CN113563475B - Bispecific antibody for resisting novel coronavirus and application thereof - Google Patents

Abstract

The present invention relates to a bispecific antibody against novel coronavirus and its application. The bispecific antibody of the present invention is obtained by transforming the new coronavirus monoclonal antibodies H4 and B38 by genetic engineering, and can recognize different sites of the new coronavirus S protein RBD at the same time, and the neutralizing activity of the new coronavirus pseudovirus is much higher than that of the parent The inhibitory activity of the original monoclonal antibody against the live virus of the new coronavirus is also higher than that of the maternal monoclonal antibody. The bispecific antibody of the present invention improves the selectivity and neutralizing activity of the maternal monoclonal antibody, improves the safety and effectiveness of the monoclonal antibody drug, and can be used as a potential drug for the preparation of diagnosis, prevention and treatment of diseases caused by novel coronavirus, The market value is huge and the application prospect is good.

Description

A kind of bispecific antibody against novel coronavirus and its application

technical field

The invention relates to the technical fields of biotechnology and immunology, in particular to a bispecific antibody against novel coronavirus and a preparation method and application thereof.

Background technique

The spike protein (Spike, S protein) on the surface of the new coronavirus (2019-nCoV) binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2) molecule during the infection of the host, thereby initiating the fusion of the viral membrane and the host cell membrane , causing the host cell to infect the virus. The S protein is divided into two parts, S1 and S2. It has been confirmed that the receptor binding domain (RBD) of the C-terminal (CTD) of S1 binds to ACE2 and mediates the membrane fusion process.

Neutralizing antibodies have so far been shown to be an effective treatment for viral diseases. Drugs currently on the market for the treatment and prevention of viral infections include palivizumab (Synagis) for the prevention of pediatric respiratory syncytial virus (RSV) infection, ibalizumab (Trogarzo) for HIV infection, and rabies Rabishield for post-exposure prophylaxis. In addition, there are multiple mAbs against different viruses in various stages of clinical research (https://clinicaltrials.gov/). Antibodies work mainly in two ways. On the one hand, antibodies with neutralizing activity can block viral infection by binding to viral envelope proteins and blocking the binding of viruses to cellular receptors. On the other hand, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) can recruit immune cells and immune molecules such as macrophages or complement to clear free viruses and be infected. cells.

Bispecific antibody (BsAb) is one of the key directions in the field of antibody drug research in recent years. Bispecific antibodies are artificial antibodies that contain two types of antibodies that can specifically recognize and bind to different antigens or different antigenic sites. If the two antigens are located on different cell surfaces, the bispecific antibody can build a bridge between the two antigen molecules, thereby forming cross-links between cells and mediating cell-directed effector functions. BsAbs have broad application prospects in biomedicine, especially in tumor immunotherapy. Bispecific antibodies for immunotherapy (immunotherapy) are artificial antibodies containing 2 specific cell receptor antigen binding sites, which can build a bridge between diseased cells (target cells) and functional cells (immune cells). , stimulate a directed immune response. The killing of tumor cells by BsAb-mediated immune cells (such as T cells, NK cells, etc.) is currently a hot spot in the application of immunotherapy. At the same time, it directly leads to the specific killing of tumor cells by immune effector cells. At present, the approved bispecific antibodies include EpCAM/CD3 and CD19/CD3, and more than 100 bispecific antibodies are still in the clinical research stage. For infectious pathogenic microorganisms, especially RNA viruses that are prone to mutation, an antibody targeting a single site is used for infection treatment. The virus is prone to mutate under the selective pressure of the antibody, and then escape the binding of the antibody, resulting in insensitivity to antibody therapy. of antibodies. Simultaneous targeting of two different sites of viral antigens can greatly reduce the probability of viral escape mutations. Not only that, but targeting different sites at the same time can significantly improve the virus neutralization effect, reduce the dosage of antibodies, and improve the therapeutic effect. For example, in the clinical treatment of Ebola virus infection, the use of three different antibodies that bind to Ebola virus surface glycoprotein (GP) can significantly reduce patient mortality and have a significant therapeutic effect on Ebola virus infection .

Bispecific antibodies can be obtained in various ways, and their preparation methods mainly include: chemical coupling method, hybrid-hybridoma method and genetic engineering antibody preparation method. The chemical coupling method is to connect two different monoclonal antibodies together by chemical coupling to prepare bispecific monoclonal antibodies, which are the earliest bispecific monoclonal antibodies. The hybrid-hybridoma method is the production of bispecific monoclonal antibodies by cell hybridization or ternary hybridomas, which are fused by established hybridomas, or established hybridomas and derived from mice. It can only be used to produce bispecific antibodies of murine origin, so its application is greatly limited. With the rapid development of molecular biology technology, various construction modes of genetically engineered humanized or fully human bispecific antibodies have emerged, mainly including bispecific minibodies, diabodies, and single-chain diabodies. , Four types of multivalent bispecific antibodies. At present, several genetically engineered bispecific antibody drugs have entered the clinical trial stage in the world, and have shown good application prospects.

Compared with monoclonal antibodies, the advantages of bispecific antibodies are: bispecific antibodies can recognize two molecules at the same time, which improves the selectivity and functionality of antibodies, and improves the safety and efficacy of drugs. Bispecific antibody drugs reduce development and clinical trial costs compared to treatment with a combination of two monoclonal antibody drugs. For infectious pathogens, acting on two different sites of the pathogen at the same time makes it less likely to develop antibody resistance during treatment, and can inhibit the generation of viral escape mutations in broad-based treatments. Therefore, the development of bispecific antibodies targeting different sites of the new coronavirus is of great significance for improving the therapeutic effect of new coronary pneumonia and reducing the generation of escape mutations.

SUMMARY OF THE INVENTION

In order to solve the technical problems existing in the prior art, the purpose of the present invention is to provide a bispecific antibody with specific targeting effect, good affinity for the new coronavirus and significant virus inhibitory activity and its application.

In order to achieve the above object, in the first aspect, the present invention provides a bispecific antibody, and its technical solution is as follows: the present invention creatively discovers that, by screening and analyzing the anti-new coronary pneumonia monoclonal antibodies published in the prior art, After the genetic modification of the monoclonal antibody B38 and the monoclonal antibody H4 according to the present invention, the specific binding ability of the original parent monoclonal antibody can be better retained, and the biological functions of the two monoclonal antibodies are at the same time. Compared with maternal monoclonal antibody, it has obvious advantages in terms of activity, safety, stability and so on.

In the present invention, the heavy chain variable region CDR1 of the monoclonal antibody B38 contains the amino acid sequence shown in SEQ ID NO.9, CDR2 contains the amino acid sequence shown in SEQ ID NO.10, and CDR3 contains the amino acid sequence shown in SEQ ID NO.11 The amino acid sequence of the monoclonal antibody B38; the light chain variable region CDR1 of the monoclonal antibody B38 contains the amino acid sequence shown in SEQ ID NO.12, CDR2 contains the amino acid sequence shown in SEQ ID NO.13, and CDR3 contains the amino acid sequence shown in SEQ ID NO.14. amino acid sequence.

The heavy chain variable region CDR1 of the monoclonal antibody H4 contains the amino acid sequence shown in SEQ ID NO.15, CDR2 contains the amino acid sequence shown in SEQ ID NO.16, and CDR3 contains the amino acid sequence shown in SEQ ID NO.17; The light chain variable region CDR1 of the monoclonal antibody H4 contains the amino acid sequence shown in SEQ ID NO.18, CDR2 contains the amino acid sequence shown in SEQ ID NO.19, and CDR3 contains the amino acid sequence shown in SEQ ID NO.20.

Different from the traditional design of monoclonal antibodies, polyclonal antibodies, single-chain antibodies, etc., the factors to be considered and the technical difficulties faced in the design of bispecific antibodies are mainly: bispecific antibodies are formed between antibodies with different structures. Antibodies with different structures bind to different antigens of two cells or different sites of the same antigen, and exert different functions. In order to make each antibody in the bispecific antibody fully exert its respective function, in the design of the antibody, the adverse effect of the mutual influence of each antibody in sequence and structure should be minimized. And when bispecific antibodies target different antigens or different sites, in order to maximize the neutralization of virus titers, the binding affinities of bispecific antibodies to different antigens or different sites of the same antigen need to be balanced and matched. It is not simply the pursuit of high affinity for both targets.

The present invention finds that in the process of selecting monoclonal antibody B38 and monoclonal antibody H4 for genetic modification to construct bispecific antibodies, the affinity, neutralization activity and stability effect of bispecific antibodies obtained by different modification methods are very different. After repeated screening and exploration, the present invention provides two bispecific antibodies with excellent effects obtained by genetic engineering methods, which are as follows:

(1) bispecific antibody BS-mAb-1, whose light chain variable region sequence is that the N-terminus of the light chain variable region of B38 monoclonal antibody and the C-terminus of the light chain variable region of H4 monoclonal antibody are connected by a linking peptide; its The sequence of the heavy chain variable region is that the N-terminus of the heavy chain variable region of the B38 monoclonal antibody and the C-terminus of the light chain variable region of the H4 monoclonal antibody are connected by a linking peptide; or

(2) bispecific antibody BS-mAb-2, including B38 monoclonal antibody unit and H4 single-chain antibody unit,

The H4 single-chain antibody unit includes two single-chain antibodies, and the single-chain antibody is a fusion of the C-terminus of the heavy chain variable region of the H4 monoclonal antibody and the N-terminus of the light chain variable region of the H4 monoclonal antibody through a connecting peptide. Peptides; the C-termini of the two single-chain antibodies are respectively connected to the N-termini of the two heavy chains of the B38 monoclonal antibody through a connecting peptide, or the N-termini of the two single-chain antibodies are respectively connected to the N-terminus of the two single-chain antibodies through a connecting peptide. The two heavy chains of the monoclonal antibody are C-terminally linked.

Further, the amino acid sequence of the linking peptide is (GGGGS)n, wherein n is a natural number of 1-4. Preferably, in the above (1), n of the connecting peptide is 2; in the above (2), n of the connecting peptide in the fusion peptide is 4, and n of the connecting peptide of the single chain antibody and the monoclonal antibody heavy chain is 1.

In one embodiment, the bispecific antibody of the present invention comprises the heavy chain variable region of monoclonal antibody B38, the light chain variable region of monoclonal antibody B38, the heavy chain variable region of monoclonal antibody H4, the light chain variable region of monoclonal antibody H4 chain variable region, wherein,

The heavy chain variable region of monoclonal antibody B38 comprises: CDR1 whose amino acid sequence is shown in SEQ ID NO.9, CDR2 whose amino acid sequence is shown in SEQ ID NO.10, and CDR3 whose amino acid sequence is shown in SEQ ID NO.11 The light chain variable region of monoclonal antibody B38 comprises: CDR1 whose amino acid sequence is shown in SEQ ID NO.12, CDR2 whose amino acid sequence is shown in SEQ ID NO.13, and CDR2 whose amino acid sequence is shown in SEQ ID NO.14 CDR3;

The heavy chain variable region of monoclonal antibody H4 comprises: CDR1 whose amino acid sequence is shown in SEQ ID NO.15, CDR2 whose amino acid sequence is shown in SEQ ID NO.16, and CDR3 whose amino acid sequence is shown in SEQ ID NO.17 The light chain variable region of monoclonal antibody H4 comprises: CDR1 whose amino acid sequence is shown in SEQ ID NO.18, CDR2 whose amino acid sequence is shown in SEQ ID NO.19, and CDR2 whose amino acid sequence is shown in SEQ ID NO.20 CDR3.

