodies based on a phage antibody library display technology, and successfully separates in vitro fully human antibodies which are combined on different epitopes of the receptor binding domain and can realize broad-spectrum neutralization on VOCs. Wherein, the affinity of the IMCAS-123 antibody to prototype strains, Alpha, Beta, Delta and Omicron variant strains reaches nM level (see Chinese patent application 202210228772.5), the neutralization to pseudoviruses of high-transmission-force Omicron variant strains reaches 0.04 mu g/ml, and the antibody can neutralize all three subtypes of Omicron BA.1BA.2BA.3. Structural biology demonstrated that IMCAS-123 binds to a similar epitope as REGN10987, but IMCAS-123 does not bind to Q498(Omicron mutated at this site), and furthermore both IMCAS-123 and S309 bind to the T345, L441 conserved site, and additionally to the Y351 conserved site. Another broad-spectrum antibody IMCAS-364 was also selected, which also has nM affinity for the prototype strain, Alpha, Beta, Delta, and Omicron (see, Chinese patent application 202210295369.4).

Through the known epitope antibody competition experiment, the fact that IMCAS-364 binds to a rare target position on the inner side of RBD and does not compete with the receptor ACE2 (different from the fact that the COVA1-16 antibody competes with ACE 2) is the first time that an antibody binding to the target position is found, and the new coronavirus can be neutralized without competing with ACE2 (different from the fact that the CR3022 antibody only neutralizes SARS). The IMCAS-364 has good potential for developing a bi-specific antibody by matching an RBM binding antibody with a receptor binding domain dorsal binding antibody, and meanwhile, the IMCAS-364 has the capacity of binding SARS virus with the affinity of nM level, so that the IMCAS-364 is a good development source of the generic SARS coronavirus neutralizing antibody.

By utilizing the characteristics of different epitopes of IMCAS-123 and IMCAS-364 antibodies, according to the characteristic of the molecular distance of 90 angstroms of three RBD epitopes on the surface S protein trimer of the new coronavirus, the span of the traditional IgG1 form antibody is considered to be 150 angstroms, and bivalent combination cannot be formed in one trimer molecule. By adopting a construction mode of single-chain diabody, the theoretical molecular length of the single-chain diabody is 60-70 angstroms, the single-chain diabody can be effectively embedded in a trimer, and in the process of combining virus and a receptor, two RBDs are combined by the bispecific molecules to lock the conformational change, so that invasion blocking is realized. In the construction process, based on a reference for constructing a Tand form tetramer molecule in an escherichia coli system, the defect that a large number of single chains are generated by a long Linker to cause double chains to be incapable of being efficiently paired is mentioned [7], and by reversely utilizing the defect of the single chain self-reverse-folding, based on two light-heavy chain variable region sequences of known IMCAS-123 and IMCAS-364, the key part of the construction is mapped as EcoRI-signal peptide-364 VH-Linker1-123VL-Linker2-123VH-Linker1-364VL-SfiI-6 xHis-stop codon-XhoI, and the construction adopts a gene synthesis mode and is connected into a pcags expression vector through EcoRI and XhoI. A single-chain bispecific antibody with the size of 55kDa which can be expressed in mammalian cells is successfully constructed, the purification is simple, and the plasmid forward yield reaches 22 mg/L.

The present invention is illustrated by the following specific examples, which are not intended to limit the invention. The experimental methods are all conventional methods as explained in Yuan Special Specification.

EXAMPLE I construction of bispecific antibody Tandab123-64

This example illustrates linking IMCAS-123 in tandem with the heavy and light chain variable regions of the IMCAS-364 antibody by a linker in the following order: 364VH-linker1-123VL-linker2-123VH-linker1-364 VL.

The specific construction steps are as follows:

by utilizing the characteristics of different epitopes of IMCAS-123 and IMCAS-364 antibodies, according to the characteristic of the molecular distance of 90 angstroms of three RBD epitopes on the surface S protein trimer of the new coronavirus, the span of the traditional IgG1 form antibody is considered to be 150 angstroms, and bivalent combination cannot be formed in one trimer molecule. According to the literature, a single-chain double-specificity construction mode is adopted, the theoretical molecular length is 60-70 angstroms, the two RBDs can be effectively embedded into a trimer, and in the process of combining the virus and a receptor, due to the fact that the two RBDs are combined by the double-specificity molecules, the conformational change is locked, and invasion blocking is achieved.

