Bispecific antibodies and preparation methods and applications thereof

Abstract

The present invention discloses a T cell engager bispecific antibody targeting a tumor antigen and a T cell antigen and its application. The bispecific antibody targets CD3 as well as CD20, CD19 or CD38. The bispecific antibody of the present invention is easy to prepare, can have the affinity of the parent antibody and can produce an ideal biological effect, thus laying a new material foundation for the concept of anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to bispecific antibodies, in particular bispecific antibodies used as T cell engagers, and preparation methods and applications thereof. Background Art

[0002] Bispecific antibodies are genetically engineered antibodies that have been artificially engineered. Bispecific antibodies can simultaneously target two different antibody binding epitopes, which can come from different antigens or the same antigen. Many current studies have shown that bispecific antibodies have great therapeutic potential in the treatment of diseases such as tumors, autoimmune diseases, and viral infections. Compared with monoclonal antibodies, the main advantage of bispecific antibodies is that they can mediate the spatial effect of two recognition epitopes and the synergistic effect of dual targeting, producing biological effects that cannot be achieved by the combined use of two antibodies.

[0003] Because of its complexity, which is different from that of monoclonal antibodies, bispecific antibodies have higher technical barriers and R&D costs. In the process of bispecific antibody research and development, it is necessary to comprehensively consider issues such as target selection, antibody engineering design, and subsequent production processes. In the process of bispecific antibody construction, problems such as low expression, poor stability, and difficulty in establishing drug efficacy models are often encountered. Traditional antibodies are generally composed of four chains, two heavy chains and two light chains. If traditional antibodies are used to construct bispecific antibodies, there will be a problem of light chain and heavy chain mismatch. In addition, it is still unknown whether the constructed bispecific antibody can have the affinity of the parent antibody and produce the desired biological effect.

[0004] Therefore, there is an urgent need in the art for bispecific antibodies that are easy to prepare and can have the affinity of the parent antibody and produce ideal biological effects. Summary of the invention

[0005] The object of the present invention is to provide a bispecific antibody, which is not only easy to prepare but also has the affinity of the original antibody and produces an ideal biological effect.

[0006] In a first aspect, the present invention provides a bispecific antibody, wherein the bispecific antibody is composed of an antibody that specifically binds to a tumor antigen and an antibody that specifically binds to a T cell antigen.

[0007] In a preferred embodiment, the bispecific antibody is a T cell engager bispecific antibody.

[0008] In a specific embodiment, the tumor antigen is CD20, CD19 or CD38 and the T cell antigen is CD3.

[0009] In a preferred embodiment, the light chains of the antibody that specifically binds to a tumor antigen and the antibody that specifically binds to a T cell antigen have a sequence similarity of more than 90%, preferably more than 95%, and more preferably more than 98%.

[0010] In a specific embodiment, the bispecific antibody comprises a heavy chain and a light chain, wherein the heavy chain is derived from an antibody that specifically binds to a tumor antigen and an antibody that specifically binds to a T cell antigen, respectively, and the light chain is derived from an antibody that specifically binds to a tumor antigen and an antibody that specifically binds to a T cell antigen, respectively;

[0011] Preferably, the heavy chain is derived from an antibody that specifically binds to a tumor antigen and an antibody that specifically binds to a T cell antigen, respectively, and the light chain is derived from an antibody that specifically binds to a tumor antigen or an antibody that specifically binds to a T cell antigen.

[0012] In a preferred embodiment, the bispecific antibody comprises two heavy chains and two light chains.

[0013] In a specific embodiment, the heavy chain variable region in the Fab fragment of the antibody is further connected to the CH1 region of the Fab fragment of the antibody that specifically binds to the tumor antigen or the CH1 region of the Fab fragment of the antibody that specifically binds to the T cell antigen via an optional linker;

[0014] Preferably, the heavy chain variable region derived from the antibody that specifically binds to a T cell antigen in the Fab fragment of the antibody is further connected to the CH1 region of the Fab fragment of the antibody that specifically binds to a tumor antigen via an optional linker.

[0015] In a preferred embodiment, the linker is (GGGS) 4 .

[0016] In a specific embodiment, the Fab fragment of the antibody is swapped with the Fab fragment of the antibody that specifically binds to a tumor antigen or the Fab fragment of the antibody that specifically binds to a T cell antigen.

[0017] In a specific embodiment, the light chain CDR1-3 of the antibody that specifically binds to CD20 are respectively shown as SEQ ID NOs: 40-42, and the heavy chain CDR1-3 are respectively shown as SEQ ID NOs: 37-39;

[0018] The light chain CDR1-3 of the antibody that specifically binds to CD19 are respectively shown in SEQ ID NOs: 52-54, and the heavy chain CDR1-3 are respectively shown in SEQ ID NOs: 49-51;

[0019] The light chain CDR1-3 of the antibody that specifically binds to CD38 are shown in SEQ ID NOs: 64-66, respectively, and the heavy chain CDR1-3 are shown in SEQ ID NOs: 61-63, respectively;

[0020] The light chain CDR1-3 of the antibody that specifically binds to CD3 are respectively shown in SEQ ID NOs:46-48 or SEQ ID NOs:58-60, and the heavy chain CDR1-3 are respectively shown in SEQ ID NOs:43-45 or SEQ ID NOs:55-57;

[0021] Preferably, the light chain CDR1-3 of the antibody that specifically binds to CD3 are respectively shown as SEQ ID NOs:46-48, and the heavy chain CDR1-3 are respectively shown as SEQ ID NOs:43-45; or the light chain CDR1-3 of the antibody that specifically binds to CD3 are respectively shown as SEQ ID NOs:58-60, and the heavy chain CDR1-3 are respectively shown as SEQ ID NOs:55-57.

[0022] In a preferred embodiment, the light chain variable region of the antibody that specifically binds to CD20 is as shown in SEQ ID NO:23, and the heavy chain variable region is as shown in SEQ ID NO:24;

[0023] The light chain variable region of the antibody that specifically binds to CD19 is shown in SEQ ID NO: 29 or 30, and the heavy chain variable region is shown in SEQ ID NO: 27 or 28;

[0024] Preferably, the light chain variable region of the antibody that specifically binds to CD19 is as shown in SEQ ID NO: 29, and the heavy chain variable region is as shown in SEQ ID NO: 27, or the light chain variable region of the antibody that specifically binds to CD19 is as shown in SEQ ID NO: 30, and the heavy chain variable region is as shown in SEQ ID NO: 28;

[0025] The light chain variable region of the antibody that specifically binds to CD38 is shown in SEQ ID NO:36, and the heavy chain variable region is shown in SEQ ID NO:35;

[0026] The light chain variable region of the antibody that specifically binds to CD3 is shown in SEQ ID NO: 26, 33 or 34, and the heavy chain variable region is shown in SEQ ID NO: 25, 31 or 32;

[0027] Preferably, the light chain variable region of the antibody that specifically binds to CD3 is shown as SEQ ID NO:26, and the heavy chain variable region is shown as SEQ ID NO:25; or the light chain variable region of the antibody that specifically binds to CD3 is shown as SEQ ID NO:33, and the heavy chain variable region is shown as SEQ ID NO:31; or the light chain variable region of the antibody that specifically binds to CD3 is shown as SEQ ID NO:34, and the heavy chain variable region is shown as SEQ ID NO:32.