Preferably, the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody B38 is shown in SEQ ID NO. 32, and the amino acid sequence of the variable region of the light chain of the monoclonal antibody B38 is shown in SEQ ID NO. 33; The amino acid sequence of the heavy chain variable region of anti-H4 is shown in SEQ ID NO.34, and the amino acid sequence of the light chain variable region of monoclonal antibody H4 is shown in SEQ ID NO.35.

Further preferably, the constant region of mAb B38 and/or the constant region of mAb H4 is also included; the constant region can be any suitable constant region known in the art.

In a preferred embodiment, the bispecific antibody is BS-mAb-1, which has the following structure (from N-terminus to C-terminus):

Heavy chain: VH _H4 -VH _B38 -CH _B38 ,

Light chain: VL _H4 -VL _B38 -CL _B38 ,

Wherein, VH _H4 and VH _B38 are connected by a connecting peptide, and VL _H4 and VL _B38 are connected by a connecting peptide;

In another preferred embodiment, the bispecific antibody is BS-mAb-2, which has the following structure (from N-terminal to C-terminal):

Heavy chain: VH _B38 -CH _B38 -VH _H4 -VL _H4 ,

Light chain: VL _B38 -CL _B38 ;

Among them, VH _H4 and VL _H4 are connected by a connecting peptide to form a single-chain antibody; CH _B38 and VH _H4 are connected by a connecting peptide.

Preferably, the bispecific antibody comprises two identical heavy chains and two identical light chains.

Wherein, the amino acid sequence of the linking peptide is (GGGGS)n, wherein n is a natural number of 1-4. Preferably, in the antibody BS-mAb-1, the n of the connecting peptide is 2; in the antibody BS-mAb-2, the n of the connecting peptide between the VH _H4 and VL _H4 is 4, and CH _B38 and VH _H4 The n of the linking peptide between is 1.

In the description above and elsewhere in this application, VH _B38 represents the heavy chain variable region of monoclonal antibody B38, which is synonymous with (VH-B38); CH _B38 represents the heavy chain constant region of monoclonal antibody B38, including CH1 to CH3; VH _H4 represents the heavy chain variable region of mAb H4, synonymous with (VH-H4); VL _H4 represents the light chain variable region of mAb H4, synonymous with (VL-H4); VL _B38 represents mAb B38 The light chain variable region of , is synonymous with (VL-B38); CL _B38 represents the light chain constant region of monoclonal antibody B38, abbreviated as CL. In some embodiments, the heavy chain constant region of mAb B38 has the same sequence as the heavy chain constant region of mAb H4; the light chain constant region of mAb B38 has the same sequence as the light chain constant region of mAb H4.

Preferably, the bispecific antibody BS-mAb-1, the amino acid sequence of its light chain is as shown in SEQ ID NO. 4 or a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids Amino acid sequence, the amino acid sequence of its heavy chain is shown in SEQ ID NO. 2 or the amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids.

The present invention expresses single-chain antibodies in the form of fusion peptides. Through specific antibody structure and sequence design, it is found that when the connection modes of single-chain antibodies and monoclonal antibodies are different, using specific single-chain antibody fusion peptide sequences can be better. Improve the stability of the antibody structure and the binding to the target.

Preferably, in the bispecific antibody BS-mAb-2, the amino acid sequence of the single-chain antibody is shown in SEQ ID NO. 21; further preferably, the bispecific antibody BS-mAb-2, which is light The amino acid sequence of the chain is shown in SEQ ID NO.8 or the amino acid sequence of a protein with the same function obtained by one or more amino acid substitution, deletion or insertion, and the amino acid sequence of its heavy chain is shown in SEQ ID NO.6 or The amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids.

The above-mentioned "amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids" refers to an organism that is different from the sequence shown at one or more amino acid residues but retains the resulting molecule. Chemically active sequences, which can be obtained by "conservatively modified variants" or by "conservative amino acid substitutions", "conservatively modified variants" or "conservative amino acid substitutions" refer to those known to those skilled in the art amino acid substitutions that generally do not alter the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Exemplary substitutions are preferably made in accordance with the substitutions shown below:

Table 1 Exemplary conservative amino acid substitution tables

original residue conservative substitution original residue conservative substitution original residue conservative substitution Ala(A) Gly, Ser Pro(P) Ala Gly(G) Ala Arg(R) Lys, His Ser(S) Thr His(H) Asn Gln Asn(N) Gln,His Thr(T) Ser Ile(I) Leu, Val Asp(D) Glu,Asn Trp(W) Tyr, Phe Lys(K) Arg,His Cys(C) Ser, Ala Tyr(Y) Trp,Phe Met(M) Leu,Ile,Tyr Gln(Q) Asn Val(V) Ile,Leu Phe(F) Tyr, Met, Leu Glu(E) Asp,Gln

In the present invention, the bispecific antibody can be a murine antibody, a humanized antibody, a chimeric antibody or a recombinant antibody.

The human or humanized antibody includes one of IgG1 antibody, IgG2 antibody, IgG3 antibody, and IgG4 antibody.

In the second aspect, based on the amino acid sequence of the bispecific antibody, the present invention also provides a gene encoding the bispecific antibody.

According to the codon coding rules and the degeneracy and preference of codons, those skilled in the art can design coding genes according to the amino acid sequences of the above-mentioned bispecific antibodies.

As a preferred embodiment of the present invention, the nucleotide sequence of the light chain of the bispecific antibody is shown in SEQ ID NO.3, and the nucleotide sequence of the heavy chain is shown in SEQ ID NO.1; or

The nucleotide sequence of the light chain of the bispecific antibody is shown in SEQ ID NO.7, and the nucleotide sequence of the heavy chain is shown in SEQ ID NO.5.

Further, the present invention also provides a biological material comprising the gene.

In the present invention, the biological material includes recombinant DNA, expression cassettes, vectors, host cells, engineered bacteria or cell lines.

In a third aspect, the present invention also provides a method for preparing the bispecific antibody, comprising: constructing a recombinant recombinant containing the heavy chain and light chain genes of the bispecific antibody BS-mAb-1 or BS-mAb-2, respectively an expression vector; the recombinant expression vector is introduced into a host cell to obtain a host cell that stably expresses the bispecific antibody; the host cell is cultured, and the bispecific antibody is obtained by separation and purification.

When preparing the bispecific antibody, those skilled in the art can select conventional host cells, expression vectors, methods for introducing expression vectors into host cells, and methods for isolation and purification of antibodies as required.

In a fourth aspect, based on the above bispecific antibody, the present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention.

Preferably, the pharmaceutical composition further includes other active ingredients or excipients permitted in the pharmaceutical field.

In a fifth aspect, the present invention also provides a diagnostic reagent or kit comprising the bispecific antibody of the present invention. Preferably, BS-mAb-1 and/or BS-mAb-2 are included.

In a sixth aspect, the present invention provides any one of the following applications of the bispecific antibody or the encoding gene of the bispecific antibody or the biological material containing the encoded gene or the pharmaceutical composition or the kit :

(1) Application in the preparation of medicines for the prevention or treatment of diseases caused by novel coronavirus infection;

(2) Application in the preparation of novel coronavirus diagnostic reagents or diagnostic kits;

(3) Application in the preparation of new coronavirus vaccine;

(4) Application in the prevention or treatment of diseases caused by SARS-CoV-2 coronavirus;

(5) Application in the detection of novel coronavirus.

Further, the present invention provides a pharmaceutical composition in a single-dose form, wherein the single-dose form contains 180mg-6000mg of the bispecific antibody of the present invention; preferably, wherein the single-dose form contains 180mg-3000mg of the bispecific antibody of the present invention more preferably, wherein the single-dose form contains 500mg-1800mg of the bispecific antibody of the present invention; further preferably, wherein the single-dose form contains 900mg-1800mg of the bispecific antibody of the present invention Bispecific antibody; further preferably, wherein the single dosage form contains 500mg-1000mg of the bispecific antibody of the present invention.

The pharmaceutical composition of the present invention in a single dose form, wherein the pharmaceutical composition is formulated for intravenous administration.

Further, the present invention provides a method for preventing or treating diseases caused by novel coronavirus infection, comprising administering 180mg-6000mg of the bispecific antibody of the present invention to a person in need; preferably, including administering 180mg-6000mg to a person in need 3000mg of the bispecific antibody of the present invention; more preferably, including administering 500mg-1800mg of the bispecific antibody of the present invention to a person in need; further preferably, including administering 900mg-1800mg of the bispecific antibody of the present invention to a person in need ; More preferably, including administering 500mg-1000mg of the bispecific antibody of the present invention to a person in need.

The present invention provides a method for preventing or treating diseases caused by novel coronavirus infection, comprising administering the bispecific antibody of the present invention to a recipient in need, 1-2 times a week, and 2-4 times continuously Week; preferably, dosing twice a week for 4 consecutive weeks.

Further, the present invention provides a method for diagnosing novel coronavirus infection, comprising using the bispecific antibody, pharmaceutical composition or preparation of the present invention to contact a sample to be tested.

In the present invention, the novel coronavirus is SARS-CoV-2 coronavirus.

The beneficial effects of the present invention are as follows: the present invention utilizes genetic engineering and antibody engineering methods to construct an anti-new coronavirus bispecific antibody comprising a single-chain antibody and a complete monoclonal antibody structure, and the bispecific antibody fusion protein retains the complete monoclonal antibody In addition, it has a highly stable symmetrical structure, and other structural protein isomers will not be produced during host expression, which greatly reduces the difficulty of extraction and purification processes, and has the advantages of simple preparation and high yield. The bispecific antibody of the present invention better retains the biological functions of the parental monoclonal antibodies B38 and H4, realizes that one bispecific antibody molecule has the biological functions of two monoclonal antibodies at the same time, and simultaneously recognizes the novel coronavirus S Different sites of protein RBD have much higher neutralization activity against 2019-nCoV pseudovirus than maternal monoclonal antibody, and higher inhibitory activity against 2019-nCoV live virus than maternal monoclonal antibody, with low toxicity and good stability. The bispecific antibody of the present invention improves the selectivity and neutralizing activity of the maternal monoclonal antibody, improves the safety and effectiveness of the monoclonal antibody drug, and can be used as a potential candidate drug for the preparation of diagnosis, prevention and treatment of diseases caused by novel coronavirus , the market value is huge, and the application prospect is good.

Description of drawings

FIG. 1 is a schematic structural diagram of the bispecific antibodies BS-mAb-1 (A) and BS-mAb-2 (B) of the present invention.

Figure 2 is a graph showing the results of molecular sieve chromatography and SDS-PAGE detection of bispecific antibodies BS-mAb-1 (A) and BS-mAb-2 (B). Among them, "-" on the gel graph indicates that DTT is not added (non-reducing SDS-PAGE); "+" indicates that DTT is added (reducing SDS-PAGE).