In the construction process, based on a reference for constructing a Tand form dimer tetravalent antibody molecule in an escherichia coli system, the defect that a large number of single-chain spontaneous folds generated by a long Linker can not be efficiently paired with double chains is mentioned, and the construction is carried out by reversely utilizing the defect of the single-chain spontaneous folds and respectively based on two light and heavy chain variable region sequences of known IMCAS-123 and IMCAS-364

Key part maps were constructed as follows: EcoRI enzyme cutting site-GCCACC-signal peptide-364 VH-linker1-123VL-linker2-123VH-linker1-364VL-SfiI-6 xHis-stop codon-enzyme cutting site XhoI, the construction adopts a gene synthesis mode, the codon optimization according to a 293F expression system is completed by Nanjing Kinshire company, the gene synthesis is completed by the same company through EcoRI and XhoI and connected into pcaggs expression vector, and the constructed plasmid is returned.

EXAMPLE two bispecific antibody Tandab123-64 purification procedure

(I) plasmid transformation

(1) Taking out Top 10 competent cells from a-80 ℃ refrigerator, and carrying out ice bath for 10 min;

(2) 0.1ug of plasmid was placed in 30 μ L of competent cells, the contents were gently swirled and mixed, ice-washed for 30 min;

(3) heat shock at 42 ℃ for 90s in ice bath for 3min without shaking the centrifuge tube;

(4) adding 500 μ L of non-resistant LB culture medium preheated at 37 ℃ into each centrifuge tube, and performing constant temperature shaking culture at 180rpm and 37 ℃ for 30 min;

(5) coating 100 mu L of bacterial liquid in an LB solid culture medium containing Amp, positively placing a flat plate for 2-3 hours at 37 ℃, and then performing inverted culture for 16-24 hours;

(6) single colonies of a suspected positive clone were selected, inoculated into an LB medium (4 mL/tube) containing Amp, and subjected to shaking culture at 37 ℃ and 200rpm for 12 to 16 hours.

(II) plasmid extraction

(1) Adding 3mL of culture solution after the positive clone culture into an LB culture medium containing 1 per mill of ampicillin, and carrying out constant temperature overnight shaking culture at 180rpm and 37 ℃;

(2) centrifuging the cultured bacterial liquid, removing the supernatant, and collecting the precipitate;

(3) carrying out large plasmid extraction by using an endotoxin-free plasmid large-plasmid extraction kit of Tiangen company according to the instruction;

(4) and (3) carrying out nucleic acid electrophoresis identification on the greatly extracted plasmid, measuring the concentration, and temporarily storing the plasmid in a refrigerator at the temperature of-20 ℃ for later use.

(III) transfection of 293F cells

(1) Transfection buffer: 20mM HAPES, 150mM NaCl, pH 7.4; transfection reagent: PEI;

(2) according to the proportion of 1ug of plasmid transfected by 1mL of cells, 500ug of plasmid was dissolved in a transfection buffer, and the total volume was 12.5mL, according to the PEI: dissolving PEI in the same solution at a ratio of 3:1 to 12.5mL, and standing for 5 min;

(3) mixing the two solutions, and standing for 15 min;

(4) the mixed solution after standing was slowly dropped into 500mL of a pipette tip with a density of 3X 10 ⁶ cells/mL 293F cells, in the process, the cells were shaken while dropping;

(5) placing the transfected cells into a CO-impermeable container ₂ The cells were cultured in the shaker for 24 hours, then taken out, added with 17.5mL of the feed solution, cultured for 48 hours, added with the feed solution again, transfected for 5 days, and then harvested for protein purification.

(IV) protein purification

1. Cell Collection and Pre-treatment

293F cells are collected by a high-speed refrigerated centrifuge, centrifuged at 6500rpm/min at 8 ℃ for 90min to discard precipitates, and a supernatant is collected and filtered by a 0.22 mu m filter to remove impurities.

2. Ni-NTA affinity chromatography column

Using a peristaltic pump, the HisTrap was equilibrated with 25mL (5 column volumes) of water and affinity chromatography buffer A (20mM Tris, 150mM NaCl, pH 8.0), respectively ^TM And (4) an HP affinity chromatographic column, wherein all supernatant flows through the chromatographic column at the flow rate of 2mL/min after the balance is good, and the lower liquid is collected. And (2) introducing the His column combined with the target protein into an AKTA chromatographic system, mixing the solution A and the solution B (20mM Tris, 150mM NaCl, 1M imidazole and pH 8.0) according to different proportions, performing gradient elution on the affinity column, wherein the concentrations of the imidazole are 0, 20, 50, 100, 300, 500mM and 1M respectively, collecting samples eluted at different imidazole concentrations respectively, and identifying by SDS-PAGE.

