CN115583995A - Bispecific antibody, preparation method and application thereof - Google Patents

Abstract

The invention discloses T cell engager bispecific antibodies targeting tumor antigens and T cell antigens and applications thereof. The bispecific antibodies target 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 parental antibody and can produce ideal biological effects, thereby laying a new material basis for the concept of antitumor drugs.

Description

Bispecific antibody and its preparation method and application

technical field

The present invention relates to the field of biotechnology. More specifically, the present invention relates to a bispecific antibody, especially a bispecific antibody used as a T cell engager, and its preparation method and application.

Background technique

Bispecific antibodies are genetically engineered antibodies that have been artificially engineered. Bispecific antibodies can simultaneously target two different antibody-binding epitopes, which can be from different antigens or from the same antigen. Many current studies have shown that bispecific antibodies have shown 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 steric effect of two recognition epitopes and the synergistic effect of dual targeting, resulting in biological effects that cannot be achieved by the combined use of two antibodies.

Due to the complexity of bispecific antibodies, which are different from monoclonal antibodies, the technical threshold and research and development costs are relatively high. In the process of developing bispecific antibodies, 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 generally consist of four chains, two heavy chains and two light chains. If traditional antibodies are used to construct bispecific antibodies, the problem of light chain and heavy chain mismatches will arise. In addition, whether the constructed bispecific antibody can have the affinity of the parental antibody and produce the desired biological effect is still unknown.

Therefore, there is an urgent need in the field for bispecific antibodies that are easy to prepare, have the affinity of the parental antibody, and produce ideal biological effects.

Contents of the invention

The purpose 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 can produce ideal biological effects at the same time.

In a first aspect, the present invention provides a bispecific antibody consisting of an antibody specifically binding to a tumor antigen and an antibody specifically binding to a T cell antigen.

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

In a specific embodiment, said tumor antigen is CD20, CD19 or CD38, and said T cell antigen is CD3.

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

In a specific embodiment, the bispecific antibody comprises a heavy chain and a light chain, the heavy chain is respectively derived from an antibody specifically binding to a tumor antigen and an antibody specifically binding to a T cell antigen, and the light chain is respectively derived from Antibodies that specifically bind to tumor antigens and antibodies that specifically bind to T cell antigens;

Preferably, the heavy chains are derived from antibodies specifically binding to tumor antigens and antibodies specifically binding to T cell antigens, respectively, and the light chains are derived from antibodies specifically binding to tumor antigens or antibodies specifically binding to T cell antigens.

In a preferred embodiment, said bispecific antibody comprises two heavy chains and two light chains.

In a specific embodiment, the heavy chain variable region in the Fab fragment of the antibody is optionally linked to the CH1 region of the Fab fragment of the antibody that specifically binds a tumor antigen or the T cell antigen specific binding The CH1 region of the Fab fragment of the antibody is further connected;

Preferably, the Fab fragment of the antibody is derived from the heavy chain variable region of the antibody specifically binding to a T cell antigen via an optional linker with the CH1 region of the Fab fragment of the antibody specifically binding to a tumor antigen Connect further.

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

In a specific embodiment, the Fab fragment of the antibody exchanges positions with the Fab fragment of the antibody specifically binding to a tumor antigen or the Fab fragment of the antibody specifically binding to a T cell antigen.

In a specific embodiment, the light chain CDR1-3 of the antibody specifically binding to CD20 is respectively shown in SEQ ID NO:40-42, and the heavy chain CDR1-3 is respectively shown in SEQ ID NO:37-39;

The light chain CDR1-3 of the antibody specifically binding to CD19 is respectively shown in SEQ ID NO:52-54, and the heavy chain CDR1-3 is respectively shown in SEQ ID NO:49-51;

The light chain CDR1-3 of the antibody specifically binding to CD38 is respectively shown in SEQ ID NO:64-66, and the heavy chain CDR1-3 is respectively shown in SEQ ID NO:61-63;

The light chain CDR1-3 of the antibody specifically binding to CD3 is shown in SEQ ID NO: 46-48 or SEQ ID NO: 58-60 respectively, and the heavy chain CDR1-3 is shown in SEQ ID NO: 43-45 or SEQ ID NO: 43-45 or SEQ ID NO: 58-60 respectively. ID NO:55-57;

Preferably, the light chain CDR1-3 of the antibody specifically binding to CD3 is respectively shown in SEQ ID NO:46-48, and the heavy chain CDR1-3 is respectively shown in SEQ ID NO:43-45; or the specific The light chain CDR1-3 of the antibody binding to CD3 is respectively shown in SEQ ID NO:58-60, and the heavy chain CDR1-3 is respectively shown in SEQ ID NO:55-57.