[0028] In a preferred embodiment, the light chain of the antibody that specifically binds to CD20 is as shown in SEQ ID NO: 2, and the heavy chain is as shown in SEQ ID NO: 1;

[0029] The light chain of the antibody that specifically binds to CD19 is shown in SEQ ID NO:6, and the heavy chain is shown in SEQ ID NO:5;

[0030] The light chain of the antibody that specifically binds to CD38 is shown in SEQ ID NO: 10, and the heavy chain is shown in SEQ ID NO: 9;

[0031] The light chain of the antibody that specifically binds to CD3 is shown in SEQ ID NO: 4 or 8, and the heavy chain is shown in SEQ ID NO: 3 or 7;

[0032] Preferably, the light chain of the antibody that specifically binds to CD3 is shown as SEQ ID NO:4, and the heavy chain is shown as SEQ ID NO:3; or the light chain of the antibody that specifically binds to CD3 is shown as SEQ ID NO:8, and the heavy chain is shown as SEQ ID NO:7.

[0033] In a second aspect, the present invention provides a bispecific antibody, characterized in that the bispecific antibody is composed of a heavy chain 1, a heavy chain 2 and a light chain, and the bispecific antibody is selected from the following antibodies:

[0034]

[0035]

[0036] In a third aspect, the present invention provides an immunoconjugate, characterized in that the immunoconjugate contains:

[0037] (a) the bispecific antibody according to the first or second aspect; and

[0038] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0039] In a fourth aspect, the present invention provides use of the bispecific antibody described in the first or second aspect or the immunoconjugate described in the third aspect in the preparation of an anti-tumor drug.

[0040] In a preferred embodiment, the tumor is a tumor that specifically expresses CD20, CD19 or CD38.

[0041] In a preferred embodiment, the tumor is lymphoma, leukemia, multiple myeloma, etc.

[0042] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody described in the first or second aspect or the immunoconjugate described in the third aspect and a pharmaceutically acceptable excipient.

[0043] In a preferred embodiment, the pharmaceutical composition further contains other drugs for treating tumors, such as cytotoxic drugs.

[0044] In a preferred embodiment, the other drugs for treating tumors include paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, and pembrolizumab.

[0045] In a preferred embodiment, the other drugs are used to block the interaction between PD-1 and PD-L1, and simultaneously block the interaction between other targets and their receptors or ligands.

[0046] In a preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0047] In a preferred embodiment, the pharmaceutical composition is used to prepare drugs for preventing and treating tumors.

[0048] In a fifth aspect, the present invention provides an isolated polynucleotide, characterized in that the polynucleotide encodes the bispecific antibody described in the first or second aspect.

[0049] In a sixth aspect, the present invention provides a vector, characterized in that the vector contains the polynucleotide described in the first or second aspect.

[0050] In a seventh aspect, the present invention provides a host cell, characterized in that the host cell contains the vector described in the sixth aspect, or the polynucleotide described in the fifth aspect is integrated into its genome;

[0051] Alternatively, the host cell expresses the bispecific antibody described in the first or second aspect.

[0052] In an eighth aspect, the present invention provides a method for producing the bispecific antibody of the first or second aspect, characterized in that it comprises the steps of:

[0053] (a) culturing the host cell according to the seventh aspect under suitable conditions to obtain a culture containing the bispecific antibody; and

[0054] (b) purifying and / or isolating the culture obtained in step (a) to obtain the bispecific antibody.

[0055] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The schematic diagram of the structure of the bispecific antibody of the present invention is shown (HC: heavy chain; LC: light chain); wherein the structures of LL206-1, LL207-1 and LL208-1 are as shown Figure 1 A; the structures of LL206-3, LL207-3 and LL208-3 are shown in Figure 1 B; the structures of LL206-5, LL207-5 and LL208-5 are shown in Figure 1 C; the structures of LL206-2, LL207-2 and LL208-2 are shown in Figure 1 D; the structures of LL206-4, LL207-4 and LL208-4 are shown in Figure 1 E; the structures of LL206-6, LL207-6 and LL208-6 are shown in Figure 1 As shown in F;

[0057] Figure 2 The results of affinity testing of the monoclonal antibody of the present invention with exchanged light chains to Raji cells and T cells are shown;

[0058] Figure 3 The results of affinity testing of the bispecific antibody of the present invention to Raji cells and T cells are shown;

[0059] Figure 4 It was shown that the bispecific antibodies of the present invention can mediate T cells to recognize tumor cells and release cytokines. DETAILED DESCRIPTION

[0060] In a large number of studies and screenings of bispecific antibodies for T cell engagers, the inventors unexpectedly discovered that specific monoclonal antibodies targeting specific tumor cell targets and T cell targets can be used as parent antibodies in bispecific antibodies, and the bispecific antibodies obtained in this way are not only easy to prepare, but also have the affinity of the parent antibody and produce ideal biological effects, thereby being able to be used as a new anti-tumor drug. On this basis, the present invention was completed.

[0061] T cell engager

[0062] The "T cell engager" used herein has the meaning conventionally understood by those skilled in the art. The T cell engager can activate endogenous T cells by bispecifically binding to targets and T cells on the surface of tumor cells, leading to the lysis of tumor cells, thereby achieving the purpose of treating tumors. German Micromet (later acquired by Amgen) is one of the earliest companies to develop the BiTE (Bispecific T cell engager) platform. BiTE is formed by connecting two scFv (Single-chain variable fragment) with a connecting peptide. Therefore, these molecules are bivalent and monovalent for each antigen, and their typical molecular weight is 50-60KDa. In 1998, Micromet developed Blinatumomab based on the BiTE platform. Since then, Blinatumomab has successfully completed Phase I, Phase II and Phase III clinical trials and became the first BiTE drug approved by the FDA. Blinatumomab can simultaneously target two proteins, CD19 and CD3. CD19 is expressed on the surface of acute lymphoblastic leukemia and lymphoma tumor cells as a target for tumor cells. CD3 includes multiple subunits (d, g and e) and is an important surface marker of T cells. CD3 subunits can bind to TCR non-covalently to form a TCR-CD3 complex. CD3 antibodies can generally recognize the ε chain of the CD3 complex, transmit the activation signal generated by the binding of TCR to the antigen into the cell, induce T cell activation, and then kill tumor cells. T cell engagers have been proven to be used to treat tumors, and subsequent T cell cell engagers targeting other targets such as CD20, BCMA and HER2 are already in the clinical research stage.

[0063] Bispecific antibodies of the present invention

[0064] Herein, "the antibody of the present invention" and "the bispecific antibody of the present invention" have the same meaning.

[0065] Based on the teachings of the present invention, those skilled in the art will understand that the bispecific antibody of the present invention is a T cell engager-type bispecific antibody; that is, a bispecific antibody that simultaneously targets tumor antigens and T cell antigens.

[0066] In a specific embodiment, the bispecific antibody of the present invention is composed of an antibody that specifically binds to a tumor antigen, such as CD20, CD19 or CD38, and an antibody that specifically binds to a T cell antigen, such as CD3.

[0067] The T cell engager bispecific antibody of the present invention is a T cell engager constructed using a common light chain antibody. Compared with other types of T cell engagers, the T cell engager of the present invention has great advantages. A bispecific antibody based on a common light chain means that two bispecific antibodies combined together have a common light chain, thus avoiding the problem of light-heavy chain mismatch. In addition, this bispecific antibody has a structure similar to that of a traditional antibody, and therefore has great advantages in terms of drugability.

[0068] Therefore, in a specific embodiment, the light chains of the antibody that specifically binds to a tumor antigen and the antibody that specifically binds to a T cell antigen in the T cell engager bispecific antibody of the present invention have a sequence similarity of more than 90%, preferably more than 95%, and more preferably more than 98%.