3. Gel filtration chromatography

According to the difference of molecular mass and shape, the separation and purification are carried out by a method of Gel filtration chromatography (GF). Hiload 16/600Superdex200 PG (200 size for short) is selected as Tandab123-64 according to the molecular weight.

Concentrating the target protein eluted by His affinity chromatography by using a 10kDa ultrafiltration tube, changing the solution into a PBS solution, centrifuging at 4 ℃ for 10min at 12000rpm/min for 3 times to remove precipitates, injecting the solution into a molecular sieve through a 2mL loop ring, flowing the solution through the molecular sieve at the flow rate of 1mL/min, and collecting a sample with the UV280 value of more than 5mAU according to the volume of 1.8mL per tube. The collected samples were run on SDS-PAGE and concentrated by selecting sample tubes based on the electrophoresis results, peak positions and peak shapes (as shown in FIG. 1). Subpackaging the concentrated protein, quickly freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C. After 120mL of buffer is sieved, 1 column volume of water and 20% ethanol are added, and the mixture is stored at room temperature.

EXAMPLE III detection of protein and antibody affinity (biacore) by surface plasmon resonance

The surface plasmon resonance phenomenon is utilized to detect the intermolecular interaction, and the detection is completed on a biomacromolecule interaction analysis system Biacore 8K produced by GE Healthcare group. The prototype strain virus receptor binding domain protein, the beta variant strain virus receptor binding domain protein, the delta variant strain virus receptor binding domain protein, the ormickron variant strain (subtypes ba.1, ba.2, ba.3 and ba.1.1) virus receptor binding domain protein and the atypical pneumovirus receptor binding domain protein were captured using biotin-streptavidin coupling (SA chip) as stationary phases, mobile phase was Tandab123-64 neo-corona bispecific neutralizing antibody protein to be detected, after which kinetic parameters were analyzed by BIA evaluation software and plotted.

The experimental steps are as follows: utilizing the coupling effect of biotin-streptavidin, firstly, the original strain virus receptor binding domain protein, the beta variant strain virus receptor binding domain protein, the delta variant strain virus receptor binding domain protein, the Onckronk variant strain (BA.1, BA.2, BA.3 and BA.1.1 subtypes) virus receptor binding domain protein and the atypical pneumonia virus receptor binding domain protein are proportionally placed with a biotinylation reagent at room temperature for 30 minutes, the protein is biotinylated and labeled, and then the liquid is changed to PBS by a concentration tube, so that the redundant biotinylation reagent is removed. Biotinylated antigenic protein prototype strain viral receptor binding domain protein, beta variant viral receptor binding domain protein, delta variant viral receptor binding domain protein, omchon variant (subtypes ba.1, ba.2, ba.3, and ba.1.1) viral receptor binding domain protein, and atypical pneumovirus receptor binding domain protein were immobilized on an SA chip (GE) at a concentration of 10 μ g/ml, and antibody Tandab123-64 was injected into the chip at a concentration gradient of 2.5nM, 5nM, 10nM, 20nM, and 40nM, and the assay was performed at a constant temperature of 25 ℃ using 0.05% PBST. The regeneration of the chip surface was carried out using a 10mM Glynice solution pH 1.7. Binding curves are shown, and curves at different concentrations make up the illustrated kinetic curves. The calculation of binding kinetic constants was performed using BIA evaluation software version 3.2(Biacore, Inc.) software.

The results are shown in FIG. 2, wherein it is known that the constructed bispecific antibody Tand ab123-64 can realize strong protection of all subtypes of the mutant strain Ornken to BA.1 (0.04. mu.g/ml), BA.2 (0.05. mu.g/ml), BA.3 (0.04. mu.g/ml) and BA.1.1 (0.08. mu.g/ml).