In a preferred embodiment, the light chain variable region of the antibody specifically binding to CD20 is shown in SEQ ID NO:23, and the heavy chain variable region is shown in SEQ ID NO:24;

The light chain variable region of the antibody specifically binding 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;

Preferably, the light chain variable region of the antibody specifically binding to CD19 is shown in SEQ ID NO: 29, and the heavy chain variable region is shown in SEQ ID NO: 27, or, the antibody specifically binding to CD19 The variable region of the light chain is shown in SEQ ID NO:30, and the variable region of the heavy chain is shown in SEQ ID NO:28;

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

The light chain variable region of the antibody specifically binding 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;

Preferably, the light chain variable region of the antibody specifically binding to CD3 is shown in SEQ ID NO: 26, and the heavy chain variable region is shown in SEQ ID NO: 25; or the antibody specifically binding to CD3 The light chain variable region is shown in SEQ ID NO:33, the heavy chain variable region is shown in SEQ ID NO:31; or the light chain variable region of the antibody specifically binding to CD3 is shown in SEQ ID NO:34 , the heavy chain variable region is shown in SEQ ID NO:32.

In a preferred embodiment, the light chain of the antibody specifically binding to CD20 is shown in SEQ ID NO: 2, and the heavy chain is shown in SEQ ID NO: 1;

The light chain of the antibody specifically binding to CD19 is shown in SEQ ID NO:6, and the heavy chain is shown in SEQ ID NO:5;

The light chain of the antibody specifically binding to CD38 is shown in SEQ ID NO: 10, and the heavy chain is shown in SEQ ID NO: 9;

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

Preferably, the light chain of the antibody specifically binding to CD3 is shown in SEQ ID NO: 4, and the heavy chain is shown in SEQ ID NO: 3; or the light chain of the antibody specifically binding to CD3 is shown in SEQ ID NO: 8, the heavy chain is shown in SEQ ID NO:7.

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

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

(a) the bispecific antibody described in the first or second aspect; and

(b) a coupling moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme, gold nanoparticles/nanorods, nanomagnetic particles, viral coat protein or VLP, or combination.

In the fourth aspect, the present invention provides the 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.

In a preferred embodiment, the tumor is a tumor specifically expressing CD20, CD19 or CD38.

In preferred embodiments, the tumor is lymphoma, leukemia, multiple myeloma, and the like.

In the 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 excipients.

In a preferred embodiment, the pharmaceutical composition also contains other drugs for treating tumors, such as cytotoxic drugs.

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

In a preferred embodiment, the other drugs are used to block the interaction between PD-1 and PD-L1, and at the same time block the interaction between other targets and their receptors or ligands.

In a preferred embodiment, the pharmaceutical composition is in the form of injection.

In a preferred embodiment, the pharmaceutical composition is used for preparing drugs for preventing and treating tumors.

In the 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.

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

In the seventh aspect, the present invention provides a host cell, which is 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;

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

In the eighth aspect, the present invention provides a method for producing the bispecific antibody described in the first or second aspect, characterized by comprising the steps of:

(a) cultivating the host cell according to the seventh aspect under suitable conditions, so as to obtain a culture containing the bispecific antibody; and

(b) purifying and/or separating the culture obtained in step (a) to obtain the bispecific antibody.

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 in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

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

Fig. 2 has shown the affinity detection result of the monoclonal antibody of the present invention that exchanges light chain to Raji cell and T cell;

Figure 3 shows the affinity test results of the bispecific antibody of the present invention to Raji cells and T cells;

Figure 4 shows that the bispecific antibody of the present invention can mediate T cells to recognize tumor cells and release cytokines.

detailed description

In the extensive research and screening of T cell engager bispecific antibodies, the inventors unexpectedly found that a specific monoclonal antibody against a specific tumor cell target and a T cell target can be used as a parent in the bispecific antibody The antibody and the bispecific antibody calculated in this way are not only easy to prepare, but also have the affinity of the parent antibody and produce ideal biological effects, so that they can be used as a new anti-tumor drug. The present invention has been accomplished on this basis.