[0069] The bispecific antibodies of the present invention comprise a heavy chain and a light chain, wherein the heavy chain may be derived from an antibody that specifically binds to a tumor antigen and an antibody that specifically binds to a T cell antigen, respectively, and the light chain may also be derived from an antibody that specifically binds to a tumor antigen and an antibody that specifically binds to a T cell antigen, respectively; preferably, the light chains are the same, i.e., the light chains may be derived from either an antibody that specifically binds to a tumor antigen or an antibody that specifically binds to a T cell antigen.

[0070] The bispecific antibody of the present invention can also be further modified to add a Fab fragment. In a specific embodiment, the heavy chain variable region in the Fab fragment of the bispecific antibody of the present invention is connected via an optional linker, such as (GGGS) 4 The CH1 region of the Fab fragment of the antibody that specifically binds to the tumor antigen or the CH1 region of the Fab fragment of the antibody that specifically binds to the T cell antigen is further connected.

[0071] In a preferred embodiment, the heavy chain variable region derived from the antibody that specifically binds to a T cell antigen in the Fab fragment of the antibody is further linked to the CH1 region of the Fab fragment of the antibody that specifically binds to a tumor antigen via an optional linker.

[0072] In another preferred embodiment, the Fab fragment of the antibody is exchanged with the Fab fragment of the antibody that specifically binds to a tumor antigen or the Fab fragment of the antibody that specifically binds to a T cell antigen.

[0073] Based on the bispecific antibody of the present invention, the present invention also provides an isolated polynucleotide encoding the bispecific antibody, an expression vector comprising the polynucleotide, and a host cell containing the expression vector or in which the polynucleotide is integrated into its genome or expresses the bispecific antibody.

[0074] Those skilled in the art are aware of the types of expression vectors that can be used to implement the present invention, including but not limited to: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof; preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof. Those skilled in the art are also aware of the types of host cells used to implement the present invention, including but not limited to: prokaryotic cells or eukaryotic cells; preferably Escherichia coli, yeast cells or mammalian cells.

[0075] Those skilled in the art know how to use the above-mentioned host cells to prepare the bispecific antibody of the present invention. For example, the host cells can be cultured under suitable conditions to obtain a culture containing the bispecific antibody. After obtaining the culture, the obtained culture can be purified and / or separated to obtain the bispecific antibody. In a preferred embodiment, the purification is a protein A affinity column purification and separation to obtain the target antibody.

[0076] The present invention includes not only complete antibodies, but also fragments, derivatives and analogs of the antibodies. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0077] The bispecific antibodies of the present invention also include variant forms of polypeptides comprising the above-mentioned CDR regions that have the same functions as the antibodies of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not change the function of the protein. For another example, adding one or several amino acids at the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention. Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0078] In addition to almost full-length polypeptides, the present invention also includes fragments of the bispecific antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0079] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0080] Table 1

[0081] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0082] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that only encodes a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0083] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences. The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0084] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biological molecules (nucleic acids, proteins, etc.) involved in the present invention include biological molecules in an isolated form.

[0085] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0086] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.

[0087] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2 Another method is to use MgCl 2 If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0088] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0089] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0090] The antibodies of the present invention can be used alone or in combination or conjugated with a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or any combination of these substances. Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing a detectable product.

[0091] Therefore, the present invention also provides an immunoconjugate, which contains the bispecific antibody of the present invention; and a coupling part. The therapeutic agent that can be combined or coupled with the antibody of the present invention includes but is not limited to: 1. Radionuclides; 2. Biotoxins; 3. Cytokines, such as IL-2, etc.; 4. Gold nanoparticles / nanorods; 5. Viral particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Drug-activated enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. Therapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc. In a preferred embodiment, the immunoconjugate is an antibody-drug conjugate (Antibody-Drug-Conjugate, ADC).

[0092] Pharmaceutical composition

[0093] The present invention also provides a pharmaceutical composition, which contains the above-mentioned bispecific antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0094] The pharmaceutical composition of the present invention can be directly used to bind to the PD-1 protein, thereby blocking the interaction between PD-1 and PD-L1. The pharmaceutical composition of the present invention can be directly used to bind to other targets, thereby blocking the binding of the other targets to their receptors or ligands. Therefore, the pharmaceutical composition of the present invention can be used to treat tumors. In a preferred embodiment, the tumor is selected from the following group: acute myeloid leukemia, chronic myeloid leukemia, multiple myelopathy, lymphocytic leukemia, lymphoma, colorectal cancer, breast cancer, gallbladder cancer, gastric cancer, liver cancer, kidney cancer, head and neck tumors, brain tumors, glioblastoma, lung cancer, small intestine cancer, bone cancer, prostate cancer, ovarian cancer, cervical cancer, adrenal tumors, bladder tumors, sarcomas, etc. In addition, the pharmaceutical composition of the present invention can also be used in combination with other therapeutic agents.

[0095] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned single domain antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0096] The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dosage is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0097] application

[0098] As described above, the bispecific antibodies of the present invention have broad biological and clinical application values, and their applications involve multiple fields such as diagnosis and treatment of diseases related to cell therapy, basic medical research, and biological research.

[0099] In a preferred embodiment, the bispecific antibodies of the present invention are used for targeted tumor therapy against CD20, CD19 or CD38 targets.

[0100] Advantages of the present invention:

[0101] 1. The bispecific antibody of the present invention is easy to prepare;

[0102] 2. The bispecific antibody of the present invention can have the affinity of the parent antibody and can produce ideal biological effects;

[0103] 3. The bispecific antibodies of the present invention are highly safe;

[0104] 4. The bispecific antibody of the present invention lays a new material foundation for the development of anti-tumor drugs.

[0105] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0106] Example

[0107] Example 1. Selection of candidate antibody sequences for bispecific antibodies

[0108] The inventors selected three membrane protein targets CD20, CD19 and CD38 as well as CD3 as the sequences of the parent antibody in the bispecific antibody.

[0109] 1. The inventors selected CD20 antibody Ofatumumab as the parent antibody, and the amino acid sequence of the heavy chain variable region is:

[0110] EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS(SEQ ID NO:23)

[0111] Among them, HCDR1-3 are: GFTFNDYAMH (SEQ ID NO: 37), TISWNSGSIGYADSVKG (SEQ ID NO: 38), DIQYGNYYYGMDV (SEQ ID NO: 39).

[0112] The amino acid sequence of its light chain variable region is:

[0113] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK(SEQ ID NO:24)

[0114] Among them, LCDR1-3 are RASQSVSSYLA (SEQ ID NO: 40), DASNRAT (SEQ ID NO: 41), and QQRSNWPIT (SEQ ID NO: 42), respectively.

[0115] The inventors screened and determined that the CD3 antibody corresponding to the CD20 antibody Ofatumumab is Foralumab. The two antibodies have very similar light chain sequences and are likely to maintain sufficient affinity and biological activity when using the same light chain.

[0116] The amino acid sequence of the heavy chain variable region of CD3 antibody, Foralumab, is:

[0117] QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS(SEQ ID NO:25)

[0118] Wherein HCDR1-3 are: GFKFSGYGMH (SEQ ID NO: 43), VIWYDGSKKYYVDSVKG (SEQ ID NO: 44), QMGYWHFDL (SEQ ID NO: 45);

[0119] The amino acid sequence of the light chain variable region of CD3 antibody, Foralumab, is:

[0120] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK(SEQ ID NO:26)

[0121] Among them, LCDR1-3 are: RASQSVSSYLA (SEQ ID NO: 46), DASNRAT (SEQ ID NO: 47), QQRSNWPPLT (SEQ ID NO: 48).