EXAMPLE four neutralization experiments of antibody Tand ab123-64 and SARS-CoV-2 pseudovirus

Firstly, a preparation part:

sample preparation: tandab123-64 bispecific neutralizing antibody

Pseudoviruses (prototype strain, alpha variant, beta variant, delta variant, gamma variant, Ormcken variant (subtypes BA.1, BA.2, BA.3 and BA.1.1), atypical pneumonia strains

Consumable material: gun head (sterile), round bottom 96-well plate, 10cm cell culture dish, flat bottom 12-well plate, flat bottom 96-well plate, flow type stationary liquid, flow type tube

Reagent: DMEM Medium + 10% FBS (044)

Secondly, pseudovirus packaging:

pCAGGS-SARS-CoV-2-S mutant plasmid 30. mu.g each, 293T cell transfection 10cm2 dish (cell amount 80% -90%), liquid exchange DMEM (10% FBS) after 4h, VSV-delta G-GFP pseudovirus 5ml after 24h transfection, liquid exchange after 2h, DMEM 10%, VSVG-containing antibody 1:1000(10mg/ml from I1 Hybridoma)

CRL2700 ^TM Cell expression, final concentration 10. mu.g/ml), supernatant was collected 20h after pseudovirus addition, centrifuged at 3000rpm for 10min, and filtered through 0.45 filter. Subpackaging the jelly-80. Cells that were not transfected with S protein, and subsequent groups to which VSV-. DELTA.G-GFP pseudovirus and antibody were also added as pseudovirus packaging controls.

Thirdly, quantification of pseudovirion:

pseudoviruses were treated with 0.5U/. mu.l BaseMauncher endonuclease (Abcam, ab270049) for 1.5 hours.

RNA was extracted and QPCR was performed using primers for L protein. And performing a unification process according to the result.

Fourthly, determination of pseudovirus titer:

spreading vero cells in a 96-well plate for 24 hours to 90 percent;

pseudovirus was diluted with DMEM (containing 10% 044FBS) 3-fold gradient, 100 μ l/well in 96-well plates, and triplicate wells were made for each sample;

after 15h, the readings were photographed using a CQ 1-dual turntable laser confocal cell imaging analysis system to calculate titer.

And fifthly, neutralization experiment:

spreading vero cells in a 96-well plate for 24 hours to 90 percent; inactivated serum was removed from-80 ℃ on the day of the experiment and thawed on ice (serum inactivated 30min at 56 ℃ in advance). Antibodies (initial 40. mu.g/ml) were diluted in DMEM medium (containing 10% 044FBS) (2 replicates, 4-fold dilution, 10 gradients) while pseudoviruses were diluted to 1000 TU/50. mu.l/assoy with the same DMEM medium.

The diluted antibody and pseudovirus were added to a 96-well plate (volume ratio 1:1, i.e. 1 replicate well 60. mu.l antibody dilution + 60. mu.l pseudovirus dilution), and mixed by pipetting 1 time. Putting the 96-well plate into a temperature of 37 ℃ for incubation for 1 h; and (3) incubating for 40-50min, taking out the vero cells prepared in the incubator in advance, completely sucking the supernatant of the vero cells, adding 100 mu l of mixed diluent of the antibody and the virus, incubating for 15h at 37 ℃, detecting green fluorescence by using a CQ 1-double turntable laser confocal cell imaging analysis system reading method, photographing, counting, and processing data by using GraphpadPrism9.3.1 software.

The experimental results are shown in fig. 3, in which the half-maximal inhibition ratios are as follows:

PT	Alpha	Beta	Delta	Gamma	BA.1	BA.2	BA.3	BA.1.1	SARS
										0.01876	0.02149	0.02560	0.1090	0.01488	0.04595	0.05193	0.03779	0.08199	18.05

therefore, the bispecific antibody Tand ab123-64 constructed by the invention can be used as an effective antibody medicament for efficiently resisting all the existing new crown mutation strains and future new crown mutation strains.

<110> institute of microbiology, national academy of sciences; shanxi institute of advanced innovation

<120> a potent anti-new crown mutation strain comprising four subtypes of human bispecific antibody of Ormcken

<160>28

<170>PatentIn version 3.5

<210> 1

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GFTFSRYG 8

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IWYDGSNK 8

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AKQEGTYCSGGSCYSGLDY 19

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QSISSY 6

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AAS 3

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QQSYSTPLT 9

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GFTFSSYA 8

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ISGSGGST 8

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AKDHLITMVQPEYFHHW 17

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QGISRW 6

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AAG 3

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CQQADSF 7

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<211> 128

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QVQLQESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKQEGTYCSGGSCYSGLDYWGQGTLVTVSSAS 128

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DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKG 108

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QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHLITMVQPEYFHHWGQGTLVTVSSAS 125

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DIQMTQSPSSVSASVGDSVTITCRASQGISRWLAWYQQRPGKAPKLLIYAAGNLETGVPSRFSGSGSGTDFTLTISDLQAEDFATYYCQQADSFPLTFGGGTKVDIKG 108

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RADAAAAGGPGS 12

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RADAAAAGGGGSGGGGSGGGGSGGGGS 27