Figure 3 is a schematic diagram of the detection of the dual-site binding properties of BS-mAb-1 (A) and BS-mAb-2 (B) binding to RBD. Among them, in Figure A, H4-BS-mAb-1 indicates that excess H4 antibody is used to saturate the RBD-immobilized probe first, and then the BS-mAb-1 antibody is bound. Compared with the H4-H4 binding curve, in There was a significant increase in the curve after the addition of the BS-mAb-1 antibody, indicating its ability to bind H4-saturated RBD. H4-H4 indicates that the H4 antibody flows through the first stage first, and then flows through the H4 antibody again in the second stage; H4-BS-mAb-1 indicates that the first stage flows through the H4 antibody first, and then flows after the second stage. BS-mAb-1 antibody; B38-BS-mAb-1 is indicated as the first stage after B38 antibody flow, and BS-mAb-1 antibody flow after second stage. In Figure B, the interpretation of legends refers to Figure A.

Figure 4 is a graph showing the results of kinetic curves of H4, B38, BS-mAb-1 and BS-mAb-2 antibodies binding to RBD protein.

Figure 5 is a graph showing the results of neutralizing activity of H4, B38, BS-mAb-1 and BS-mAb-2 antibodies against 2019-nCoV pseudovirus at different concentrations.

Figure 6 is a graph showing the results of neutralizing activity of BS-mAb-1 and BS-mAb-2 antibodies against 2019-nCoV live virus at different concentrations.

FIG. 7 is a graph showing the results of SDS-PAGE detection of double-antibody 1#, 6# and 12# antibodies.

Figure 8 is a graph showing the results of neutralizing activity of 1#, 6# and 12# antibodies against 2019-nCoV live virus at different concentrations.

FIG. 9 is a schematic diagram of the detection results of viral load in throat swabs of rhesus monkeys in Example 10. FIG.

FIG. 10 is a schematic diagram of the viral load detection results of nasal swabs of rhesus monkeys in Example 10. FIG.

FIG. 11 is a schematic diagram of the viral load detection results of the rhesus monkey anal swab in Example 10. FIG.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and spirit of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Bispecific Antibody Construction

In the construction of bispecific antibodies, the arrangement of the variable regions of the two antibodies will affect their ability to bind to the antigen, and different constructions may lead to differences in antiviral activity. Therefore, whether the bispecific antibody constructed based on antibodies at two different sites can retain its dual-site binding activity and whether its antiviral activity can be improved remains uncertain. Bispecific antibodies are fusion proteins formed between antibodies with different structures. Antibodies with different structures bind to different antigens of two cells or different sites of the same antigen to exert different functions. In order to make each antibody in the bispecific antibody fully exert its respective function, a large number of alternative bispecific antibodies were designed in the antibody design in this example, in order to minimize the mutual influence of each antibody in sequence and structure on their respective functions. adverse effects caused.

1. BS-mAb-1 construction

In the construction of BS-mAb-1, the V regions of B38 and H4 were connected in tandem with the GS flexible hinge region (Fig. 1A). Since the relative distance between the antigen-binding sites is the shortest, it is speculated that the binding of adjacent epitopes may have Certain advantages, its construction features are:

The light chain sequence is: (VL-H4)-(GGGGS) _2- (VL-B38)-CL;

The heavy chain sequence is: (VH-H4)-(GGGGS) _2- (VH-B38)-CH1-CH2-CH3.

The light chain and heavy chain DNA sequences (shown as SEQ ID NO: 3 and SEQ ID NO: 1, respectively) were cloned into the expression vector pCAGGS (purchased from Addgene), respectively, to obtain recombination encoding antibody heavy and light chains, respectively Expression vector. The construction scheme for constructs expressing heavy and light chains is as follows:

Heavy chain coding sequence (5'-3'): CMV promoter-EcoR I restriction site-leader sequence gene (sequence is SEQID NO.22) (VH-H4)-(GGGGS) _2- (VH-B38) -CH1-CH2-CH3 gene-Xho I restriction site;

Light chain (κ) coding sequence (5'-3'): CMV promoter-Sac I restriction site-leader sequence gene (SEQ ID NO. 23)-(VL-H4)-(GGGGS) _2- ( VL-B38)-CL gene-Xho I restriction site;

2. BS-mAb-2 construction

In the construction of BS-mAb-2, the VL and VH regions of the H4 antibody were connected with the GS flexible hinge region to construct a single-chain antibody (scFv), and then the H4-scFv was constructed to the C-terminus of the heavy chain of the B38 antibody to form B38 The bispecific antibody with H4-scFv was constructed (Fig. 1B), and its construction structure is:

The light chain sequence is: (VL-B38)-CL

The heavy chain sequence is: (VH-B38)-CH1-CH2-CH3-GGGS-(VH-H4)-(GGGGS) _4- (VL-H4)

The light chain and heavy chain DNA sequences (shown in SEQ ID NO: 7 and SEQ ID NO: 5, respectively) were cloned into the expression vector pCAGGS (purchased from Addgene) to obtain recombination encoding antibody heavy and light chains, respectively Expression vector. The construction scheme for constructs expressing heavy and light chains is as follows:

Heavy chain coding sequence (5'-3'): CMV promoter-EcoR I restriction site-leader sequence gene (sequence is SEQID NO.24)-(VH-B38)-CH1-CH2-CH3-GGGS-( VH-H4)-(GGGGS) _4- (VL-H4) gene-Xho I restriction site;

Light chain (κ) coding sequence (5'-3'): CMV promoter-Sac I restriction site-leader sequence gene (sequence is SEQID NO.25)-(VL-B38)-CL gene-Xho I enzyme cut point.

Example 2 Recombinant expression construction and protein expression purification of dual-characteristic antibodies

In this example, two different bispecific antibodies, BS-mAb-1 and BS-mAb-2, were constructed on the basis of two novel coronavirus S antigen-specific antibodies, H4 and B38, whose heavy chain amino acid sequences As shown in SEQ ID NO.2 and SEQ ID NO.6, its light chain amino acid sequence is shown in SEQ ID NO.4 and SEQ ID NO.8, respectively.

HEK293T cells (ATCC CRL-3216) were cultured in DMEM with 10% FBS. HEK 293T cells were co-transfected with the above-mentioned BS-mAb-1 and BS-mAb-2 dual-characterized recombinant expression vectors encoding antibody heavy and light chains, respectively. 4-6 hours after transfection, the cell culture medium was changed to serum-free DMEM, and the culture was continued for 3 days. The supernatant was collected, then supplemented with DMEM, and the culture was continued for 4 days, and then the supernatant was collected again.

The collected supernatant was centrifuged at 5000 rpm for 30 min, then mixed with an equal volume of buffer containing 20 mM sodium phosphate (pH 7.0), filtered through a 0.22 μm filter, and loaded into a prepacked column with protein A (5 mL, GE Healthcare) . Protein bound to the prepacked column was eluted with 10 mM glycine (pH 3.0). The eluted fractions were concentrated and purified by molecular sieve chromatography. Subsequently, the purified target protein was detected by SDS-PAGE (reducing and non-reducing). The results are shown in Figure 2. The results in Figure 2 show that purified BS-mAb-1 and BS-mAb-2 antibodies were obtained after purification. The antibodies showed a single band in SDS-PAGE under non-reducing conditions, and the antibodies showed a single band in SDS-PAGE under reducing conditions. The Fc region disulfide bond was opened to show two bands, and the antibody was more than 95% pure.

Example 3 Evaluation of the double-site binding ability of BS-mAb-1 and BS-mAb-2 antibodies to S protein RBD

In this example, the ForteBio Octet RED 96 biofilm surface interference technology was used to analyze the binding of the purified antibody to the S protein RBD, and to evaluate whether the BS-mAb-1 and BS-mAb-2 antibodies have dual-site binding ability .

Histidine-tagged S protein RBD (Science. 2020 Jun 12; 368(6496): 1274-1278) was immobilized on a probe (Fortebio). To evaluate the dual-site binding ability of the BS-mAb-1 antibody, the RBD protein-immobilized probe was first bound to the H4 antibody (Science. 2020 Jun 12; 368(6496): 1274-1278) and saturated. A mixture of BS-mAb-1 and equal concentrations of H4 antibody was then passed through the H4 antibody saturation-bound probe. The results showed that, compared with the binding level of H4-H4 antibody to RBD, the saturated-bound RBD of H4 antibody was still able to bind BS-mAb-1, indicating that the non-competitive BS38 antibody with H4 antibody played a binding role (Fig. 3 of A). Likewise, the RBD protein-immobilized probe was first bound to the B38 antibody and saturated. Then a mixture of BS-mAb-1 and an equal concentration of B38 antibody (Science. 2020 Jun 12; 368(6496): 1274-1278) was passed through the B38 antibody saturation-binding probe. The results showed that, compared with the binding level of B38-B38 antibody to RBD, the saturated-bound RBD of B38 antibody was still able to bind BS-mAb-1, indicating that the H4 antibody contained in it non-competitive with B38 antibody played a binding role (Fig. 3 of A). The double-site binding ability of the BS-mAb-2 antibody was tested by the above-mentioned similar experiments. The results showed that the BS-mAb-2 antibody also had double-site binding ability, indicating that the scFv of the H4 and B38 antibodies contained were both. Antigen-binding ability was exerted (B of FIG. 3 ).

Example 4 Evaluation of the binding ability of BS-mAb-1 and BS-mAb-2 antibodies to RBD

First, an anti-human IgG antibody (purchased from GE Healthcare) was immobilized on a channel (flow cell, Fc) of a CM5 chip in an amino-coupling manner. The fixed amount is controlled at around 8,000 response units (RU). Then, the purified B38, H4, BS-mAb-1 or BS-mAb-2 antibodies were bound, respectively, in the manner of antibody capture. Additionally, RBD protein was serially fold diluted in a 20 mM HEPES, 150 mM NaCl, pH 7.4 solution. Then, serial dilutions of RBD protein (6.25nM–100nM) were passed through each channel in turn (loaded one by one starting from the lowest concentration). The kinetic curves of the binding of each antibody to RBD protein were recorded, and the kinetic constants were calculated using BIAevaluation software 8K (Biacore, Inc.) (FIG. 4).

The results in Figure 4 show that the affinity of H4 antibody and B38 antibody to RBD is 8.25nM and 22.5nM, respectively, while the affinities of bispecific antibody BS-mAb-1 and BS-mAb-2 to RBD are 4.14nM and 21.7nM, respectively . This indicates that the affinity of the bispecific antibody to RBD is maintained at the same level as the B38 and H4 antibodies.

Example 5 Evaluation of the ability of BS-mAb-1 and BS-mAb-2 antibodies to neutralize 2019-nCoV pseudovirus

The hACE2 full-length gene (UNIPRO: Q9BYF1) was constructed into a Pcdna4.0 expression plasmid (purchased from Invitrogen), transferred to Hela cells, and cultured under the selection pressure of 2 μg/ml puromycin (purchased from Gibco), and Single cells were sorted into monoclonal cells, and Hela stably transfected cell line (Hela-hACE2) stably expressing hACE2 gene was prepared for future use.