[0122] 2. The inventors selected the CD19-targeting antibody clone FMC63 as a candidate antibody. The amino acid sequence of the heavy chain variable region of the CD19 antibody is:

[0123] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:27)

[0124] HCDR1-3 are: GVSLPDYGVS (SEQ ID NO: 49), VIWGSETTYYNSALKS (SEQ ID NO: 50), HYYYGGSYA (SEQ ID NO: 51)

[0125] The heavy chain variable region sequence of the CD19 antibody was humanized by CDR transplantation. The amino acid sequence after humanization is:

[0126] QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID NO:28)

[0127] The amino acid sequence of the CD19 antibody light chain variable region is:

[0128] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT(SEQ ID NO:29)

[0129] LCDR1-3 are: RASQDISKYLN (SEQ ID NO: 52), HTSRLHS (SEQ ID NO: 53), QQGNTLPYT (SEQ ID NO: 54)

[0130] The light chain variable region sequence of the CD19 antibody was humanized by CDR transplantation. The amino acid sequence after humanization is:

[0131] DIQMTQSPSSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPYTFGQGTKVEIK(SEQ ID NO:30)

[0132] The CD3 antibody corresponding to the CD19 antibody clone FMC63 is clone UCHT-1. The amino acid sequence of the heavy chain variable region of the CD3 antibody UCHT-1 is derived from PDB: 1XIW_D

[0133] >UCHT-1-VH anti-CD3

[0134] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS(SEQ ID NO:31)

[0135] HCDR1-3 are: GYSFTGYTMN (SEQ ID NO: 55), LINPYKGVSTYNQKFKD (SEQ ID NO: 56), SGYYGDSDWYFDV (SEQ ID NO: 57)

[0136] The amino acid sequence of the heavy chain variable region sequence of the UCHT-1 antibody clone after sequence humanization is:

[0137] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS(SEQ ID NO:32)

[0138] The amino acid sequence of the light chain variable region of the CD3 antibody is from PDB:1XIW_C

[0139] >UCHT-VL

[0140] DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK(SEQ ID NO:33)

[0141] LCDR1-3 are: RASQDIRNYLN (SEQ ID NO: 58), YTSRLHS (SEQ ID NO: 59), QQGNTLPWT (SEQ ID NO: 60)

[0142] The amino acid sequence of the light chain variable region sequence of the UCHT-1 antibody clone after sequence humanization is:

[0143] DIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIK(SEQ ID NO:34)

[0144] 3. The inventors selected Daratumumab, an antibody targeting CD38, as a candidate antibody. The amino acid sequence of the heavy chain variable region of the CD38 antibody Daratumumab is:

[0145] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS(SEQ ID NO:35)

[0146] HCDR1-3 are: GFTFNSFAMS (SEQ ID NO: 61), AISGSGGGTYYADSVKG (SEQ ID NO: 62), DKILWFGEPVFDY (SEQ ID NO: 63)

[0147] The amino acid sequence of the light chain variable region of the CD38 antibody Daratumumab is:

[0148] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK(SEQ ID NO:36)

[0149] LCDR1-3 are: RASQSVSSYLA (SEQ ID NO: 64), DASNRAT (SEQ ID NO: 65), QQRSNWPPT (SEQ ID NO: 66)

[0150] Foralumab was selected as the CD3 antibody corresponding to the CD38 antibody Daratumumab for combination.

[0151] Example 2. Design of bispecific antibody sequences

[0152] 1. Design of bispecific antibodies targeting CD20 and CD3

[0153] First, a monoclonal antibody was designed in which the light chains of CD20 antibody Ofatumumab and CD3 antibody Foralumab were exchanged to verify whether CD20 antibody and CD3 antibody still maintained affinity for the target after exchanging the light chains.

[0154] The monoclonal antibody composed of CD20 heavy chain and CD3 light chain was named as LL206-BM3, and its amino acid sequence is:

[0155] Heavy chain: SEQ ID NO: 1

[0156] EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0157] Light chain: SEQ ID NO: 4

[0158] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0159] The monoclonal antibody composed of CD3 heavy chain and CD20 light chain was named as LL206-BM4, and its amino acid sequence is:

[0160] Heavy chain: SEQ ID NO: 3

[0161] QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0162] Light chain: SEQ ID NO: 2

[0163] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0164] At the same time, CD20 antibody Ofatumumab and CD3 antibody Foralumab were prepared as control antibodies. CD20 antibody Ofatumumab was named: LL206-BM1. CD3 antibody Foralumab was named: LL206-BM2.

[0165] By exchanging light chains, bispecific antibodies targeting CD20 and CD3 were constructed. Knobs-into-holes technology (JB Ridgway, LG Presta, P Carter. Protein Eng. 1996 Jul; 9(7): 617-21.) was used to construct bispecific antibodies targeting CD20 and CD3 with "1+1 structure" and "2+1 structure".

[0166] The bispecific antibodies with a "1+1 structure" are named LL206-1 and LL206-2. The heavy chain of Ofatumumab is a knot chain, and the heavy chain of Foralumab is a hole chain. LL206-1 uses the light chain of Ofatumumab, and LL206-2 uses the light chain of Foralumab.

[0167] The amino acid sequence of LL206-1 is:

[0168] Heavy chain 1: SEQ ID NO: 11

[0169] EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0170] Heavy chain 2: SEQ ID NO: 12

[0171] QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0172] Light chain: SEQ ID NO: 2

[0173] The amino acid sequence of LL206-2 is:

[0174] Heavy chain 1: SEQ ID NO: 11

[0175] Heavy chain 2: SEQ ID NO: 12

[0176] Light chain: SEQ ID NO: 4

[0177] In the "2+1 structure", two antigen binding fragments (Fab) of Ofatumumab are used, and the two variable regions are connected by linker: 4*(GGGS). Among them, the bispecific antibodies of the "2+1 structure" constructed by connecting the Fab end of CD20 to the Fab end of CD3 based on LL206-1 and LL206-2 are named: LL206-3 and LL206-4 respectively.

[0178] The bispecific antibodies with a "2+1 structure" constructed by placing the Fab end of CD3 on the outside, that is, connecting the Fab end of CD20 to the Fc end, and then connecting the Fab end of CD3 to the Fab end of CD20 were named: LL206-5 and LL206-6 respectively.

[0179] The amino acid sequence of LL206-3 is as follows:

[0180] Heavy chain 1: SEQ ID NO:11

[0181] Heavy chain 2: SEQ ID NO:13

[0182] EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0183] Light chain: SEQ ID NO:2

[0184] The amino acid sequence of LL206-4 is as follows:

[0185] Heavy chain 1: SEQ ID NO:11

[0186] Heavy chain 2: SEQ ID NO:13

[0187] Light chain: SEQ ID NO:4

[0188] The amino acid sequence of LL206-5 is:

[0189] Heavy chain 1: SEQ ID NO:11

[0190] Heavy chain 2: SEQ ID NO:14

[0191] QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0192] Light chain: SEQ ID NO:2

[0193] The amino acid sequence of LL206-6 is:

[0194] Heavy chain 1: SEQ ID NO:11

[0195] Heavy chain 2: SEQ ID NO: 14

[0196] Light chain: SEQ ID NO:4.

[0197] 2. Design of bispecific antibodies targeting CD19 and CD3

[0198] First, a monoclonal antibody was designed by swapping the light chains of CD19 antibody clone FMC63 and CD3 clone UCHT-1 to verify whether CD20 antibody and CD3 antibody still maintain affinity to the target after swapping the light chains. At the same time, the light chain with better affinity was selected as the candidate light chain for constructing a bispecific antibody.

[0199] The monoclonal antibody composed of CD19 heavy chain and CD3 light chain was named as LL207-BM3, and the heavy chain was SEQ ID NO: 5.