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QVQLQESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKQEGTYCSGGSCYSGLDYWGQGTLVTVSSASRADAAAAGGPGSDIQMTQSPSSVSASVGDSVTITCRASQGISRWLAWYQQRPGKAPKLLIYAAGNLETGVPSRFSGSGSGTDFTLTISDLQAEDFATYYCQQADSFPLTFGGGTKVDIKGRADAAAAGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHLITMVQPEYFHHWGQGTLVTVSSASRADAAAAGGPGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKGTKGPSVTSGQAGQ 532

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METDTLLLWVLLLWVPGSTGD 21

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METDTLLLWVLLLWVPGSTGDQVQLQESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKQEGTYCSGGSCYSGLDYWGQGTLVTVSSASRADAAAAGGPGSDIQMTQSPSSVSASVGDSVTITCRASQGISRWLAWYQQRPGKAPKLLIYAAGNLETGVPSRFSGSGSGTDFTLTISDLQAEDFATYYCQQADSFPLTFGGGTKVDIKGRADAAAAGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHLITMVQPEYFHHWGQGTLVTVSSASRADAAAAGGPGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKGTKGPSVTSGQAGQHHHHHH 559

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<212> DNA

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ATGCAGGTGCAGCTGCAAGAGAGCGGCGGCGGTGTCGTGCAGCCTGGCCGGAGCCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTTTCTAGATACGGCATGCACTGGGTGAGACAGGCTCCTGGCAAGGGGCTGGAATGGGTGGCCGTGATCTGGTACGACGGCTCCAACAAGTACTACGCCGATAGCGTGAAGGGCAGATTCACCATCTCCCGGGACAACAGCAAAAATACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGACGACACGGCCGTGTACTACTGCGCCAAACAGGAGGGCACATATTGCAGCGGCGGAAGCTGCTACAGCGGACTGGACTACTGGGGACAGGGCACACTGGTGACCGTGAGCAGCGCTTCTCGGGCCGATGCCGCTGCTGCCGGCGGCCCCGGCTCTGATATCCAGATGACCCAGTCCCCAAGCTCTGTGAGCGCCTCTGTTGGCGACAGCGTGACAATCACATGCCGCGCCAGCCAGGGCATCAGCAGATGGCTGGCCTGGTATCAGCAAAGACCTGGCAAGGCCCCTAAACTGCTGATCTACGCCGCTGGGAACCTGGAAACCGGCGTGCCTAGCAGGTTCAGCGGCTCCGGTAGCGGCACCGACTTTACACTTACCATCAGCGACCTGCAAGCAGAGGACTTCGCCACATACTATTGTCAGCAGGCCGACTCATTCCCCCTGACCTTCGGCGGCGGCACAAAGGTGGACATTAAGGGCAGAGCCGACGCCGCGGCCGCCGGCGGAGGAGGCTCGGGCGGCGGAGGTAGCGGCGGCGGAGGTTCTGGAGGCGGCGGCTCACAGGTGCAGCTGGTCGAGAGCGGCGGCGGCCTGGTGCAGCCTGGCGGCAGCCTCAGACTGAGCTGTGCCGCCAGCGGATTCACCTTTAGTAGCTACGCCATGAGCTGGGTTAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGTCCGCCATCAGCGGCTCCGGAGGCAGCACCTACTACGCCGACAGCGTCAAGGGCAGATTTACCATCAGCAGAGATAATAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTCCGGGCCGAAGATACCGCTGTGTACTACTGTGCCAAGGACCACCTGATCACCATGGTGCAGCCCGAGTACTTCCACCACTGGGGCCAAGGAACCCTGGTGACAGTGTCTTCCGCCAGCAGAGCCGATGCCGCCGCCGCCGGCGGACCCGGCGACATTCAGATGACACAGAGCCCTTCTTCTCTGTCTGCTTCTGTCGGCGATCGGGTGACCATCACCTGCAGAGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAAAAGCCCGGCAAGGCCCCAAAGCTGCTGATCTACGCGGCCTCTAGCCTGCAGAGCGGAGTGCCCAGCCGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCCTGCAACCTGAGGACTTCGCCACATACTACTGCCAGCAGAGTTATAGCACACCTCTGACCTTCGGCGGTGGAACAAAGGTGGACATCAAGGGTACCAAGGGCCCTAGCGTGACCAGCGGCCAGGCCGGC 1596