The purified B38, H4, BS-mAb-1 or BS-mAb-2 antibodies were fold-diluted from 200 μg/mL to the 10th gradient, and then mixed with pseudoviruses expressing the new coronavirus S antigen (wild-type VSV-SARS). -CoV-2 pseudovirus) (obtained from the Chinese Academy of Food and Drug Control) and incubated with mixing at 37°C for 2 hours. After incubation, the virus was added to a 96-well plate pre-seeded with Hela stably transfected cell line (Hela-hACE2) expressing human ACE2, and incubated at 37°C in a 5% _CO2 incubator for 24 hours. The level of activated luciferase substrate response after infection was used to calculate neutralizing titers of B38, H4, BS-mAb-1 or BS-mAb-2 antibodies. The results are shown in Figure 5. Figure 5 shows the neutralizing activity of different concentrations of B38, H4, BS-mAb-1 or BS-mAb-2 antibodies against the 2019-nCoV pseudovirus. The results showed that the neutralizing titer (half neutralizing concentration, NC ₅₀ ) of BS-mAb-1 antibody to 2019-nCoV pseudovirus was 0.119 μg/mL, which was higher than that of H4 (NC ₅₀ =0.718 μg/mL) and B38 ( _NC50 = 0.681 [mu]g/mL) antibody increased 6-7 fold. The neutralizing titer of BS-mAb-2 antibody against 2019-nCoV pseudovirus was 0.048 μg/mL, which was 14-15 times higher than that of H4 and B38 antibodies. Therefore, the bispecific antibodies BS-mAb-1 and BS-mAb-2 were constructed with higher neutralizing activity relative to the H4 and B38 monoclonal antibodies.

Example 6 Evaluation of the ability of BS-mAb-1 and BS-mAb-2 antibodies to neutralize live 2019-nCoV virus

The BS-mAb-1 and BS-mAb-2 antibodies purified in Example 1 were double-diluted from 100 μg/mL to the 10th gradient, and then mixed with half the tissue culture infectious dose (TCID ₅₀ ) of BetaCoV/Shenzhen/SZTH respectively. -003/2020 virus (GISAID number: EPI_ISL_406594) was incubated with mixing at 37°C for 2 hours. After incubation, the virus was added to a 96-well plate pre-seeded with Vero cells and incubated for 4 days in a 37°C, 5% _CO2 incubator to observe the cytopathic effect (CPE) and calculate the BS-mAb-1 and neutralizing titers of BS-mAb-2 antibodies. The results are shown in Figure 6. Figure 6 shows the neutralizing activity of different concentrations of BS-mAb-1 and BS-mAb-2 antibodies against live 2019-nCoV virus. The results showed that the neutralization titer (semi-inhibitory concentration, IC50) of BS-mAb-1 antibody to 2019-nCoV live virus was 1.725 μg/mL, and the neutralization titer of BS-mAb-2 antibody to 2019-nCoV live virus (50% inhibitory concentration, IC50) was 0.656μg/mL, with good neutralizing activity.

Example 7 Construction and verification of other double antibodies

In order to prove that the variable region arrangement of the bispecific antibody or the linking peptide connecting the two monoclonal antibody parts will affect its ability to bind to the antigen, the applicant also constructed other bispecific antibodies based on the monoclonal antibodies B38 and H4. Antibodies are validated. Other bispecific antibodies are: 1#, 6# and 12#, and their structures are:

1#:

Light chain: (VL-B38)-GQPKAAP-(VL-H4)-(TKQPS)-CL, the amino acid sequence is as shown in SEQ ID NO.26;

Heavy chain: (VH-H4)-S-(VH-B38)-RT-CH1-CH2-CH3, the amino acid sequence is as shown in SEQ ID NO.27;

6#:

Light chain: (VL-B38)-(G) _10- (VL-H4)-CL, the amino acid sequence is as shown in SEQ ID NO.28;

Heavy chain: (VH-H4)-(G) _7- (VH-B38)-S-CH1-CH2-CH3, the amino acid sequence is as shown in SEQ ID NO.29;

12#:

The light chain sequence is: (VL-H4)-(G) _10- (VL-B38)-CL, and the amino acid sequence is as shown in SEQ ID NO.30;

The heavy chain sequence is: (VH-B38)-(G) _7- (VH-H4)-S-CH1-CH2-CH3, and the amino acid sequence is shown in SEQ ID NO.31.

(1) Antibody Construction

The three antibodies were prepared according to the construction of BS-mAb-1 in Example 1, and the antibodies were purified and checked for purity as described in Example 2.

The results in FIG. 7 show that purified 1#, 6# and 12# antibodies were obtained, the disulfide bonds of the Fc region of the antibodies were opened in SDS-PAGE under reducing conditions, and showed as two bands, and the antibodies were pure more than 95%.

(2) Assessment of the ability to neutralize 2019-nCoV live virus

The above-purified 1#, 6# and 12# antibodies were diluted from 100 μg/mL to the 10th gradient, and then mixed with BetaCoV/Shenzhen/SZTH-003/2020 virus at half the tissue culture infectious dose (TCID ₅₀ ) respectively. (GISAID number: EPI_ISL_406594) Incubate with mixing at 37°C for 2 hours. After incubation, the virus was added to a 96-well plate pre-seeded with Vero cells, and cultured for 4 days in a 37°C, 5% _CO2 incubator to observe the cytopathic effect (CPE), and calculate 1#, 6# and the neutralizing titer of 12# antibody. The results are shown in Figure 8, showing the neutralizing activity of 1#, 6# and 12# antibodies against 2019-nCoV live virus at different concentrations. The results showed that the neutralizing titer (semi-inhibitory concentration, IC50) of 1# antibody to 2019-nCoV live virus was 5004 μg/mL, and the neutralizing titer (semi-inhibitory concentration, IC50) of 6# antibody to 2019-nCoV live virus The neutralization titer (semi-inhibitory concentration, IC50) of 12# antibody against 2019-nCoV live virus was 54304 μg/mL, which was 102.6 μg/mL.

Example 8: In vitro neutralization activity experiment of bispecific antibodies against new coronavirus wild strain/variant pseudovirus

This experiment verifies the neutralizing activity of the BS-mAb-2 antibody constructed in Example 2 to 40 new coronavirus pseudovirus strains.

1)Material

Huh7 cells (JCRB, Cat#0403), a total of 31 strains of SARS-Cov-2 pseudovirus from the China National Inspection and Quarantine Institute, DMEM high glucose medium, firefly luciferase detection reagent, sterile PBS (pH7.2), fetal bovine serum, 1% (m/v) dual antibodies (penicillin/streptomycin antibiotics), 0.25% (m/v) trypsin-EDTA.

The 31 new coronavirus pseudoviruses constructed by the China Food and Drug Research Institute are shown in Table 2 below.

Table 2: 31 pseudoviruses

2) Dilution method: Dilute the stock solution (30.7g/L) by 30 times; then as shown in Table 3 below, take a 96-well plate and add it to the second column (CC: cell control, only add cells and medium, see Table 3) DMEM complete medium (1% double antibody, 25mM HEPES, 10% FBS) 150μl/well, in the 3rd, 6th, and 9th columns (the 3rd, 6th, and 9th columns are the pseudovirus control in turn VC1, VC2, VC3, and so on for the next 96-well plate number; VC: pseudovirus control, only add pseudovirus, cells and culture medium) add 100 μl/well of DMEM complete medium to C4-G5, C7-G8, 100 μl/well of DMEM complete medium was added to wells C10-G11, and 142.5 μl/well of DMEM complete medium was added to wells B4-B5, B7-B8, and B10-B11.

Sample loading: Add the antibody to be tested to wells B4-B5, B7-B8, B10-B11: 7.5 μl/well (30-fold initial dilution of working solution)...and so on.

3) Sample dilution: adjust the multi-channel pipette to 50 μl, gently blow and suck the liquid in wells B4-B5, B7-B8, B10-B11 for 6 to 8 times to mix thoroughly, and then transfer 50 μl of the liquid to the corresponding hole. C4-C5, C7-C8, C10-C11 holes, gently blow and suck 6 to 8 times and then transfer to D4-D5, D7-D8, D10-D11 holes, and so on, and finally from G4-G5, G7- Aspirate and discard 50 μl of liquid in G8 and G10-G11, and refer to Table 3 for the loading position of the 96-well cell culture plate. Put the sample back when the loading is complete.

4) Dilute each strain of pseudovirus to 1.3 × 10 ⁴ (1 × 10 ⁴ ~ 2 × 10 ⁴ ) TCID ₅₀ /ml with DMEM complete medium (diluted by the dilution factor provided), and add pseudovirus in columns 3 to 5. Virus 1, add pseudovirus 2 to columns 6 to 8, add pseudovirus 3 to columns 9 to 11, and so on, add 50 μl to each well, so that the amount of pseudovirus is 650 (500-1000)/well. After virus addition, the above 96-well plate was placed in a cell culture incubator (37°C, 5% CO ₂ ) and incubated for 1 hour.

Table 3: 96-well plate loading position table

When the incubation time reaches half an hour, take out the pre-prepared cells from the incubator (the confluence rate is 80% to 90%), aspirate the medium in the bottle, add 5ml PBS buffer to wash the cells, pour out the PBS, add 3ml 0.25% trypsin-EDTA, submerge the cells for 1 minute, pour off the trypsin, place in a cell incubator to digest for 5 minutes, gently tap the side wall of the culture flask to dislodge the cells, add 10 ml of medium to neutralize the trypsin, After several pipetting, cells were counted and cells were diluted to ² x 105/ml with DMEM complete medium. Incubate for 1 hour and add 100 μl of cells to each well of a 96-well plate to make 2×10 ⁴ cells per well. Gently shake the 96-well plate back and forth to make the cells evenly dispersed in the wells. Place the 96-well plate in a cell culture incubator at 37°C, 5% CO ₂ for 20 to 28 hours. Take out the 96-well plate from the cell incubator, remove 150 μl of supernatant from each loading well with a multi-channel pipette, add 100 μl of luciferase detection reagent, and react at room temperature for 2 min in the dark. After the reaction, use a multi-channel pipette to repeatedly blow and suck the liquid in the reaction well for 6 to 8 times to fully lyse the cells. Aspirate 150 μl of the liquid from each well, add it to the corresponding 96-well chemiluminescence detection plate, and place it in the corresponding 96-well chemiluminescence detection plate. Read the luminescence value in a multi-function microplate reader.

5) Calculate neutralization inhibition rate: inhibition rate=[1-(mean value of luminescence intensity of sample group-mean value of blank control CC)/(mean value of luminescence intensity of negative group-mean value of blank control value CC)]×100%. According to the results of neutralization inhibition rate, _IC50 value was calculated by Reed-Muench method. Among them, the negative group is normal human serum + pseudovirus + cells + culture medium without anti-new coronavirus antibodies.

Repeated experiment: The detection of each variant strain was repeated twice according to the above method, and the mean IC ₅₀ of the antibody and pseudovirus variant strains measured three times was the final IC ₅₀ value of the antibody against a certain variant strain.

6) Results

The antibody concentration was diluted to 100 μg/mL for detection, and the detection results are shown in Table 4.