T cell engager

"T cell engager" as used herein has the meaning commonly understood by those skilled in the art. The T cell engager (T cell engager) can activate endogenous T cells by bispecifically binding targets on the surface of tumor cells and T cells, leading to the lysis of tumor cells, thereby achieving the purpose of treating tumors. Germany's Micromet (later acquired by Amgen) was one of the first companies to develop the BiTE (Bispecific T cell engager) platform. BiTE is formed by linking two scFv (Single-chain variable fragment) with a linker peptide. Thus, these molecules are bivalent, monovalent for each antigen, and typically have a molecular weight of 50-60 KDa. 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, becoming the first BiTE drug approved by the FDA. Blinatumomab targets both CD19 and CD3 proteins. CD19 is expressed on the surface of tumor cells in acute lymphoblastic leukemia and lymphoma, and serves as a target for tumor cells. CD3 includes multiple subunits (d, g and e), which are important surface markers of T cells. CD3 subunits can bind to TCR non-covalently to form a TCR-CD3 complex. The CD3 antibody can generally recognize the ε chain of the CD3 complex, transmit the activation signal generated after the TCR binds to the antigen into the cell, induce the activation of T cells, and then kill tumor cells. T cell engagers have been proven to be used to treat tumors, and subsequent T cell engagers targeting other targets: CD20, BCMA and HER2 are already in the clinical research stage.

Bispecific antibody of the present invention

Herein, "antibody of the present invention" has the same meaning as "bispecific antibody of the present invention".

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 targeting both tumor antigens and T cell antigens.

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

The T cell engager bispecific antibody of the present invention is a T cell engager constructed by 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 combined bispecific antibodies 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 traditional antibodies, so it has great advantages in druggability.

Therefore, in a specific embodiment, the light chain of the antibody specifically binding to a tumor antigen and the antibody specifically binding to a T cell antigen in the T cell engager bispecific antibody of the present invention has more than 90%, preferably 95%. Above, more preferably above 98% sequence similarity.

The bispecific antibody of the present invention comprises a heavy chain and a light chain. The heavy chain can be derived from an antibody specifically binding to a tumor antigen and an antibody specifically binding to a T cell antigen, and the light chain can also be derived from an antibody specifically binding to a tumor antigen. The antibody and the antibody that specifically binds a T cell antigen; preferably the light chains are the same, ie the light chain is either derived from an antibody that specifically binds a tumor antigen or from an antibody that specifically binds a T cell antigen.

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 optionally linked with CH1 of the Fab fragment of the antibody specifically binding to a tumor antigen, for example (GGGS) ₄ region or the CH1 region of the Fab fragment of the antibody that specifically binds a T cell antigen is further linked.

In a preferred embodiment, the Fab fragment of the antibody is derived from the heavy chain variable region of the antibody that specifically binds to a T cell antigen via an optional linker with the Fab of the antibody that specifically binds to a tumor antigen The CH1 regions of the fragments are further ligated.

In another preferred embodiment, the Fab fragment of the antibody exchanges positions with the Fab fragment of the antibody specifically binding to a tumor antigen or the Fab fragment of the antibody specifically binding to a T cell antigen.

On the basis of 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 an expression vector or A host cell whose genome integrates the polynucleotide or expresses the bispecific antibody.

Those skilled in the art know 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 Vectors include viral vectors, such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof. Those skilled in the art are also aware of the types of host cells used to practice the present invention, including but not limited to: prokaryotic cells or eukaryotic cells; preferably E. coli, yeast cells or mammalian cells.

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, they can culture the host cells under appropriate conditions to obtain a culture containing the bispecific antibody. After obtaining the culture, the obtained culture can be purified and/or isolated, so as to obtain the bispecific antibody. In a preferred embodiment, the purification is protein A affinity column purification and separation to obtain the target antibody.

The present invention includes not only whole antibodies, but also fragments, derivatives and analogs of said antibodies. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag). These fragments, derivatives and analogs are within the purview of those skilled in the art based on the teachings herein.

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

In addition to substantially full-length polypeptides, the invention also includes fragments of the bispecific antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.

In the present invention, "conservative variants of the antibody of the present invention" refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acid sequences compared with the amino acid sequence of the antibody of the present invention. An amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1

initial residue representative replacement preferred substitution 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 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; Leu

The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof. A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. A polynucleotide encoding a mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences .

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

The full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods. The biomolecules (nucleic acid, protein, etc.) involved in the present invention include biomolecules in an isolated form.