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ATGGAAACTGACACCCTGCTGCTCTGGGTGCTGCTGCTGTGGGTGCCAGGCTCTACCGGAGATCAGGTGCAGCTGCAAGAGAGCGGCGGCGGTGTCGTGCAGCCTGGCCGGAGCCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTTTCTAGATACGGCATGCACTGGGTGAGACAGGCTCCTGGCAAGGGGCTGGAATGGGTGGCCGTGATCTGGTACGACGGCTCCAACAAGTACTACGCCGATAGCGTGAAGGGCAGATTCACCATCTCCCGGGACAACAGCAAAAATACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGACGACACGGCCGTGTACTACTGCGCCAAACAGGAGGGCACATATTGCAGCGGCGGAAGCTGCTACAGCGGACTGGACTACTGGGGACAGGGCACACTGGTGACCGTGAGCAGCGCTTCTCGGGCCGATGCCGCTGCTGCCGGCGGCCCCGGCTCTGATATCCAGATGACCCAGTCCCCAAGCTCTGTGAGCGCCTCTGTTGGCGACAGCGTGACAATCACATGCCGCGCCAGCCAGGGCATCAGCAGATGGCTGGCCTGGTATCAGCAAAGACCTGGCAAGGCCCCTAAACTGCTGATCTACGCCGCTGGGAACCTGGAAACCGGCGTGCCTAGCAGGTTCAGCGGCTCCGGTAGCGGCACCGACTTTACACTTACCATCAGCGACCTGCAAGCAGAGGACTTCGCCACATACTATTGTCAGCAGGCCGACTCATTCCCCCTGACCTTCGGCGGCGGCACAAAGGTGGACATTAAGGGCAGAGCCGACGCCGCGGCCGCCGGCGGAGGAGGCTCGGGCGGCGGAGGTAGCGGCGGCGGAGGTTCTGGAGGCGGCGGCTCACAGGTGCAGCTGGTCGAGAGCGGCGGCGGCCTGGTGCAGCCTGGCGGCAGCCTCAGACTGAGCTGTGCCGCCAGCGGATTCACCTTTAGTAGCTACGCCATGAGCTGGGTTAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGTCCGCCATCAGCGGCTCCGGAGGCAGCACCTACTACGCCGACAGCGTCAAGGGCAGATTTACCATCAGCAGAGATAATAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTCCGGGCCGAAGATACCGCTGTGTACTACTGTGCCAAGGACCACCTGATCACCATGGTGCAGCCCGAGTACTTCCACCACTGGGGCCAAGGAACCCTGGTGACAGTGTCTTCCGCCAGCAGAGCCGATGCCGCCGCCGCCGGCGGACCCGGCGACATTCAGATGACACAGAGCCCTTCTTCTCTGTCTGCTTCTGTCGGCGATCGGGTGACCATCACCTGCAGAGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAAAAGCCCGGCAAGGCCCCAAAGCTGCTGATCTACGCGGCCTCTAGCCTGCAGAGCGGAGTGCCCAGCCGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCCTGCAACCTGAGGACTTCGCCACATACTACTGCCAGCAGAGTTATAGCACACCTCTGACCTTCGGCGGTGGAACAAAGGTGGACATCAAGGGTACCAAGGGCCCTAGCGTGACCAGCGGCCAGGCCGGCCAGCACCATCACCACCACCACTGA 1680

<210> 24

<211> 17

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AKDHLSTMVQPEYFHHW 17

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<211> 17

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<400> 25

AKDHLMTMVQPEYFHHW 17

<210> 26

<211> 17

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<400> 26

AKDHLITLVQPEYFHHW 17

<210> 27

<211> 17

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<400> 27

AKDHFITMVQPEYFHHW 17

<210> 28

<211> 17

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AKDHFITMVQPEYFYHW 17

Claims

1. A strong double-specific humanized antibody of anti-new crown mutation strain is formed by connecting a first antibody heavy chain, a second antibody light chain and a first antibody heavy chain by a connecting peptide linker;

wherein the heavy chain variable region of said first antibody comprises the amino acid sequence of CDR 1: SEQ ID NO. 1: GFTFSRYG; the CDR2 amino acid sequence is SEQ ID NO. 2: IWYDGSNK; the CDR3 amino acid sequence is SEQ ID No. 3: AKQEGTYCSGGSCYSGLDY, respectively; the variable region of the light chain of the first antibody (IMCAS-364) comprises the CDR1 amino acid sequence of SEQ ID No. 4: QSISSY; the CDR2 amino acid sequence is SEQ ID No. 5: AAS; the CDR3 amino acid sequence is SEQ ID NO. 6: QQSYSTPLT, respectively;