Table 4: The neutralization activity detection results of the antibodies of the present invention to different virus strains

It can be seen that the antibody of the present invention can neutralize and inhibit various mutant pseudovirus strains in vitro to varying degrees, indicating that the antibody of the present invention is effective against the new coronavirus and its various mutant strains, and has a wide range of applications. Among them, the B.1.617-1 strain first discovered in India is the delta strain, which is one of the most infectious respiratory viruses known at present, and spreads rapidly around the world. The antibody of the present invention has neutralizing activity against it. Very high.

Example 9: Toxicity test of bispecific antibodies

(1) Repeated dosing experiments

This experiment evaluates the possible toxic reactions, toxic target organs, toxicokinetic properties and immunogenicity after repeated intravenous infusion of BS-mAb-2 antibody to cynomolgus monkeys twice a week for 4 consecutive weeks , and the recovery of toxic reactions or possible delayed toxic reactions 4 weeks after the last dose.

Experimental animals and groups: A total of 40 cynomolgus monkeys (20/sex) were used (Source: Guangxi Xiongsen Primate Experimental Animal Breeding and Development Co., Ltd.; Experimental Animal Production License No.: SCXK (Gui) 2016-0003; Experiment Animal quality certificate number: 0002914, 0002923, 0002931; production license issuing unit: Guangxi Zhuang Autonomous Region Science and Technology Department; age: 2.8-4 years old), randomly divided into 4 groups (5 animals/sex/group), the first Group animals were given sodium chloride injection as a negative control substance (0mg/kg), animals in groups 2, 3, and 4 were given 25, 50, and 150 mg/kg of BS-mAb-2 antibody, respectively, which were low, medium, and low for the test. and high-dose groups. The drug was administered twice a week for 4 consecutive weeks for a total of 9 doses (D1, D5, D8, D12, D15, D19, D22, D26 and D29). The drug was administered by subcutaneous intravenous infusion in the hind limbs of cynomolgus monkeys using an infusion pump, the administration volume was 10 mL/kg, and the administration rate was about 0.5 mL/kg/min. The day of first dosing for males and females was defined as test D1 for animals of that sex, respectively.

Detection indicators: During the experiment, the animals were clinically observed, and the body weight, food intake, body temperature, electrocardiogram (vest telemetry), respiratory function (vest telemetry), electrocardiogram (extremity lead II ECG), blood pressure, ophthalmological examination, Blood counts, coagulation, blood chemistry and urinalysis. Blood concentration and toxicokinetic analysis were performed before and after administration on D1 and D26, and before administration on D15, D19 and D22. To evaluate the immune response, lymphocyte subsets (CD3 ⁺ , CD3 ⁺ CD4 ⁺ , CD3 ⁺ CD8 ⁺ , CD20 ⁺ , CD3 ⁺ CD4 ⁺ /CD3 ⁺ CD8 ⁺ ), cytokines (TNF-α, IFN- gamma, IL-2, IL-4, IL-5, IL-6), immunoglobulins (IgG, IgM, IgA), complement (C3, C4) and BS-mAb-2 antibodies. After the dosing period, the first 3 animals/sex/group in groups 1-4 were euthanized as planned at 4 weeks post-dose (D30), and the remaining 2 animals/sex/group in groups 1-4 recovered at 4 weeks At the end of the period (D57), euthanasia was carried out as planned. Animals in groups 1-4 were systematically dissected, and the organ weight, gross anatomy and histopathology were examined.

result:

1) During the experiment, all animals in groups 1-4 were not found dead or dying. No abnormal changes related to the test article were found in the clinical observation of the animals in the test article 25, 50 and 150 mg/kg dose groups.

2) During the experiment, the body weight and body weight increase, food intake, body temperature, ECG indicators (vest telemetry), respiratory function (vest telemetry), electrocardiogram (limb lead II ECG) of the test article 25, 50 and 150 mg/kg dose groups ), blood pressure, ophthalmological examination, blood cell count, coagulation function, blood biochemistry, urinalysis, lymphocyte subsets, cytokines, immunoglobulins and complements showed no test-related or toxicologically significant changes.

3) After repeated intravenous infusion of BS-mAb-2 antibody to cynomolgus monkeys at doses of 25, 50 and 150 mg/kg, one animal in each of the 50 and 150 mg/kg dose groups developed anti-drug antibodies on the 29th day after administration (ADA), ADA titers were 1:160 and 1:1280, respectively, the total positive rate of samples in the test group was 1.4%, and the total positive rate of individuals was 6.7%. The time, incidence and titer of antibody production showed no significant gender differences and dose correlation; it indicated that the immunogenicity of cynomolgus monkeys to BS-mAb-2 antibody was weak.

4) after repeated intravenous infusion of the test product is given to the cynomolgus monkey, the results of the animal toxicokinetic parameters of each test product group are as shown in Table 5:

Table 5: Animal toxicokinetic parameter results of antibody BS-mAb-2:

Remarks: n is the number of animals; accumulation factor AF=AUC _{last, 8th} /AUC _{last, 1st}

After repeated intravenous infusion of the test product to cynomolgus monkeys, only the _Cmax and AUC _last of the low-dose group D26 showed a difference between males and females (p<0.05, but the difference was not significant, the female/male ratios were 1.24 and 1.37, respectively), and the other There was no statistical difference in the pharmacokinetic parameters of male and female animals in each group (p>0.05), indicating that the parameters of different genders of animals in each group were basically the same. After the 8th (D26) administration, the accumulation factor (AF=AUC _{last, 8th} /AUC _{last, 1st} ) of male and female animals in the test group was between 1.69 and 2.28, and no obvious drug accumulation was found.

5) The test product BS-mAb-2 antibody was administered to cynomolgus monkeys by repeated intravenous infusion within the dose range of 25-150 mg/kg. The time change trend of serum drug concentration among animals of different genders in each group was basically the same, and the peak serum drug concentration and Serum drug exposures were positively correlated with dose.

6) Euthanasia at the end of administration (D30) and at the end of the observation period (D57), the organ weights, gross anatomy and histopathological examinations of the animals in the test substance 25, 50 and 150 mg/kg dose groups were not found to be consistent with the test. product-related pathological changes.

7) No abnormal changes related to the test product were found in the macroscopic observation, gross anatomical examination and histopathological examination of the administration site (ie the injection site) of the animals in the test product 25, 50 and 150 mg/kg dose groups.

Conclusion: In summary, under the conditions of this experiment, BS-mAb-2 antibody was administered to cynomolgus monkeys by repeated intravenous infusion at doses of 25, 50 and 150 mg/kg, twice a week for 4 consecutive weeks (9 administrations in total), and the recovery period was 4 weeks. There were no systemic toxic reactions and toxic target organs in animals in each dose group. The dose level (NOAEL) at which no clinical adverse reactions were observed in this experiment was 150 mg/kg. After the 8th (D26) dose at this dose, the C _max and AUC _last of male animals were 4460 ng/mL and 234 h·mg/mL, respectively, and the C _max and AUC _last of female animals were 4480 ng/mL and 218 h·mg/mL, respectively .

(2) Single-dose experiment

This experiment evaluated the possible toxic reactions, toxic target organs and in vivo metabolic characteristics of cynomolgus monkeys after a single intravenous infusion of BS-mAb-2 antibody, and observed the toxic reactions 14 days after the end of the administration period.

6 cynomolgus monkeys were randomly divided into 3 groups (1/sex/group), the first group was the negative control group (sodium chloride injection, 0 mg/kg), and the second and third groups were given 50 and 300 mg/kg, respectively. kg of BS-mAb-2 antibody. Animals were given a single dose on the first day (D1), which was administered by subcutaneous intravenous infusion in the hind limbs of animals using a syringe pump. During the experiment, the animals were continuously observed for about 4 hours after administration, and the clinical observation, body weight, body temperature, electrocardiogram, blood cell count, coagulation function, blood biochemistry, T lymphocyte subsets, cytokines and toxicokinetic indexes of the animals were carried out regularly. examine.

During the experiment, there was no death or moribund phenomenon in each group of animals. There were no clinical abnormalities related to the test article in the 50mg/kg dose group and 300mg/kg dose group of BS-mAb-2 antibody. There were no abnormal changes related to the test article in the lymphocyte subsets. After the observation period (D15), the general observation of the animals in the 50 and 300 mg/kg dose groups of BS-mAb-2 antibody showed no abnormality, and no histopathological examination was performed. The results of the toxicity test showed that the change trend of serum drug concentration among animals of different genders in each group was basically the same, and the serum drug concentration was positively correlated with the administration dose.

In conclusion, under the conditions of this experiment, BS-mAb-2 antibody was administered to cynomolgus monkeys by a single intravenous infusion at a dose of 50 and 300 mg/kg, and no definite BS-mAb-2 antibody-related toxicity was observed. The observed adverse effect level (NOEL) was 300 mg/kg.

Example 10: Antibody Therapeutic Experiment

In this example, the rhesus monkey model was infected with SARS-CoV-2 virus, and the therapeutic effect of BS-mAb-2 antibody in rhesus monkeys was evaluated.

1) Experimental materials:

(1) SARS-CoV-2 strain: derived from clinical samples of sputum from COVID-19 patients in Kunming City, Yunnan Province (transferred with the coordination and approval of Yunnan Provincial Health Commission). The BSL-3 laboratory (the laboratory has been accredited by the national CNAS and approved by the National Health Commission to engage in SARS-CoV-2 research) was isolated, and after the Vero cells were adapted, the virus species identification, sequencing and preservation (SARS-CoV-2) were completed. -2-KMS1/2020/GenBank accessionnumber:MT226610.1).

(2) Cells: African green monkey kidney cell line (Vero) was purchased from ATCC (Manassas, VA, USA), cultured in MEM containing 100 U/mL penicillin, 100 μg/mL streptomycin, 10% neonatal bovine serum based on 5% CO ₂ , cultured at 37°C.

(3) Experimental animals: Rhesus monkeys, 1.5-1.7 years old, weighing 1.5-2.5kg, 16, male, provided by the Primate Laboratory Animal Center, Institute of Medical Biology, Chinese Academy of Medical Sciences (Experimental Animal Production License No.: SCXK (Dian) K2020-0005). The use of all experimental animals was in accordance with the relevant regulations of Yunnan Province and the Laboratory Animal Management Committee of the Institute, and was approved by the Laboratory Animal Ethics Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences (approval number DWSP 202104010). All animal experiments were carried out under the conditions of a biosafety protection level 3 laboratory (ABSL-3) certified by China National Accreditation Service for Conformity Assessment (CNAS), and were engaged in experiments on SARS-CoV-2 virus The work has been approved by the National Health Commission.