[0200] QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0201] Light chain: SEQ ID NO: 8

[0202] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0203] The monoclonal antibody selected with the combination of CD3 heavy chain and CD19 light chain is named LL207 - BM4,

[0204] The heavy chain is: SEQ ID NO:7

[0205] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0206] The light chain is: SEQ ID NO:6

[0207] DIQMTQSPSSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0208] At the same time, CD19 antibody clone FMC63 and CD3 antibody clone UCHT-1 were prepared as control antibodies. CD19 antibody clone FMC63 was named: LL207-BM1. CD3 antibody clone UCHT-1 was named: LL207-BM2.

[0209] By exchanging light chains, bispecific antibodies targeting CD19 and CD3 were constructed. At the same time, Knobs-into-holes technology was used to construct "1+1 structure" and "2+1 structure" bispecific antibodies targeting CD19 and CD3.

[0210] Among them, the bispecific antibodies of "1+1 structure" are named: LL207-1 and LL207-2, the heavy chain of CD19 antibody clone FMC63 is knot chain, and the heavy chain of antibody clone UCHT-1 is hole chain. LL207-1 uses the light chain of CD19 antibody clone FMC63, and LL207-2 uses the light chain of antibody clone UCHT-1.

[0211] The amino acid sequence of LL207-1 is:

[0212] Heavy chain 1: SEQ ID NO: 15

[0213] QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0214] Heavy chain 2: SEQ ID NO:16

[0215] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0216] Light chain: SEQ ID NO: 8

[0217] The amino acid sequence of LL207-2 is:

[0218] Heavy chain 1: SEQ ID NO: 15

[0219] Heavy chain 2: SEQ ID NO: 16

[0220] Light chain: SEQ ID NO: 6

[0221] In the "2+1 structure", two antigen binding fragments (Fab) of FMC63 clones are used, and the two variable regions are connected by linker: 4*(GGGS). Among them, on the basis of LL207-1 and LL207-2, the bispecific antibodies of the "2+1 structure" constructed by connecting the Fab end of CD19 to the Fab end of CD3 are named: LL207-3 and LL207-4. The bispecific antibodies of the "2+1 structure" constructed by placing the Fab end of CD3 on the outside, that is, connecting the Fab end of CD19 to the Fc end, and then connecting the Fab end of CD3 to the Fab end of CD19 are named: LL207-5 and LL207-6.

[0222] The amino acid sequence of LL207-3 is:

[0223] Heavy chain 1: SEQ ID NO: 15

[0224] Heavy chain 2: SEQ ID NO: 17

[0225] QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0226] Light chain: SEQ ID NO:8

[0227] The amino acid sequence of LL207-4 is:

[0228] Heavy chain 1: SEQ ID NO:15

[0229] Heavy chain 2: SEQ ID NO:17

[0230] Light chain: SEQ ID NO:6

[0231] The amino acid sequence of LL207-5 is:

[0232] Heavy chain 1: SEQ ID NO:15

[0233] Heavy chain 2: SEQ ID NO:18

[0234] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0235] Light chain: SEQ ID NO:8

[0236] The amino acid sequence of LL207-6 is:

[0237] Heavy chain 1: SEQ ID NO:15

[0238] Heavy chain 2: SEQ ID NO:18

[0239] Light chain: SEQ ID NO:6

[0240] 3. Design of bispecific antibodies targeting CD38 and CD3

[0241] First, a monoclonal antibody was designed in which the CD38 antibody Daratumumab and the CD3 antibody Foralumab exchanged light chains to verify whether the CD38 antibody and the CD3 antibody still maintained affinity for the target after exchanging light chains. At the same time, the light chain with better affinity was selected as the candidate light chain for constructing a bispecific antibody.

[0242] The monoclonal antibody composed of CD38 heavy chain and CD3 light chain was named as LL208-BM2.

[0243] The heavy chain is: SEQ ID NO: 9

[0244] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0245] Light chain: SEQ ID NO: 4

[0246] The monoclonal antibody composed of CD3 heavy chain and CD38 light chain was named as LL208-BM3.

[0247] The heavy chain is: SEQ ID NO: 3

[0248] Light chain: SEQ ID NO: 10

[0249] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0250] Meanwhile, CD38 antibody Daratumumab was prepared as a control antibody and named as LL208-BM1.

[0251] By exchanging light chains, bispecific antibodies targeting CD38 and CD3 were constructed. At the same time, Knobs-into-holes technology was used to construct "1+1 structure" and "2+1 structure" bispecific antibodies targeting CD38 and CD3.

[0252] The bispecific antibodies with a "1+1 structure" are named LL208-1 and LL208-2. The heavy chain of the CD38 antibody Daratumumab is a knot chain, and the heavy chain of the antibody Foralumab is a hole chain. LL208-1 uses the light chain of the CD38 antibody Daratumumab, and LL208-2 uses the light chain of the antibody Foralumab.

[0253] The amino acid sequence of LL208-1 is:

[0254] Heavy chain 1: SEQ ID NO: 19

[0255] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0256] Heavy chain 2: SEQ ID NO:20

[0257] QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0258] Light chain: SEQ ID NO: 10

[0259] The amino acid sequence of LL208-2 is:

[0260] Heavy chain 1: SEQ ID NO: 19

[0261] Heavy chain 2: SEQ ID NO: 20

[0262] Light chain: SEQ ID NO: 4

[0263] In the "2+1 structure", two antigen binding fragments (Fab) of the CD38 antibody Daratumumab are used, and the two variable regions are connected by a linker: 4*(GGGS). Among them, on the basis of LL208-1 and LL208-2, the Fab end of CD38 is connected to the Fab end of CD3 to construct the 2+1 structure bispecific antibodies named: LL208-3 and LL208-4. The "2+1 structure" bispecific antibodies constructed by placing the Fab end of CD3 on the outside, that is, connecting the Fab end of CD38 to the Fc end, and then connecting the Fab end of CD3 to the Fab end of CD38 are named: LL208-5 and LL208-6.

[0264] The amino acid sequence of LL208-3 is:

[0265] Heavy chain 1: SEQ ID NO: 19

[0266] Heavy chain 2: SEQ ID NO: 21

[0267] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0268] Light chain: SEQ ID NO: 10

[0269] The amino acid sequence of LL208-4 is:

[0270] Heavy chain 1: SEQ ID NO: 19

[0271] Heavy chain 2: SEQ ID NO: 21

[0272] Light chain: SEQ ID NO: 4

[0273] The amino acid sequence of LL208-5 is:

[0274] Heavy chain 1: SEQ ID NO: 19

[0275] Heavy chain 2: SEQ ID NO:22

[0276] QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0277] Light chain: SEQ ID NO:10

[0278] The amino acid sequence of LL208-6 is as follows:

[0279] Heavy chain 1: SEQ ID NO:19

[0280] Heavy chain 2: SEQ ID NO:22

[0281] Light chain: SEQ ID NO:4.

[0282] The structure of the above bispecific antibody is as Figure 1 shown.

[0283] Example 3. Expression and purification of bispecific antibodies

[0284] According to the nucleic acid sequence table of the above-mentioned bispecific antibody, the relevant bispecific antibody gene sequence is synthesized, and the gene sequence of the bispecific antibody is constructed into the antibody expression vector using molecular cloning technology. After the vector is successfully constructed, the above-mentioned bispecific antibody is expressed using the 293 protein expression system.