the variable region of the second antibody heavy chain comprises the amino acid sequence of CDR 1: SEQ ID NO. 7: GFTFSSYA; the CDR2 amino acid sequence is SEQ ID NO. 8: ISGSGGST; the CDR3 amino acid sequence is SEQ ID NO. 9: AKDHLITMVQPEYFHHW, SEQ ID NO. 24: AKDHLSTMVQPEYFHHW，SEQ ID NO.25：AKDHLMTMVQPEYFHHW，SEQ ID NO.26：AKDHLITLVQPEYFHHW, SEQ ID NO. 27: AKDHFITMVQPEYFHHW, SEQ ID NO. 28: AKDHFITMVQPEYFYHW, respectively; the light chain variable region of the second antibody comprises the CDR1 amino acid sequence of SEQ ID No. 10: QGISRW; the CDR2 amino acid sequence is SEQ ID NO. 11: AAG; the CDR3 amino acid sequence is SEQ ID No. 12: CQADSSF.

2. The bispecific humanized antibody against a neocrown mutant strain according to claim 1, wherein more preferably the variable region amino acid sequence of the heavy chain of the first antibody is SEQ ID No. 13: QVQLQESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKQEGTYCSGGSCYSGLDYWGQGTLVTVSSAS, respectively; the variable region amino acid sequence of the light chain of the second antibody is SEQ ID No. 14: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKG, respectively;

the variable region amino acid sequence of the heavy chain of the second antibody is SEQ ID No. 15: QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHLITMVQPEYFHHWGQGTLVTVSSAS; the variable region amino acid sequence of the light chain of the second antibody is SEQ ID No. 16: DIQMTQSPSSVSASVGDSVTITCRASQGISRWLAWYQQRPGKAPKLLIYAAGNLETGVPSRFSGSGSGTDFTLTISDLQAEDFATYYCQQADSFPLTFGGGTKVDIKG are provided.

3. The bispecific humanized antibody against a new crown mutant strain of claim 1 or 2, wherein the connection mode is formed by connecting a first antibody heavy chain, a linker1, a second antibody light chain, a linker2, a second antibody heavy chain, a linker1 and the first antibody light chain in sequence; preferably, the amino acid sequence of Linker1 is SEQ ID NO. 17: RADAAAAGGPGS, respectively; the amino acid sequence of Linker2 is SEQ ID NO. 18: RADAAAAGGGGSGGGGSGGGGSGGGGS are provided.

4. The bispecific humanized antibody against a novel crown mutation strain according to claim 3, wherein the amino acid sequence of the bispecific humanized antibody against a novel crown mutation strain is SEQ ID No. 19:

QVQLQESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKQEGTYCSGGSCYSGLDYWGQGTLVTVSSASRADAAAAGGPGSDIQMTQSPSSVSASVGDSVTITCRASQGISRWLAWYQQRPGKAPKLLIYAAGNLETGVPSRFSGSGSGTDFTLTISDLQAEDFATYYCQQADSFPLTFGGGTKVDIKGRADAAAAGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHLITMVQPEYFHHWGQGTLVTVSSASRADAAAAGGPGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKGTKGPSVTSGQAGQ。

5. the bispecific humanized antibody against the novel crown mutation strain of claim 3, which further comprises a signal peptide, wherein the signal peptide, the first antibody heavy chain, the linker1, the second antibody light chain, the linker2, the second antibody heavy chain, the linker1 and the first antibody light chain are connected in sequence; preferably, the amino acid sequence of the signal peptide is SEQ ID No. 20: METDTLLLWVLLLWVPGSTGD; even more preferably, it further comprises a tag sequence for isolation and purification, such as a 6 × His tag.

6. The bispecific humanized antibody against the novel crown mutation strain of claim 5, wherein the amino acid sequence of the bispecific humanized antibody against the novel crown mutation strain is SEQ ID NO. 21:

METDTLLLWVLLLWVPGSTGDQVQLQESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKQEGTYCSGGSCYSGLDYWGQGTLVTVSSASRADAAAAGGPGSDIQMTQSPSSVSASVGDSVTITCRASQGISRWLAWYQQRPGKAPKLLIYAAGNLETGVPSRFSGSGSGTDFTLTISDLQAEDFATYYCQQADSFPLTFGGGTKVDIKGRADAAAAGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHLITMVQPEYFHHWGQGTLVTVSSASRADAAAAGGPGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKGTKGPSVTSGQAGQHHHHHH。