2) Virus titer detection

Half cell culture infection dose method (50% Cell culture infections does, CCID50): Vero cells were added to a 96-well cell culture plate at a concentration of 1×10 ⁴ to 5×10 ⁴ cells/well one day before the assay, and the culture medium was added to the 96-well cell culture plate. It is a complete MEM culture medium containing 10% newborn bovine serum, with a total volume of 100 μL, the original culture medium is poured out the next day, and the cells are washed once with 200 μL PBS to remove the remaining serum and dead cells. Before the experiment, 100 μL of MEM complete medium containing 3% newborn calf serum was added to each well, and the cells were cultured at 37°C for later use. At the same time, the virus solution to be tested was taken out from -80°C, thawed at room temperature or 37°C and then vortexed and mixed, and the virus solution was diluted 10 times with serum-free MEM medium ( ^10-1 , ^10-2 , ^10-3 , ..., 10 ^-8 ). When diluting the virus, the pipette tip that absorbs the virus solution is suspended in the air, and the virus solution is poured into the diluent (do not put the pipette tip into the diluent). After fully vortexing and mixing, continue to the next step of dilution, and replace the pipette at each dilution step. gun head once. Afterwards, the diluted virus solution was added to Vero cells in 96-well plate in turn, with 8 parallel wells for each dilution, and a positive control well of virus stock solution and a negative control well of MEM culture fluid were set. Afterwards, the cells were cultured in a 37°C, 5% CO ₂ environment, and the cytopathic effect (CPE) was observed on day 6-7. The results were calculated by Karber's method: 1gCCID50=(1g lowest dilution ratio)-(dilution group distance)×(sum of positive lesion hole ratio-0.5) CCID50=10lgCCID50/0.1mL.

3) SARS-CoV-2 fluorescent PCR identification

Primers, probes and reagents: All primers were synthesized by BGI, dissolved in double distilled water to a concentration of 10 μmol/μl working solution, and stored at -20°C for later use.

Table 6: Sequences of primers for SARS-CoV-2 fluorescent PCR identification

4) Experimental design

The monkeys used in the experiment were tested negative for SARS-CoV-2 antibody after routine quarantine and were selected for this experiment. A control group, a low-dose group, a high-dose group and a preventive administration group were set, with 4 animals in each group.

Experimental program:

(1) Challenge: The challenge dose of each experimental group was 5.0logCCID50/injection, 100 μL of intranasal infection;

(2) Grouping:

Prevention group: antibody was injected 12 hours before challenge, 50 mg/kg body weight, intravenous injection;

Low-dose group: 12 hours after challenge, the first injection of antibody, 25 mg/kg body weight, intravenous injection; 72 hours after the first injection of antibody, the second injection of antibody, the dose is the same as the first.

High-dose group: 12 hours after challenge, the first injection of antibody, 50 mg/kg body weight, intravenous injection; 72 hours after the first injection of antibody, the second injection of antibody, the dose is the same as the first.

Blank control group: no treatment.

(3) Observe the clinical manifestations of animals, measure body temperature, body weight, collect nasal swabs, throat swabs, and anal swabs every day for virus qPCR detection, and on the 1st, 3rd, 5th, and 7th days, conduct nasal swabs, throat swabs, and anal swab samples for virus qPCR , subgenomic RNA and CCID50 detection; the experimental animals were sacrificed after anesthesia on the 7th day, and the main tissues were collected for pathological and viral load detection and analysis.

Test content:

(1) Detoxification and viral load analysis in tissues after infection

Pharyngeal swabs, nasal swabs, anal swabs, and tissues were used to extract RNA from samples, and then q-PCR was performed, and the viral load of the samples was calculated using the standard curve and Ct value.

(2) Histopathological examination

Animal tissue samples were fixed with 4% formaldehyde solution and embedded in paraffin.

(3) Statistical analysis

Data statistics were performed using Graphad Prism 8.0 software, and the results were expressed as mean, or mean±SD (neutralizing antibodies were expressed as GMT or GMT±SD).

5) Experimental results:

5.1) Clinical manifestations:

(1) All experimental monkeys had no obvious changes in spirit and diet from 1 to 7 days; no animal death occurred; 1 experimental monkey in the control group had mild diarrhea from the second day, and there was no obvious change in diet.

(2) Body temperature: Except that the body temperature of the experimental monkeys in the control group and the prevention group showed a relatively obvious decrease trend, all the experimental monkeys did not show obvious increase or decrease in body temperature from 1 to 7 days, and the body temperature of all the experimental monkeys was within the normal reference range. within the value.

(3) Body weight: There was no obvious increase or decrease in body weight of all experimental monkeys from 1 to 7 days.

5.2) Virus detection

In the challenge test, the dose of virus was 5.0 log CCID50 per animal, and the infection was administered with 100 μl intranasally. Pharyngeal, nasal and anal swabs were collected on days 1-7 to detect the number of virus copies. Throat swab, nasal swab and anal swab viral load results are shown below. (1) Throat swab results

In the control group, detoxification occurred in the pharynx from the 3rd day, and obvious detoxification occurred in the 4-5th day, of which the highest on the 4th day, reaching 4.98log (copy/100μL), and it was not detected from the 6th day; while the prevention group and BS In the -mAb-2 antibody treatment group, except for individual monkeys whose viral load was detected within a certain number of days, the average detection of the rest of the experimental monkeys was below the detection threshold on days 1-7, as shown in Figure 9 for details.

(2) Nasal swab results

The results of nasal swab virus detection showed that the control group had detoxification from the first day to the seventh day, and a clear peak of detoxification occurred in the 4th to the 5th day. While the prevention group, BS-mAb-2 antibody low-dose group and BS-mAb-2 antibody high-dose group all had detoxification peaks on the first day of 4.32log (copy/100μL), 5.07log (copy/100μL) and 4.89log (copy/100μL), among which, the viral load of the prevention group and the BS-mAb-2 antibody low-dose group decreased by more than 2 log values compared with the control group on the 4-7th day; Viral load decreased by more than 2 log values on days 6-7, as shown in Figure 10.

(3) Anal swab results

No fecal excretion was detected in all experimental monkeys on days 1-7, as shown in Figure 11.

5.3) General inspection

(1) General results

The gross pathological observation results showed that the volume of the lungs of the experimental monkeys in the antibody prevention group and the high and low dose groups was basically normal, except that the color of the lungs showed different degrees of gray-red interlaced changes, and there were scattered bleeding spots on the surface of the lungs, and there was no serious pulmonary hemorrhage or nodules. .

(2) Tissue viral load results

The results of tissue viral load detection showed that the viral loads in nasal mucosa, trachea and lung tissue of all experimental monkeys in the prevention group were lower than the detection threshold, and no viral loads were detected in cervical lymph nodes and lung lymph nodes. In the BS-mAb-2 antibody low-dose group and high-dose group, except for a small amount of viral nucleic acid detected in the lung, pulmonary lymphoma, cervical lymphoma, trachea, and nasal mucosa of individual monkeys, no SARS-CoV-2 was detected in other tissues. 2 viruses.

The above experimental results show that the rhesus monkeys were challenged with SARS-CoV-2 virus after prophylactic and therapeutic administration of BS-mAb-2 antibody, and the experimental monkeys in the low-dose group, high-dose group and prevention group had the virus in the early and middle stages. Proliferation and detoxification can be inhibited to a certain extent.

According to the above experiments, rhesus monkeys were injected with 25mg/kg of BS-mAb-2 at a dose of 5.0log CCID50/mouse, 100μl intranasally, and 12 hours after virus infection, which was significantly lower than that of the control group. Nasal and pharyngeal swab virus gene copy and virus infection ability, the effective dose of rhesus monkeys is 25mg/kg. According to the "Guidelines for Estimation of the Maximum Recommended Initial Dose of Drugs in the First Clinical Trial of Healthy Adult Volunteers" issued by the NMPA, "Other situations where the dose is not converted by ^mg /m2 between species: intravascular drugs with a molecular weight greater than 100,000 Daltons. The administered protein should be converted in mg/kg", and the calculated human equivalent dose (HED) is 8.6 mg/kg. Based on an adult body weight of 60kg, the estimated effective dose in humans is about 500mg. Considering the safety, 1/2 to 1/3 of this dose is selected as the first human dose, and the effective dose range for humans is 180 to 250 mg.

According to the dose-exploration test given to cynomolgus monkeys by a single intravenous infusion, the no observable adverse effect level (NOEL) is 300mg/kg, and the corresponding human equivalent dose (HED) is 100mg/kg, calculated based on a human body weight of 60kg, the corresponding human dose It is 6000mg; according to the toxicology study results of repeated administration of cynomolgus monkeys for 4 weeks, the dose level (NOAEL) of no clinical adverse reactions is 150mg/kg, and the corresponding human equivalent dose (HED) is 50mg/kg, according to human body weight 60kg calculation, the corresponding human dose is 3000mg. The maximum human dose currently supported by preclinical studies is 6000 mg. The single administration dose is 180-6000 mg, such as 180, 250, 500, 900, 1000, 1800 mg, 3000 mg or 6000 mg, or the above numerical range. The mode of administration is single/multiple administration.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