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

The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein. The host cell may 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 are: 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.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

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

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The antibodies of the invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these. Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or substances capable of producing a detectable product. enzyme.

Therefore, the present invention also provides an immunoconjugate comprising the bispecific antibody of the present invention; and a coupling moiety. Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biotoxin; 3. Cytokines, such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. 6. Liposomes; 7. Nanomagnetic particles; 8. Drug-activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); 9. Therapeutic agents ( For example, cisplatin) or any form of nanoparticles, etc. In a preferred embodiment, the immunoconjugate is an antibody-drug conjugate (Antibody-Drug-Conjugate, ADC).

pharmaceutical composition

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 materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated 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 topical administration.

The pharmaceutical composition of the present invention can be directly used to bind PD-1 protein, thereby blocking the interaction between PD-1 and PD-L1, and the pharmaceutical composition of the present invention can be directly used to bind other targets, thereby blocking the Binding of 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 group consisting of acute myeloid leukemia, chronic myelogenous 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 gland tumors, bladder tumors, sarcoma, etc. In addition, the pharmaceutical composition of the present invention can also be used in combination with other therapeutic agents.

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 pharmaceutical acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.

The active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day. When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

application

As mentioned above, the bispecific antibody of the present invention has a wide range of biological and clinical application values, and its application involves the diagnosis and treatment of diseases related to cell therapy, basic medical research, biological research and other fields.

In a preferred embodiment, the bispecific antibody of the present invention is used for targeted tumor therapy targeting CD20, CD19 or CD38.

Advantages of the present invention:

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

2. The bispecific antibody of the present invention can have the affinity of the parental antibody and can produce ideal biological effects;

3. The bispecific antibody of the present invention has a high degree of safety;

4. The bispecific antibody of the present invention has laid a new material basis for the development of antitumor drugs.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Example.

Example 1. Selection of Bispecific Antibody Candidate Antibody Sequences

The inventors selected three membrane protein targets CD20, CD19, CD38 and CD3 as the sequence of the parent antibody in the bispecific antibody.

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

EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS (SEQ ID NO: 23)

Wherein HCDR1-3 are respectively: GFTFNDYAMH (SEQ ID NO: 37), TISWNSGSIGYADSVKG (SEQ ID NO: 38), DIQYGNYYYGMDV (SEQ ID NO: 39).

The amino acid sequence of the light chain variable region is:

EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK (SEQ ID NO: 24)

Wherein LCDR1-3 are respectively RASQSVSSYLA (SEQ ID NO: 40), DASNRAT (SEQ ID NO: 41), QQRSNWPIT (SEQ ID NO: 42).

The present inventor screened and determined that the CD3 antibody corresponding to the CD20 antibody Ofatumumab was Foralumab. The two antibodies have very similar light chain sequences and are likely to be able to maintain sufficient affinity and biological activity using the same light chain.

CD3 antibody, the amino acid sequence of the heavy chain variable region of Foralumab is:

QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (SEQ ID NO: 25)

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

CD3 antibody, the amino acid sequence of the light chain variable region of Foralumab is:

EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO: 26)

Wherein LCDR1-3 are respectively: RASQSVSSYLA (SEQ ID NO: 46), DASNRAT (SEQ ID NO: 47), QQRSNWPPLT (SEQ ID NO: 48).

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

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 27)

Wherein HCDR1-3 are respectively: GVSLPDYGVS (SEQ ID NO: 49), VIWGSETTYYNSALKS (SEQ ID NO: 50), HYYYGGSYA (SEQ ID NO: 51)

The heavy chain variable region sequence of the CD19 antibody was humanized by CDR transplantation, and the amino acid sequence after humanization was:

QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS (SEQ ID NO: 28)

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

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 29)

Wherein LCDR1-3 are respectively: RASQDISKYLN (SEQ ID NO:52), HTSRLHS (SEQ ID NO:53), QQGNTLPYT (SEQ ID NO:54)

The light chain variable region sequence of the CD19 antibody was humanized by CDR transplantation, and the amino acid sequence after humanization was:

DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPYTFGQGTKVEIK (SEQ ID NO: 30)

The CD3 antibody corresponding to 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

>UCHT-1-VH anti-CD3

EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 31)

Wherein HCDR1-3 are respectively: GYSFTGYTMN (SEQ ID NO: 55), LINPYKGVSTYNQKFKD (SEQ ID NO: 56), SGYYGDSDWYFDV (SEQ ID NO: 57)