7. nucleic acid encoding the bispecific humanized antibody against a novel crown mutation strain according to any one of claims 1 to 6; preferably, the nucleotide sequence is SEQ ID No. 22:

ATGCAGGTGCAGCTGCAAGAGAGCGGCGGCGGTGTCGTGCAGCCTGGCCGGAGCCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTTTCTAGATACGGCATGCACTGGGTGAGACAGGCTCCTGGCAAGGGGCTGGAATGGGTGGCCGTGATCTGGTACGACGGCTCCAACAAGTACTACGCCGATAGCGTGAAGGGCAGATTCACCATCTCCCGGGACAACAGCAAAAATACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGACGACACGGCCGTGTACTACTGCGCCAAACAGGAGGGCACATATTGCAGCGGCGGAAGCTGCTACAGCGGACTGGACTACTGGGGACAGGGCACACTGGTGACCGTGAGCAGCGCTTCTCGGGCCGATGCCGCTGCTGCCGGCGGCCCCGGCTCTGATATCCAGATGACCCAGTCCCCAAGCTCTGTGAGCGCCTCTGTTGGCGACAGCGTGACAATCACATGCCGCGCCAGCCAGGGCATCAGCAGATGGCTGGCCTGGTATCAGCAAAGACCTGGCAAGGCCCCTAAACTGCTGATCTACGCCGCTGGGAACCTGGAAACCGGCGTGCCTAGCAGGTTCAGCGGCTCCGGTAGCGGCACCGACTTTACACTTACCATCAGCGACCTGCAAGCAGAGGACTTCGCCACATACTATTGTCAGCAGGCCGACTCATTCCCCCTGACCTTCGGCGGCGGCACAAAGGTGGACATTAAGGGCAGAGCCGACGCCGCGGCCGCCGGCGGAGGAGGCTCGGGCGGCGGAGGTAGCGGCGGCGGAGGTTCTGGAGGCGGCGGCTCACAGGTGCAGCTGGTCGAGAGCGGCGGCGGCCTGGTGCAGCCTGGCGGCAGCCTCAGACTGAGCTGTGCCGCCAGCGGATTCACCTTTAGTAGCTACGCCATGAGCTGGGTTAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGTCCGCCATCAGCGGCTCCGGAGGCAGCACCTACTACGCCGACAGCGTCAAGGGCAGATTTACCATCAGCAGAGATAATAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTCCGGGCCGAAGATACCGCTGTGTACTACTGTGCCAAGGACCACCTGATCACCATGGTGCAGCCCGAGTACTTCCACCACTGGGGCCAAGGAACCCTGGTGACAGTGTCTTCCGCCAGCAGAGCCGATGCCGCCGCCGCCGGCGGACCCGGCGACATTCAGATGACACAGAGCCCTTCTTCTCTGTCTGCTTCTGTCGGCGATCGGGTGACCATCACCTGCAGAGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAAAAGCCCGGCAAGGCCCCAAAGCTGCTGATCTACGCGGCCTCTAGCCTGCAGAGCGGAGTGCCCAGCCGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCCTGCAACCTGAGGACTTCGCCACATACTACTGCCAGCAGAGTTATAGCACACCTCTGACCTTCGGCGGTGGAACAAAGGTGGACATCAAGGGTACCAAGGGCCCTAGCGTGACCAGCGGCCAGGCCGGC；

or SEQ ID NO. 23:

8. an expression vector or recombinant cell comprising the nucleic acid of claim 7.

9. The use of the bispecific humanized antibody against a novel crown mutation strain according to any one of claims 1 to 6 for the preparation of a medicament for the treatment of a disease caused by a novel coronavirus selected from the group consisting of SARS-CoV-2, SARS strain; more preferably, said SARS-CoV-2 is selected from the group consisting of a prototype strain, an alpha variant, a beta variant, a delta variant, a gamma variant, an Onckronk variant (more specifically subtypes BA.1, BA.2, BA.3 and BA.1.1).

10. Pharmaceutical composition for the treatment of diseases caused by a new coronavirus containing the bispecific humanized antibody against a new crown mutation strain as potent as described in any one of claims 1 to 6, preferably said new coronavirus is selected from the group consisting of SARS-CoV-2, SARS strain; more preferably, said SARS-CoV-2 is selected from the group consisting of a prototype strain, an alpha variant, a beta variant, a delta variant, a gamma variant, an Onckronk variant (more specifically subtypes BA.1, BA.2, BA.3 and BA.1.1).