sequence listing

<110> Institute of Microbiology, Chinese Academy of Sciences

<120> A bispecific antibody against novel coronavirus and its application

<130> DSP1F211340JW

<150> 202010763140.X

<151> 2020-08-01

<160> 35

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1743

<212> DNA

<213> Artificial Sequence

<400> 1

caggttcagt tagttcagag tggagcagaa gtgaagaagc ccggtgcctc tgtgaaagtg 60

tcatgcaaag catcaggata cacatttaca ggatactaca tgcactgggt taggcaggcg 120

cccggtcaag gcctggagtg gatgggcaga atcaacccta actctggcgg gacgaattat 180

gcacagaagt tccagggaag agtgacaatg acaagagata catcaatctc aacagcatac 240

atggaacttt caagacttag atcagatgac actgctgtct actactgcgc acgagtcccg 300

tactgctcat caacatcatg ccacagagat tggtactttg atctttgggg gagaggtaca 360

cttgtgacag tctcatcggg tggaggcgga tcagggggtg gtggctccga agtccagctc 420

gtcgagagtg gtggaggact ggttcaacct ggaggatcac ttagactttc atgcgcagca 480

tcaggattta tcgtgtcatc aaactacatg tcatgggtac gccaggctcc cgggaagggt 540

ctggagtggg tctcggtgat ctactcagga ggatcaacat actacgcaga ttcagtgaaa 600

ggaagattta caatctcaag acacaactca aagaatactc tttaccttca aatgaactca 660

cttagagcag aagacactgc ggtttactac tgcgcaagag aagcgtacgg aatggatgtg 720

tggggccagg gtacaacagt tactgtttcg tctgcgtcca ctaaaggccc ttccgtcttc 780

ccgctggcac cttcatcaaa gtctacttct ggtggaacgg cggctcttgg atgtctggtt 840

aaggactatt tcccggagcc tgtaactgta tcgtggaact caggagcact tacatcagga 900

gtgcacacat ttcctgcagt gcttcaatca tcaggacttt actcactttc atcagtggtg 960

acagtgcctt catcatcact tggaacacaa acatacatct gcaacgtgaa ccacaaacct 1020

tcaaacacaa aggttgacaa gaaggtcgag cctaaatcat gcgataagac tcatacatgc 1080

cctccttgcc ctgcacctga acttcttgga ggcccgtcgg tgtttttatt cccgccaaaa 1140

ccgaaggata cacttatgat ctcaagaaca cctgaagtga catgcgtggt ggtggatgtg 1200

tcacacgaag atcctgaagt gaaatttaac tggtacgtgg atggagtgga agtgcacaac 1260

gcaaagacta agcctagaga agaacaatac aactcaacat acagagtggt gtcagtgctt 1320

acagtgcttc accaagattg gcttaacgga aaggagtata aatgcaaagt gtcaaacaaa 1380

gcacttcctg cacctatcga gaagactata tcaaaagcaa aaggacaacc tagagaacct 1440

caagtgtaca cacttcctcc ttcaagagat gaacttacaa agaatcaggt gtcacttaca 1500

tgtttagtaa agggcttcta cccatctgac atcgcagtgg aatgggaatc aaacggacaa 1560

cctgagaata attataagac cactcctcct gtacttgatt cagatggatc attcttcctc 1620

tattcaaagt taaccgtgga taaatcaaga tggcaacaag gaaacgtgtt ctcctgttca 1680

gtgatgcacg aagcacttca caaccactac acacagaaga gtctatcact ttcacctgga 1740

tga 1743

<210> 2

<211> 580

<212> PRT

<213> Artificial Sequence

<400> 2

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr

100 105 110

Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

130 135 140

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

145 150 155 160

Ser Gly Phe Ile Val Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala

165 170 175

Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ser

180 185 190

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg His

195 200 205

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

210 215 220

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Ala Tyr Gly Met Asp Val

225 230 235 240

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

245 250 255

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

260 265 270

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

275 280 285

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

290 295 300

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

305 310 315 320

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

325 330 335

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

340 345 350

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

355 360 365

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

370 375 380

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

385 390 395 400

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

405 410 415

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

420 425 430

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

435 440 445

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

450 455 460

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

465 470 475 480

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

485 490 495

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

500 505 510

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

515 520 525

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

530 535 540

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

545 550 555 560

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

565 570 575

Leu Ser Pro Gly

580

<210> 3

<211> 1017

<212> DNA

<213> Artificial Sequence

<400> 3

gatatccaaa tgacccagag cccattgagc cttcctgtga cacctggaga acctgcatca 60

atctcatgca gatcatcaca atcacttctt gattcagatg atggaaacac ataccttgat 120

tggtaccttc agaagcccgg tcagtctcct cagctactaa tttatactct atcatacaga 180

gcatcgggcg taccggaccg cttctccgga tcagggtcag ggactgattt caccctaaag 240

atatccagag tggaagcaga agatgtggga gtgtactact gcatgcaaag aatcgaattt 300

cctcttacat ttggaggagg aacaaaggta gagatcaaag gtggaggagg atccggagga 360

ggtggatccg atatcgtgat gacgcagagc cctagctttc tttctgctag tgtaggcgac 420

agagtgacaa tcacatgcag agcatcacaa ggaatctcat cataccttgc atggtaccaa 480

cagaagccgg gaaaggcgcc caagctgctc atatatgctg cctcaactct gcagagtggc 540

gtgccgtctc gcttcagcgg tagcggatcg ggaacagagt ttacacttac aatctcatca 600

cttcaacctg aagatttcgc cacgtactac tgccaacaac ttaactcata ccctccttac 660

acatttggac aaggaacaaa gttagagatc aagcgaaccg tggcagcacc ttcagtgttt 720

atctttcctc cttcagatga acaacttaaa tcaggaactg cgtctgtagt ttgtcttctt 780

aacaacttct atccaagaga agcaaaggta cagtggaaag tggataacgc cctgcagtct 840

ggtaatagcc aagaatcagt gacagaacaa gattcaaagg acagtacata ctcactttca 900

tcaacactta cactttcaaa ggcggactac gagaagcata aggtgtacgc atgcgaagtg 960

acacaccaag gactttcatc acctgtgaca aagtccttca acagaggaga atgctga 1017

<210> 4

<211> 338

<212> PRT

<213> Artificial Sequence

<400> 4

Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr

115 120 125

Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

130 135 140

Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala Trp Tyr Gln

145 150 155 160

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr

165 170 175

Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

180 185 190

Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

195 200 205

Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Pro Tyr Thr Phe Gly Gln

210 215 220

Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe

225 230 235 240

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

245 250 255

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

260 265 270

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

275 280 285

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

290 295 300

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

305 310 315 320

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

325 330 335

Glu Cys

<210> 5

<211> 2121

<212> DNA

<213> Artificial Sequence

<400> 5

gaggtccagc tcgttgagtc cggtggggga ttggtgcaac ctggaggatc acttagactt 60

tcatgcgcag catcaggatt tatcgtgtca tcaaactaca tgtcatgggt acggcaggcg 120

ccagggaaag gcctagagtg ggtttcggtg atctactcag gaggatcaac atactacgca 180

gattcagtga aaggaagatt tacaatctca agacacaact caaagaatac tctttacctt 240

caaatgaact cacttagagc agaagacact gcggtgtact actgcgcacg ggaggcgtac 300

ggaatggatg tgtgggggca gggaacaaca gtcacggtct ctagcgccag caccaaaggc 360

ccgtctgtct ttccactcgc accttcatca aagagcacgt cgggagggac ggctgcgctt 420

ggatgtttag ttaaggacta tttcccagag ccggtcacag ttagctggaa ctcaggagca 480

cttacctcgg gcgttcacac ctttcctgca gtgcttcaat catcaggact ttactcactt 540

tcatcagtgg tgacagtgcc ttcatcatca cttggaacac aaacatacat ctgcaacgtg 600

aaccacaaac cttcaaacac aaaggtagac aagcgagtag aacctaaatc atgcgataag 660

acccatacat gccctccttg ccctgcacct gaacttcttg gaggtccatc ggtattctta 720

tttccgccaa agccgaagga tacacttatg atctcaagaa cacctgaagt gacatgcgtg 780

gtggtggatg tgtcacacga agatcctgaa gtgaaattta actggtacgt ggatggagtg 840

gaagtgcaca acgcaaagac gaagcctaga gaagaacaat acaactcaac atacagagtg 900

gtgtcagtgc ttacagtgct tcaccaagat tggcttaacg gaaaggagta taaatgcaaa 960

gtgtcaaaca aagcacttcc tgcacctatc gagaagacga tatcaaaagc aaaaggacaa 1020

cctagagaac ctcaagtgta cacacttcct ccttcaagag atgaacttac aaagaatcag 1080

gtgtcactta catgtttagt caagggcttc tacccttcag atatcgcagt ggaatgggaa 1140

tcaaacggac aacctgagaa taattataag actacaccac cggtgttgga cagtgacggg 1200

agcttcttcc tgtattcaaa gctcactgtg gataaatcaa gatggcaaca aggaaacgtg 1260

ttctcgtgtt cagtgatgca cgaagcactt cacaaccact acacacagaa gtcgctgtca 1320

ctttcacctg gtggtggaag tcaagttcag ttggtacaga gcggagcaga agtgaagaag 1380

cccggtgcga gtgtgaaagt gtcatgcaaa gcatcaggat acacatttac aggatactac 1440

atgcactggg tacgacaggc tccgggtcaa ggtttagagt ggatgggcag aatcaaccct 1500

aactctggag gcaccaacta cgcacagaag ttccagggaa gagtgacaat gacaagagat 1560

acatcaatct caacagcata catggaactt tcaagactta gatcagatga cactgcggtc 1620

tattattgtg cacgggtacc ttactgctca tcaacatcat gccacagaga ttggtacttt 1680

gatctttggg gaagaggaac acttgtcact gtgtcgtcag gaggcggagg atcagggggt 1740

gggggcagcg gcggtggcgg ttctggtgga ggtggcagcg acatccaaat gacacaatca 1800

cctctttcac ttcctgtgac acctggagaa cctgcatcaa tctcatgcag atcatcacaa 1860

tcacttctgg actcggacga cggcaacaca taccttgatt ggtaccttca gaagccgggt 1920

caatcacctc aacttcttat ctacacactt tcatacagag cgtccggagt cccggacaga 1980

ttcagcggtt caggatcagg aacagatttc acgttgaaga tttctagagt ggaagcagaa 2040

gatgtgggag tgtactactg catgcaaaga atcgaatttc ctcttacatt tggaggtggc 2100

acgaaagtgg agatcaaatg a 2121

<210> 6

<211> 706

<212> PRT

<213> Artificial Sequence

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Ser Gln

435 440 445

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

450 455 460

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr

465 470 475 480

Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

485 490 495

Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

500 505 510

Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met

515 520 525

Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala

530 535 540

Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr Phe

545 550 555 560

Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

565 570 575

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

580 585 590

Ser Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

595 600 605

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp

610 615 620

Ser Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly

625 630 635 640

Gln Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly

645 650 655

Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

660 665 670

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met

675 680 685

Gln Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu

690 695 700

Ile Lys

705

<210> 7

<211> 648

<212> DNA

<213> Artificial Sequence

<400> 7

gacatcgtga tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gggcattagc agttatttag cctggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagag ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag cttaatagtt accctccgta cacttttggc 300

caggggacca agctggagat caaacgaact gtggctgcac caagcgtgtt tatcttccct 360

cccagcgacg agcagctgaa gagcggcacc gccagcgtgg tctgtctcct gaacaacttc 420

tatcccaggg aggccaaggt ccagtggaaa gtggacaacg ccctgcaaag cggcaatagc 480

caggagtccg tcacagagca ggacagcaag gacagcacct acagcctgtc cagcaccctg 540

accctcagca aggccgacta cgagaagcac aaggtgtacg cttgcgaggt gacccatcag 600

ggcctgtcca gccccgtgac caagtccttc aacaggggcg aatgcagc 648

<210> 8

<211> 216

<212> PRT

<213> Artificial Sequence

<400> 8

Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys Ser

210 215

<210> 9

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 9

Gly Phe Ile Val Ser Ser Asn Tyr

1 5

<210> 10

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 10

Ile Tyr Ser Gly Gly Ser Thr

1 5

<210> 11

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 11

Ala Arg Glu Ala Tyr Gly Met Asp Val

1 5

<210> 12

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 12

Gln Gly Ile Ser Ser Tyr

1 5

<210> 13

<211> 3

<212> PRT

<213> Artificial Sequence

<400> 13

Ala Ala Ser

1

<210> 14

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 14

Gln Gln Leu Asn Ser Tyr Pro Pro Tyr Thr

1 5 10

<210> 15

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 15

Gly Tyr Thr Phe Thr Gly Tyr Tyr

1 5

<210> 16

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 16

Ile Asn Pro Asn Ser Gly Gly Thr

1 5

<210> 17

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 17

Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr

1 5 10 15

Phe Asp Leu

<210> 18

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 18

Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr

1 5 10

<210> 19

<211> 3

<212> PRT

<213> Artificial Sequence

<400> 19

Thr Leu Ser

1

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 20

Met Gln Arg Ile Glu Phe Pro Leu Thr

1 5

<210> 21

<211> 259

<212> PRT

<213> Artificial Sequence

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr

100 105 110

Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr

145 150 155 160

Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu

165 170 175

Asp Ser Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro

180 185 190

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser

195 200 205

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

210 215 220

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

225 230 235 240

Met Gln Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val

245 250 255

Glu Ile Lys

<210> 22

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 22

atgggatggt catgcatcat cctattccta gtagcaacag caacaggagt gcactca 57

<210> 23

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 23

atggatatga gagtgcctgc acaacttctt ggacttcttc ttctttggct tcctggagca 60

agatgc 66

<210> 24

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 24

atgggatggt catgcatcat cctcttccta gtagcaacag caacaggagt gcactca 57

<210> 25

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 25

atggagacgg atacgctgct cctgtgggtt ttgctgctgt gggttccagg ttccactggt 60

gac 63

<210> 26

<211> 362

<212> PRT

<213> Artificial Sequence

<400> 26

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Pro Gly Ala Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Phe