[0285] Basic steps: Clone the bispecific antibody gene into the expression vector pCDNA4 (Invitrogen, CatV86220). Use the transient method to transfect the vectors carrying the bispecific antibody gene sequence into HEK293 suspension cells. Place the cells in a cell culture incubator for suspension culture. After the expression is completed, the cell culture supernatant is collected, and the bispecific antibody is purified using a Protein A affinity chromatography column. After the bispecific antibody is purified, the protein concentration and total protein amount of different antibodies are detected by ultraviolet spectrophotometry, and then the expression amount of each bispecific antibody is calculated based on the expression volume. Specifically, NanoDrop 1000 is used to read the sample solution absorbance value A280 at a wavelength of 280nm, and the protein concentration of different samples is calculated by the formula C (mg / mL) = A280 / ε (ε is 1.482mL / mg·cm-1).

[0286] The purity of different antibodies was evaluated by using gel electrophoresis SDS-PAGE. The basic steps are as follows: The sample was separated by electrophoresis. The sample was diluted to about 1 mg / mL, and an appropriate amount of loading buffer was added. After mixing, it was heated at 90°C for about 3 minutes. The loading amount was 2 to 10 μg. After the electrophoresis was completed, staining and decolorization were performed according to the SDS-PAGE standard process. After decolorization, a gel imaging system was used to take pictures and analyze and calculate the purity of the main band.

[0287] In addition, the purity of different antibodies was evaluated by using molecular exclusion high performance liquid chromatography (SEC-HPLC). The basic steps are as follows: dilute the sample to about 1.0 mg / mL, use a TSKgel G3000SWXL column, set the column temperature to 25°C, inject a volume of 30 to 50 μL, and elute isocratically for 20 min at a flow rate of 1.0 mL / min. Detect at a wavelength of 280 nm, and use the peak area normalization method to obtain the monomer content.

[0288] The expression level and purity information of CD3×CD20 bispecific antibodies are shown in Table 1. LL206-BM1 is ofatumumab, and LL206-BM2 is foralumab. The results of SEC-HPLC and SDS-PAGE showed that the structures of the two newly assembled monoclonal antibodies LL206-BM3, which were assembled with the light chain of foralumab and the heavy chain of ofatumumab, or the monoclonal antibody LL206-BM4, which were assembled with the light chain of ofatumumab and the heavy chain of foralumab, were complete, and the purity and expression level were close to the original parent antibody molecules. Subsequently, ofatumumab and the heavy chain of foralumab were assembled using knob into hole, and then assembled with the light chain of ofatumumab or foralumab to form the common light chain bispecific antibodies LL206-1 and LL206-2, respectively. The results of SEC-HPLC and SDS-PAGE showed that the structures of LL206-1 and LL206-2 were complete and the purity was high, but the expression level of LL206-2 was low. The above results demonstrate that the CD3×CD20 common light chain bispecific antibody of the present invention has a correct structure and a high purity.

[0289] Table 1. Expression level and purity of CD3×CD20 bispecific antibodies

[0290]

[0291] The expression level and purity information of CD3×CD19 bispecific antibodies are shown in Table 2. The results of SEC-HPLC and SDS-PAGE of the monoclonal antibody LL207-BM3 assembled with the light chain of UCHT and the heavy chain of FMC or the monoclonal antibody LL206-BM4 assembled with the light chain of FMC and the heavy chain of UCHT prove that the structures of the two newly assembled monoclonal antibodies are intact, and the purity and expression level are close to the original parent antibody molecules. Subsequently, the heavy chains of FMC and UCHT were assembled using knob into hole, and then assembled with the light chains of FMC or UCHT to form common light chain bispecific antibodies LL207-1 and LL207-2, respectively. The results of SEC-HPLC and SDS-PAGE prove that the structures of LL207-1 and LL207-2 are intact and have high expression levels, but SEC-HPLC is slightly reduced. Therefore, these results prove that the CD3×CD19 shared light chain bispecific antibody of the present invention has a correct structure and a high expression level.

[0292] Table 2. Expression level and purity of CD3×CD19 bispecific antibodies

[0293]

[0294] The expression level and purity information of CD3×CD38 bispecific antibodies are shown in Table 3. For the monoclonal antibody LL208-BM2 assembled with the light chain of Foralumab and the heavy chain of Daratumumab, the results of SEC-HPLC and SDS-PAGE proved that the structures of the two newly assembled monoclonal antibodies were intact, and the purity and expression level were close to the original parent antibody molecules. Therefore, Daratumumab and Foralumab heavy chains were assembled using knob into hole, and Foralumab light chain could be assembled with them to form a CD3×CD38 shared light chain bispecific antibody.

[0295] Table 3. Expression level and purity of CD3×CD38 bispecific antibodies

[0296]

[0297] From the above results, it can be seen that the expression level and purity of the bispecific antibodies targeting CD3 and CD20, CD3 and CD19, and CD3 and CD38 constructed by us are good, and can be used for further biological function screening and evaluation.

[0298] Example 4. Detection of target binding activity of bispecific antibodies

[0299] Flow cytometry was used to detect the activity of different antibodies binding to targets at the cellular level and evaluate the binding ability of bispecific antibodies to protein antigens expressed on the cell surface. The basic steps are as follows:

[0300] 1. Prepare cells: Take appropriate amount of Raji (cell bank of Shanghai Institute of Biochemistry and Cell Biology) and T cells (Shanghai Miaotong Biotechnology), adjust the cell density to 2E6 / ml, and dispense into 1.5ml EP tubes, 50ul (about 1E5 cells) per tube;

[0301] 2. Antibody dilution: Determine the starting concentration of the antibody according to the experimental requirements (the starting concentration of LL-206-BM1, LL206-BM2, LL-206-BM3, LL206-BM4, LL207-BM1, LL207-BM2, LL-206-BM3, LL206-BM4 is 76.8ug / ml, and the starting concentration of LL206-1, LL206-2, LL207-1, LL207-2 is 153.6ug / ml), adjust the original antibody to the starting concentration with PBS, dilute it 2 times, and set up 8 concentration gradients (LL-206-BM1, LL206-BM2, LL-206-BM3, LL206- The concentration gradient of BM4, LL207-BM1, LL207-BM2, LL-206-BM3, and LL206-BM4 is 4.8ug / ml, 2.4ug / ml, 1.2ug / ml, 0.6ug / ml, 0.3ug / ml, 0.15ug / ml, 0.075ug / ml, and 0.0375ug / ml; the concentration gradient of LL206-1, LL206-2, LL207-1, and LL207-2 is 9.6ug / ml, 4.8ug / ml, 2.4ug / ml, 1.2ug / ml, 0.6ug / ml, 0.3ug / ml, 0.15ug / ml, and 0.075ug / ml);

[0302] 3. Antibody incubation: Take 50ul of diluted antibody and add it to the previously aliquoted cells, mix well by pipetting, and incubate at 4℃ in the dark for 30min;

[0303] 4. Antibody washing: Add 1 ml of wash buffer (1x PBS + 0.5% FBS) to the incubated antibody-cell mixture, centrifuge at 2000 rpm for 5 min, and discard the supernatant;

[0304] 5. Secondary antibody incubation: dilute the secondary antibody anti-human IgG Fc-APC (BD Biosciences) at 1:200 with PBS, take 50ul and add it to the cell pellet after discarding the supernatant, pipette and mix, and incubate at 4℃ in the dark for 30min;

[0305] 6. Secondary antibody washing: add 1 ml wash buffer (1x PBS + 0.5% FBS) to the incubated antibody-cell mixture, centrifuge at 2000 rpm for 5 min, and discard the supernatant;

[0306] 7. Detection on the flow cytometer: Add 200ul wash buffer to each tube for resuspending, transfer to the flow cytometer tube, and detect on the flow cytometer.