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

QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 32)

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

>UCHT-VL

DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK (SEQ ID NO: 33)

Wherein LCDR1-3 are respectively: RASQDIRNYLN (SEQ ID NO:58), YTSRLHS (SEQ ID NO:59), QQGNTLPWT (SEQ ID NO:60)

The amino acid sequence of the light chain variable region sequence of the UCHT-1 antibody clone after sequence humanization is as follows:

DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIK (SEQ ID NO: 34)

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:

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS (SEQ ID NO: 35)

Wherein HCDR1-3 are respectively: GFTFNSFAMS (SEQ ID NO: 61), AISGSGGGTYYADSVKG (SEQ ID NO: 62), DKILWFGEPVFDY (SEQ ID NO: 63)

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

EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK (SEQ ID NO: 36)

Wherein LCDR1-3 are respectively: RASQSVSSYLA (SEQ ID NO: 64), DASNRAT (SEQ ID NO: 65), QQRSNWPPT (SEQ ID NO: 66)

The CD3 antibody corresponding to the CD38 antibody Daratumumab was combined with Foralumab.

Example 2. Design of bispecific antibody sequences

1. Design of bispecific antibodies targeting CD20 and CD3

Firstly, monoclonal antibodies with exchanged light chains of the CD20 antibody Ofatumumab and the CD3 antibody Foralumab were designed to verify whether the CD20 antibody and CD3 antibody still maintain the affinity for the target after the exchange of light chains.

The monoclonal antibody selected from the combination of CD20 antibody heavy chain and CD3 light chain is named: LL206-BM3, and the amino acid sequence is:

Heavy chain: SEQ ID NO: 1

EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain: SEQ ID NO:4

EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQSGNSQESVTEQDSKDSTYSLSSTLLFSKADYEKHKVGLRYACEVSSTT

The monoclonal antibody selected from the combination of CD3 antibody heavy chain and CD20 light chain is named: LL206-BM4, and the amino acid sequence is:

Heavy chain: SEQ ID NO:3

QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain: SEQ ID NO:2

EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLNFGLRRASKADGYEKHKVYACEVKQS

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

A bispecific antibody targeting CD20 and CD3 was constructed by exchanging light chains. At the same time, Knobs-into-holes technology (J B Ridgway, L G Presta, P Carter. Protein Eng. 1996Jul; 9 (7): 617-21.) was used to construct "1+1 structure" and "2+1 structure" targeting Bispecific antibody to CD20 and CD3.

Among them, the "1+1 structure" bispecific antibodies are named: LL206-1 and LL206-2, the heavy chain of Ofatumumab is the knot chain, and the heavy chain of Foralumab is the hole chain. LL206-1 uses the light chain of Ofatumumab, and LL206-2 uses the light chain of Foralumab.

The amino acid sequence of LL206-1 is:

Heavy chain 1: SEQ ID NO: 11

EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Heavy chain 2: SEQ ID NO: 12

QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Light chain: SEQ ID NO:2

The amino acid sequence of LL206-2 is:

Heavy chain 1: SEQ ID NO: 11

Heavy chain 2: SEQ ID NO: 12

Light chain: SEQ ID NO:4

In the "2+1 structure", two ofatumumab antigen binding regions (Antigen binding fragments; Fab) are used, and a linker: 4* (GGGS) is used between the two variable regions. Among them, on the basis of LL206-1 and LL206-2, the "2+1 structure" bispecific antibodies constructed by linking the Fab end of CD20 to the Fab end of CD3 are named: LL206-3 and LL206-4, respectively.

Put the Fab end of CD3 on the outside, that is, connect the Fab end of CD20 to the Fc end, and then connect the Fab end of CD3 to the Fab end of CD20 to construct the "2+1 structure" bispecific antibody named respectively For: LL206-5 and LL206-6.

The amino acid sequence of LL206-3 is:

Heavy chain 1: SEQ ID NO: 11

Heavy chain 2: SEQ ID NO: 13

EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Light chain: SEQ ID NO:2

The amino acid sequence of LL206-4 is:

Heavy chain 1: SEQ ID NO: 11

Heavy chain 2: SEQ ID NO: 13

Light chain: SEQ ID NO:4

The amino acid sequence of LL206-5 is:

Heavy chain 1: SEQ ID NO: 11

Heavy chain 2: SEQ ID NO: 14

QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Light chain: SEQ ID NO:2

The amino acid sequence of LL206-6 is:

Heavy chain 1: SEQ ID NO: 11

Heavy chain 2: SEQ ID NO: 14

Light chain: SEQ ID NO:4.