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Gly Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Leu Asn Ser Tyr Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

115 120 125

Ile Lys Gly Gln Pro Lys Ala Ala Pro Asp Ile Gln Met Thr Gln Ser

130 135 140

Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys

145 150 155 160

Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu

165 170 175

Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr

180 185 190

Thr Leu Ser Tyr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu

210 215 220

Asp Val Gly Val Tyr Tyr Cys Met Gln Arg Ile Glu Phe Pro Leu Thr

225 230 235 240

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Lys Gln Pro Ser Arg

245 250 255

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

260 265 270

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

275 280 285

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

290 295 300

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

305 310 315 320

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

325 330 335

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

340 345 350

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

355 360

<210> 27

<211> 592

<212> PRT

<213> Artificial Sequence

<400> 27

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala

65 70 75 80

Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg

115 120 125

Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser

130 135 140

Ser Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

145 150 155 160

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val Ser

165 170 175

Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

180 185 190

Trp Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

195 200 205

Val Lys Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu

210 215 220

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

225 230 235 240

Cys Ala Arg Glu Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

245 250 255

Val Thr Val Ser Ser Arg Thr Ala Ser Thr Lys Gly Pro Ser Val Phe

260 265 270

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

275 280 285

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

290 295 300

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

305 310 315 320

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

325 330 335

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

340 345 350

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

355 360 365

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

370 375 380

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

385 390 395 400

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

405 410 415

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

420 425 430

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

435 440 445

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

450 455 460

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

465 470 475 480

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

485 490 495

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

500 505 510

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

515 520 525

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

530 535 540

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

545 550 555 560

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

565 570 575

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

580 585 590

<210> 28

<211> 360

<212> PRT

<213> Artificial Sequence

<400> 28

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Pro Gly Ala Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Phe

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Gly Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Leu Asn Ser Tyr Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

115 120 125

Ile Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asp Ile Gln Met

130 135 140

Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser

145 150 155 160

Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn

165 170 175

Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu

180 185 190

Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val Pro Asp Arg Phe

195 200 205

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val

210 215 220

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Arg Ile Glu Phe

225 230 235 240

Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val

245 250 255

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

260 265 270

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

275 280 285

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

290 295 300

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

305 310 315 320

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

325 330 335

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

340 345 350

Lys Ser Phe Asn Arg Gly Glu Cys

355 360

<210> 29

<211> 598

<212> PRT

<213> Artificial Sequence

<400> 29

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala

65 70 75 80

Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg

115 120 125

Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser

130 135 140

Ser Gly Gly Gly Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Phe Ile Val Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala

180 185 190

Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ser

195 200 205

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg His

210 215 220

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

225 230 235 240

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Ala Tyr Gly Met Asp Val

245 250 255

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Leu Ala Ser Thr

260 265 270

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

275 280 285

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

290 295 300

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

305 310 315 320

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

325 330 335

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

340 345 350

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

355 360 365

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

370 375 380

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

385 390 395 400

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

405 410 415

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

420 425 430

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

435 440 445

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

450 455 460

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

465 470 475 480

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

485 490 495

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

500 505 510

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

515 520 525

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

530 535 540

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

545 550 555 560

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

565 570 575

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

580 585 590

Leu Ser Leu Ser Pro Gly

595

<210> 30

<211> 360

<212> PRT

<213> Artificial Sequence

<400> 30

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Leu Ser

20 25 30

Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser

35 40 45

Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr

50 55 60

Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser

65 70 75 80

Tyr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

85 90 95

Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly

100 105 110

Val Tyr Tyr Cys Met Gln Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly

115 120 125

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly

130 135 140

Gly Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val

145 150 155 160

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser

165 170 175

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

180 185 190

Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

195 200 205

Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

210 215 220

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro

225 230 235 240

Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val

245 250 255

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

260 265 270

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

275 280 285

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

290 295 300

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

305 310 315 320

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

325 330 335

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

340 345 350

Lys Ser Phe Asn Arg Gly Glu Cys

355 360

<210> 31

<211> 596

<212> PRT

<213> Artificial Sequence

<400> 31

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val

35 40 45

Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

65 70 75 80

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr

85 90 95

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Glu Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Gly Gly Gly Gln Val Gln

130 135 140

Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys

145 150 155 160

Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His

165 170 175

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile

180 185 190

Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly Arg

195 200 205

Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu

210 215 220

Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val

225 230 235 240

Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr Phe Asp Leu

245 250 255

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

260 265 270

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

275 280 285

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

290 295 300

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

305 310 315 320

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

325 330 335

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

340 345 350

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

355 360 365

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

370 375 380

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

385 390 395 400

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

405 410 415

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

420 425 430

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

435 440 445

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

450 455 460

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

465 470 475 480

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

485 490 495

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

500 505 510

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

515 520 525

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

530 535 540

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

545 550 555 560

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

565 570 575

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

580 585 590

Leu Ser Pro Gly

595

<210> 32

<211> 115

<212> PRT

<213> Artificial Sequence

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 33

<211> 108

<212> PRT

<213> Artificial Sequence

<400> 33

Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 34

<211> 126

<212> PRT

<213> Artificial Sequence

<400> 34

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr

100 105 110

Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 35

<211> 113

<212> PRT

<213> Artificial Sequence

<400> 35

Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

Claims (19)

1. A bispecific antibody against a novel coronavirus, which comprises the heavy chain variable region of mAb B38, the light chain variable region of mAb B38, the heavy chain variable region of mAb H4, and the light chain variable region of mAb H4, wherein,

the heavy chain variable region of mab B38 comprises: CDR1 with an amino acid sequence shown as SEQ ID NO.9, CDR2 with an amino acid sequence shown as SEQ ID NO.10 and CDR3 with an amino acid sequence shown as SEQ ID NO. 11; the light chain variable region of mab B38 comprises: CDR1 with an amino acid sequence shown in SEQ ID NO.12, CDR2 with an amino acid sequence shown in SEQ ID NO.13 and CDR3 with an amino acid sequence shown in SEQ ID NO. 14;

the heavy chain variable region of mab H4 comprises: CDR1 with an amino acid sequence shown as SEQ ID NO.15, CDR2 with an amino acid sequence shown as SEQ ID NO.16, and CDR3 with an amino acid sequence shown as SEQ ID NO. 17; the light chain variable region of mab H4 comprises: CDR1 with an amino acid sequence shown in SEQ ID NO.18, CDR2 with an amino acid sequence shown in SEQ ID NO.19 and CDR3 with an amino acid sequence shown in SEQ ID NO. 20;

wherein the bispecific antibody is BS-mAb-1 having the following structure from N-terminus to C-terminus:

heavy chain: VH_H4-VH_B38-CH_B38，

Light chain: VL_H4-VL_B38-CL_B38，

Wherein VH_H4And VH_B38By a linker peptide, VL_H4And VL_B38Linked by a linker peptide; or,

the bispecific antibody is BS-mAb-2, which has the following structure from N-terminus to C-terminus:

heavy chain: VH_B38-CH_B38-VH_H4-VL_H4，

Light chain: VL_B38-CL_B38；

Wherein VH_H4And VL_H4Connecting through connecting peptide to form single-chain antibody; CH (CH)_B38And VH_H4Linked by a linker peptide;

the amino acid sequence of the connecting peptide is (GGGGS) n, wherein n is a natural number of 1-4.

2. The bispecific antibody of claim 1, wherein in antibody BS-mAb-1, n of the linking peptide is 2; in antibody BS-mAb-2, the VH_H4And VL_H4N of the connecting peptide between is 4, CH_B38And VH_H4N of the connecting peptide therebetween is 1.

3. The bispecific antibody of claim 2, wherein the amino acid sequence of the heavy chain variable region of mab B38 is set forth in SEQ ID No.32, and the amino acid sequence of the light chain variable region of mab B38 is set forth in SEQ ID No. 33; the amino acid sequence of the heavy chain variable region of the monoclonal antibody H4 is shown as SEQ ID NO.34, and the amino acid sequence of the light chain variable region of the monoclonal antibody H4 is shown as SEQ ID NO. 35.

4. The bispecific antibody of claim 3, wherein the bispecific antibody BS-mAb-1 has the amino acid sequence of its light chain as shown in SEQ ID No.4 and the amino acid sequence of its heavy chain as shown in SEQ ID No. 2; or,

the amino acid sequence of the light chain of the bispecific antibody BS-mAb-2 is shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain of the bispecific antibody BS-mAb-2 is shown in SEQ ID NO. 6.

5. The bispecific antibody of any one of claims 1 to 4, wherein said bispecific antibody is a murine antibody, a humanized antibody, a chimeric antibody or a recombinant antibody.

6. The bispecific antibody of any one of claims 1-4, wherein the antibody is one of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody.

7. A gene encoding the bispecific antibody of any one of claims 1 to 6.

8. The gene of claim 7, wherein the nucleotide sequence of the light chain of the bispecific antibody is shown as SEQ ID No.3, and the nucleotide sequence of the heavy chain is shown as SEQ ID No. 1; or

The nucleotide sequence of the bispecific antibody light chain is shown as SEQ ID NO.7, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 5.

9. A biological material comprising the gene of claim 7 or 8, which is a recombinant DNA, an expression cassette, a vector, a host cell, an engineered bacterium or a cell line.

10. A process for the preparation of a bispecific antibody according to any one of claims 1 to 6, comprising: respectively constructing recombinant expression vectors containing heavy chain and light chain genes of the bispecific antibody BS-mAb-1 or BS-mAb-2; introducing the recombinant expression vector into a host cell to obtain a host cell for stably expressing the bispecific antibody; culturing host cells, and separating and purifying to obtain the bispecific antibody.

11. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 6.

12. A kit comprising the bispecific antibody according to any one of claims 1 to 6.

13. Use of the bispecific antibody of any one of claims 1 to 6 or the gene of claim 7 or 8 or the biological material of claim 9 or the pharmaceutical composition of claim 11 or the kit of claim 12 for any one of the following:

(1) the application in preparing the medicine for preventing or treating the diseases caused by the novel coronavirus infection;

(2) the application in preparing a novel coronavirus diagnostic reagent or a diagnostic kit.

14. A pharmaceutical composition in single dose form, wherein the single dose form contains 180mg-6000mg of the bispecific antibody of any one of claims 1 to 6.

15. The pharmaceutical composition of claim 14, wherein the single dose form contains 180mg to 3000mg of the bispecific antibody of any one of claims 1 to 6.

16. The pharmaceutical composition of claim 15, wherein the single dose form contains 500mg to 1800mg of the bispecific antibody of any one of claims 1 to 6.

17. The pharmaceutical composition of claim 16, wherein the single dose form contains 900mg to 1800mg of the bispecific antibody of any one of claims 1 to 6.

18. The pharmaceutical composition of claim 14, wherein the single dose form contains 500mg to 1000mg of the bispecific antibody of any one of claims 1 to 6.

19. The pharmaceutical composition of any one of claims 14 to 18, wherein the pharmaceutical composition is formulated in a form suitable for intravenous administration.

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