[0307] from Figure 2It can be seen that LL206-BM1, LL207-BM1 and the antibodies with interchanged light chains LL206-BM3 and LL207-BM3 can all bind to Raji cells very well. The binding abilities of LL206-BM1 and LL206-BM3 to Raji cells are similar, and the binding abilities of LL207-BM1 and LL207-BM3 to Raji cells are similar, indicating that the interchange of light chains has little effect on the binding of CD20 and CD19 targets.

[0308] from Figure 3 It can be seen that the bispecific antibody LL206-1 can bind to Raji cells very well, with an affinity of about 1 / 3 of the parent monoclonal antibody LL206-BM1, and EC50s of 3.50 and 1.12 (ug / ml), respectively. However, LL206-2 has a very weak binding ability to Raji cells. This indicates that different light chain combinations seriously affect the affinity of bispecific antibodies to CD20. Both bispecific antibodies LL207-1 and LL207-2 can bind to Raji cells very well, with EC50s of 2.23 and 2.41 (ug / ml), respectively, which is about half of the parent monoclonal antibody LL207-BM1. This indicates that for the binding of LL207-1 and LL207-2 to CD19, the light chains of the two antibodies are not much different.

[0309] from Figure 2 It can be seen that in terms of binding to T cells, LL206-BM2, LL207-BM2 and the antibodies with interchanged light chains LL206-BM4 and LL207-BM4 can all bind to T cells very well.

[0310] from Figure 3 It can be seen that the affinity EC50 of LL206-BM2, LL206-1 and LL206-2 for T cells is approximately 1.81, 12.1 and 12.8 (ug / ml), respectively. The affinity of LL206-1 and LL206-2 for T cells is similar, which is about 6 times lower than that of the parent antibody, reducing the probability of non-specific activation of T cells to a certain extent, suggesting that the two bispecific antibodies have better safety. The affinity EC50 of LL207-BM2, LL207-1 and LL207-2 for T cells is approximately 1.92, 4.65 and 9.32 (ug / ml), respectively. The affinity of LL207-1 for T cells is still very high, which is about 2 times lower than that of the parent antibody. The affinity of LL207-2 is about 5 times lower than that of the parent antibody, which reduces the probability of non-specific activation of T cells to a certain extent, suggesting better safety.

[0311] In summary, the bispecific antibodies LL206-1 and LL207-2 of the present invention have the advantage of better affinity for both targets and T cells, and have greater potential for subsequent development.

[0312] Example 5. Detection of bispecific antibody-mediated T cell secretion of cytokines

[0313] The bispecific antibody of the present invention can bind to T cells and target cells simultaneously, mediate T cells to recognize and kill target cells and secrete cytokines such as IL-2 and IFN-g.

[0314] The basic steps for testing bispecific antibody-mediated T cell cytokine secretion are as follows:

[0315] 1. Cell co-culture. Use 1640 complete medium to dilute antibodies LL206-BM2, LL206-1, LL206-2, LL207-BM2, LL207-1, LL207-2 to 4 concentration gradients: 9.6ug / ml, 2.4ug / ml, 0.6ug / ml, 0.15ug / ml for later use. Adjust the T cell concentration to 5×10 5 / ml, then seed 100ul of T cells into each well of a 96-well plate and temporarily incubate at 37°C. Use the diluted antibodies of four concentration gradients to adjust the Raji cell concentration to 5×10 5 / ml, and then seed Raji cells into 96-well plates containing T cells at 100ul per well. The final ratio of T cells to tumor cells is 1:1. The final concentrations of each antibody are: 4.8ug / ml, 1.2ug / ml, 0.3ug / ml, and 0.075ug / ml. After thorough mixing, centrifuge at 500rpm for 3 minutes. Incubate the cells at 37°C for 24 hours;

[0316] 2. Cytokine secretion detection. After incubation, the supernatant was taken and transferred to a new round-bottom 96-well plate. The secretion of IFN-γ and IL-2 cytokines in T cells was detected using an ELISA kit (Thermo Fisher Scientific; Cat. No. 88-7316). Plate preparation and supernatant cytokine detection were performed according to the procedures provided in the kit.

[0317] The results showed that both bispecific antibodies LL206-1 and LL206-2 could effectively mediate T cell killing of Raji tumor cells and secrete a large amount of cytokines ( Figure 4 ). The pure maternal antibody does not have this function. Taking 0.3ug / ml as an example, the IFN-g secretion in the LL206-1, LL206-2, LL207-1 and LL207-2 groups were 2130, 450, 568 and 223 (pg / ml), respectively. The negative control group did not secrete IFN-g ( Figure 4A). The results show that LL206-1 has better efficacy, and LL206-2 requires a higher dose to achieve the same effect as LL206-1. LL207-1 and LL207-2 can also mediate T cell killing of Raji tumor cells, but the effect is weaker than LL206-1, and a larger dose is required to achieve a better efficacy.

[0318] Example 6. Bispecific Antibodies Mediate T Cell Killing of Tumor Cells

[0319] 1. Cell co-culture. Raji cells were transfected with luciferase-carrying lentivirus to prepare a Raji cell line labeled with luciferase, labeled as Raji.luc. Raji.luc was cultured at a cell concentration of 1×10 5 / ml, 50ul / well were plated in a 96-well flat-bottom opaque white plate and incubated at 37°C. Antibodies LL206-BM2, LL206-1, LL206-2, LL207-BM2, LL207-1, and LL207-2 were diluted with culture medium to 4 concentration gradients: 9.6ug / ml, 2.4ug / ml, 0.6ug / ml, and 0.15ug / ml. The T cell concentration was adjusted to 1×10 5 / ml, and then seed 50ul of T cells per well into a 96-well flat-bottom opaque white plate containing Raji.luc cells. Mix thoroughly and centrifuge at 500rpm for 3 minutes. Incubate the cells at 37℃ for 24 hours.

[0320] 2. Killing detection. After 24 hours of cell co-culture, the remaining luciferase activity (relative light unit, RLU) of the target cells was measured to detect the killing ability of T cells on target cells in the presence of different specific antibodies. The specific steps are:

[0321] After co-culture, the cells were centrifuged at 800 rpm for 5 min, the supernatant was removed, 100 ul of D-luciferin substrate (Thermo Fisher Scientific: 88293) was added, mixed and protected from light for 5 min, and the fluorescence intensity was detected in the chemiluminescence mode of the microplate reader. Since luciferase is only expressed in target cells, the remaining luciferase activity in the well is directly related to the number of live target cells in the well. In the absence of effector cells and antibodies, the maximum luciferase activity was obtained by adding culture medium to target cells as a control.