2. Design of bispecific antibodies targeting CD19 and CD3

First, CD19 antibody clone FMC63 and CD3 clone UCHT-1 monoclonal antibodies were designed to exchange light chains to verify whether CD20 antibodies and CD3 antibodies still maintain the affinity for the target after the light chains were exchanged. At the same time, the light chain with better affinity among the two light chains is selected as the candidate light chain for constructing the bispecific antibody.

The monoclonal antibody selected from the combination of CD19 antibody heavy chain and CD3 light chain is named: LL207-BM3, and the heavy chain is: SEQID NO:5

QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The light chain is: SEQ ID NO:8

DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLGSKADYEVECKQGLS

The monoclonal antibody selected from the combination of CD3 antibody heavy chain and CD19 light chain is named: LL207-BM4,

The heavy chain is: SEQ ID NO:7

QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The light chain is: SEQ ID NO:6

DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFLTLSSLQPEDFATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEVKQHKGLS

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

A bispecific antibody targeting CD19 and CD3 was constructed by exchanging light chains. At the same time, Knobs-into-holes technology is used to construct "1+1 structure" and "2+1 structure" bispecific antibodies targeting CD19 and CD3.

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

The amino acid sequence of LL207-1 is:

Heavy chain 1: SEQ ID NO: 15

QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Heavy chain 2: SEQ ID NO: 16

QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Light chain: SEQ ID NO:8

The amino acid sequence of LL207-2 is:

Heavy chain 1: SEQ ID NO: 15

Heavy chain 2: SEQ ID NO: 16

Light chain: SEQ ID NO:6

In the "2+1 structure", the antigen binding regions (Antigen binding fragments; Fab) of two FMC63 clones are used, and the linker: 4* (GGGS) is used between the two variable regions. Among them, on the basis of LL207-1 and LL207-2, the "2+1 structure" bispecific antibodies constructed by linking the Fab end of CD19 to the Fab end of CD3 are named: LL207-3 and LL207-4, respectively. Put the Fab end of CD3 on the outside, that is, connect the Fab end of CD19 to the Fc end, and then connect the Fab end of CD3 to the Fab end of CD19 to construct the "2+1 structure" bispecific antibody named respectively For: LL207-5 and LL207-6.

The amino acid sequence of LL207-3 is:

Heavy chain 1: SEQ ID NO: 15

Heavy chain 2: SEQ ID NO: 17

QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Light chain: SEQ ID NO:8

The amino acid sequence of LL207-4 is:

Heavy chain 1: SEQ ID NO: 15

Heavy chain 2: SEQ ID NO: 17

Light chain: SEQ ID NO:6

The amino acid sequence of LL207-5 is:

Heavy chain 1: SEQ ID NO: 15

Heavy chain 2: SEQ ID NO: 18

QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Light chain: SEQ ID NO:8

The amino acid sequence of LL207-6 is:

Heavy chain 1: SEQ ID NO: 15

Heavy chain 2: SEQ ID NO: 18

Light chain: SEQ ID NO:6

3. Design of bispecific antibodies targeting CD38 and CD3

Firstly, monoclonal antibodies with CD38 antibody Daratumumab and CD3 antibody Foralumab were designed to exchange light chains to verify whether the CD38 antibody and CD3 antibody still maintain the affinity for the target after the light chain exchange. At the same time, the light chain with better affinity among the two light chains is selected as the candidate light chain for constructing the bispecific antibody.

The monoclonal antibody selected from the combination of CD38 antibody heavy chain and CD3 light chain was named: LL208-BM2,

The heavy chain is: SEQ ID NO:9

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The light chain is: SEQ ID NO:4

The monoclonal antibody selected from the combination of CD3 antibody heavy chain and CD38 light chain is named: LL208-BM3,

The heavy chain is: SEQ ID NO:3

The light chain is: SEQ ID NO: 10

EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESGNSQESVTEQDSKDSTYSLSSTLNTLSKADGYEKHKVYACEVTHS

At the same time, the CD38 antibody Daratumumab was prepared as a control antibody. The CD38 antibody Daratumumab is named: LL208-BM1.