[0322] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Liling Biopharmaceuticals (Suzhou) Co., Ltd. <120> Bispecific antibodies and preparation methods and applications thereof <130> P2021-2780 <160> 66 <170> PatentIn version 3.5 <210> 1 <211> 452 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Pro Gly Lys Serum 450 <210> 2 <211> 214 <212> PRT <213> artificial sequence <220> <223> Synthesized multipeptide <400> 2 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 3 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 4 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val 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 210 215 <210> 5 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Light 450 <210> 6 <211> 214 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 7 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 7 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 Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 8 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 9 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Pro Gly Lys Serum 450 <210> 10 <211> 214 <212> PRT <213> artificial sequence <220> <223> Synthesized multipeptide <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 11 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 12 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 12 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 355 360 365 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 13 <211> 684 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser 210 215 220 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu 225 230 235 240 Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu 245 250 255 Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr Gly Met His Trp 260 265 270 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp 275 280 285 Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe 290 295 300 Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 305 310 315 320 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Met 325 330 335 Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val 340 345 350 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 355 360 365 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 370 375 380 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 385 390 395 400 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 405 410 415 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 420 425 430 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 435 440 445 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 450 455 460 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 465 470 475 480 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 485 490 495 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 500 505 510 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 515 520 525 Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr 530 535 540 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 545 550 555 560 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 565 570 575 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 580 585 590 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly 595 600 605 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 610 615 620 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 625 630 635 640 Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 645 650 655 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg 660 665 670 Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 675 680 <210> 14 <211> 684 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 14 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser Gly Gly Gly Ser 210 215 220 Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 225 230 235 240 Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala 245 250 255 Ser Gly Phe Thr Phe Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala 260 265 270 Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly 275 280 285 Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 290 295 300 Asp Asn Ala Lys Lys Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 305 310 315 320 Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn 325 330 335 Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 340 345 350 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 355 360 365 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 370 375 380 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 385 390 395 400 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 405 410 415 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 420 425 430 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 435 440 445 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 450 455 460 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 465 470 475 480 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 485 490 495 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 500 505 510 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 515 520 525 Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr 530 535 540 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 545 550 555 560 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 565 570 575 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 580 585 590 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly 595 600 605 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 610 615 620 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 625 630 635 640 Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 645 650 655 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg 660 665 670 Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 675 680 <210> 15 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 15 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 16 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 16 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 Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 17 <211> 686 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser Gly Gly 210 215 220 Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu Val Gln 225 230 235 240 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 245 250 255 Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg 260 265 270 Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr 275 280 285 Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Ala Thr Leu 290 295 300 Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu 305 310 315 320 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr 325 330 335 Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val 340 345 350 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 355 360 365 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 370 375 380 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 385 390 395 400 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 405 410 415 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 420 425 430 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 435 440 445 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 450 455 460 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 465 470 475 480 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 485 490 495 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 500 505 510 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 515 520 525 Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val 530 535 540 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 545 550 555 560 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 565 570 575 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 580 585 590 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 595 600 605 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 610 615 620 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 625 630 635 640 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 645 650 655 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 660 665 670 Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 675 680 685 <210> 18 <211> 686 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 18 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 Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser 210 215 220 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu 225 230 235 240 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu 245 250 255 Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp 260 265 270 Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp 275 280 285 Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr 290 295 300 Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu Lys Leu Ser Ser 305 310 315 320 Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr 325 330 335 Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 340 345 350 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 355 360 365 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 370 375 380 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 385 390 395 400 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 405 410 415 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 420 425 430 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 435 440 445 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 450 455 460 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 465 470 475 480 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 485 490 495 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 500 505 510 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 515 520 525 Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val 530 535 540 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 545 550 555 560 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 565 570 575 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 580 585 590 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 595 600 605 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 610 615 620 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 625 630 635 640 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 645 650 655 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 660 665 670 Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 675 680 685 <210> 19 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 19 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 20 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 20 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 355 360 365 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 21 <211> 684 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 21 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser 210 215 220 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu 225 230 235 240 Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu 245 250 255 Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr Gly Met His Trp 260 265 270 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp 275 280 285 Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe 290 295 300 Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 305 310 315 320 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Met 325 330 335 Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val 340 345 350 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 355 360 365 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 370 375 380 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 385 390 395 400 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 405 410 415 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 420 425 430 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 435 440 445 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 450 455 460 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 465 470 475 480 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 485 490 495 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 500 505 510 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 515 520 525 Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr 530 535 540 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 545 550 555 560 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 565 570 575 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 580 585 590 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly 595 600 605 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 610 615 620 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 625 630 635 640 Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 645 650 655 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg 660 665 670 Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 675 680 <210> 22 <211> 684 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 22 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser Gly Gly Gly Ser 210 215 220 Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly 225 230 235 240 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val 245 250 255 Ser Gly Phe Thr Phe Asn Ser Phe Ala Met Ser Trp Val Arg Gln Ala 260 265 270 Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly 275 280 285 Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 290 295 300 Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 305 310 315 320 Glu Asp Thr Ala Val Tyr Phe Cys Ala Lys Asp Lys Ile Leu Trp Phe 325 330 335 Gly Glu Pro Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 340 345 350 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 355 360 365 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 370 375 380 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 385 390 395 400 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 405 410 415 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 420 425 430 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 435 440 445 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 450 455 460 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 465 470 475 480 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 485 490 495 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 500 505 510 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 515 520 525 Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr 530 535 540 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 545 550 555 560 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 565 570 575 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 580 585 590 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly 595 600 605 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 610 615 620 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 625 630 635 640 Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 645 650 655 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg 660 665 670 Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 675 680 <210> 23 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 24 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 24 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 25 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 25 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 26 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 26 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 27 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 27 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 28 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 28 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 29 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 29 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 100 105 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 30 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 31 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 31 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Phe Ser 115 120 <210> 32 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 32 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 Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 33 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 33 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 34 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 35 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 35 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 36 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 37 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 37 Gly Phe Thr Phe Asn Asp Tyr Ala Met His 1 5 10 <210> 38 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 38 Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 39 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 39 Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val 1 5 10 <210> 40 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 40 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 41 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 42 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 42 Gln Gln Arg Ser Asn Trp Pro Ile Thr 1 5 <210> 43 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 43 Gly Phe Lys Phe Ser Gly Tyr Gly Met His 1 5 10 <210> 44 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 44 Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val Lys 1 5 10 15 Gly <210> 45 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 45 Gln Met Gly Tyr Trp His Phe Asp Leu 1 5 <210> 46 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 46 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 47 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 47 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 48 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 48 Gln Gln Arg Ser Asn Trp Pro Pro Leu Thr 1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 49 Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 1 5 10 <210> 50 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 50 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 <210> 51 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 51 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 1 5 <210> 52 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 52 Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn 1 5 10 <210> 53 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 53 His Thr Ser Arg Leu His Ser 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 54 Gln Gln Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 55 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 55 Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn 1 5 10 <210> 56 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 56 Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 57 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 57 Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val 1 5 10 <210> 58 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 58 Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn 1 5 10 <210> 59 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 59 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 60 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 60 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 61 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 61 Gly Phe Thr Phe Asn Ser Phe Ala Met Ser 1 5 10 <210> 62 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 62 Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 63 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 63 Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr 1 5 10 <210> 64 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 64 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 65 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 65 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 66 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 66 Gln Gln Arg Ser Asn Trp Pro Pro Thr 1 5

Claims

1. A bispecific antibody, characterized in that: The bispecific antibody is composed of a heavy chain 1, a heavy chain 2 and a light chain, and the bispecific antibody is selected from the following antibodies: 。 2. An immunoconjugate, characterized in that: The immunoconjugate is composed of: (a) the bispecific antibody according to claim 1; and (b) a conjugation moiety selected from the group consisting of a detectable label, gold nanoparticles / nanorods, nanomagnetic particles or a combination thereof.

3. The immunoconjugate according to claim 2, characterized in that The detectable label is a radionuclide.

4. Use of the bispecific antibody according to claim 1 or the immunoconjugate according to claim 2 or 3 in the preparation of an anti-tumor drug, wherein the tumor is lymphoma, leukemia, or multiple myeloma.

5. A pharmaceutical composition comprising the bispecific antibody according to claim 1 or the immunoconjugate according to claim 2 or 3 and a pharmaceutically acceptable excipient.

6. An isolated polynucleotide, characterized in that The polynucleotide encodes the bispecific antibody according to claim 1.

7. A carrier, characterized in that The vector contains the polynucleotide according to claim 6.

8. A host cell, characterized in that The host cell contains the vector according to claim 7, or the polynucleotide according to claim 6 is integrated into its genome; Alternatively, the host cell expresses the bispecific antibody according to claim 1.

9. A method for producing the bispecific antibody according to claim 1, characterized in that: Includes steps: (a) culturing the host cell according to claim 8 under suitable conditions to obtain a culture containing the bispecific antibody; and (b) purifying and / or isolating the culture obtained in step (a) to obtain the bispecific antibody.

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