A bispecific antibody targeting CD38 and CD3 was constructed by exchanging light chains. At the same time, Knobs-into-holes technology is used to construct "1+1 structure" and "2+1 structure" bispecific antibodies targeting CD38 and CD3.

Among them, the "1+1 structure" bispecific antibodies are named: LL208-1 and LL208-2, the heavy chain of the CD38 antibody Daratumumab is the knot chain, and the heavy chain of the antibody Foralumab is the hole chain. LL208-1 uses the light chain of the CD38 antibody Daratumumab, and LL208-2 uses the light chain of the antibody antibody Foralumab.

The amino acid sequence of LL208-1 is:

Heavy chain 1: SEQ ID NO: 19

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Heavy chain 2: SEQ ID NO:20

QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Light chain: SEQ ID NO: 10

The amino acid sequence of LL208-2 is:

Heavy chain 1: SEQ ID NO: 19

Heavy chain 2: SEQ ID NO:20

Light chain: SEQ ID NO:4

In the "2+1 structure", two CD38 antibody Daratumumab antigen binding regions (Antigen binding fragments; Fab) are used, and a linker: 4* (GGGS) is used between the two variable regions. Among them, on the basis of LL208-1 and LL208-2, the bispecific antibodies with a 2+1 structure constructed by linking the Fab end of CD38 to the Fab end of CD3 are named: LL208-3 and LL208-4, respectively. Put the Fab end of CD3 on the outside, that is, connect the Fab end of CD38 to the Fc end, and then connect the Fab end of CD3 to the Fab end of CD38 to construct the "2+1 structure" bispecific antibody named respectively For: LL208-5 and LL208-6.

The amino acid sequence of LL208-3 is:

Heavy chain 1: SEQ ID NO: 19

Heavy chain 2: SEQ ID NO:21

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Light chain: SEQ ID NO: 10

The amino acid sequence of LL208-4 is:

Heavy chain 1: SEQ ID NO: 19

Heavy chain 2: SEQ ID NO:21

Light chain: SEQ ID NO:4

The amino acid sequence of LL208-5 is:

Heavy chain 1: SEQ ID NO: 19

Heavy chain 2: SEQ ID NO:22

QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Light chain: SEQ ID NO: 10

The amino acid sequence of LL208-6 is:

Heavy chain 1: SEQ ID NO: 19

Heavy chain 2: SEQ ID NO:22

Light chain: SEQ ID NO:4.

The structure of the above bispecific antibody is shown in FIG. 1 .

Example 3. Expression and purification of bispecific antibodies

According to the nucleic acid sequence list of the above-mentioned bispecific antibody, the related bispecific antibody gene sequence was synthesized, and the gene sequence of the bispecific antibody was constructed into an antibody expression vector using molecular cloning technology. After the vector was successfully constructed, the above-mentioned bispecific antibody was expressed using the 293 protein expression system.

Basic steps: clone the bispecific antibody gene into the expression vector pCDNA4 (Invitrogen, CatV86220). The vectors carrying the bispecific antibody gene sequences were transfected into HEK293 suspension cells by transient method. 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, use UV spectrophotometry to detect the protein concentration and total protein amount of different antibodies, and then calculate the expression amount of each double antibody based on the expression volume. Specifically, use NanoDrop 1000 to read the absorbance value A280 of the sample solution at a wavelength of 280nm, and calculate the protein concentration of different samples by the formula C (mg/mL) = A280/ε (ε is 1.482mL/mg cm-1).

The purity of the different antibodies was assessed by using gel electrophoresis SDS-PAGE. The basic steps are as follows: the sample is separated by electrophoresis. Dilute the sample to about 1 mg/mL, add an appropriate amount of loading buffer, mix, heat at 90°C for about 3 minutes, and load the sample at 2-10 μg. After electrophoresis, refer to the SDS-PAGE standard procedure for staining and decolorization. After the decolorization was completed, the gel imaging system was used to take pictures and analyze and calculate the purity of the main band.

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

The expression level and purity of the CD3×CD20 bispecific antibody are shown in Table 1. LL206-BM1 is ofatumumab and LL206-BM2 is foralumab. The monoclonal antibody LL206-BM3 assembled with the light chain of foralumab and the heavy chain of ofatumumab or the monoclonal antibody LL206-BM4 assembled with the light chain of ofatumumab and the heavy chain of foralumab, the results of SEC-HPLC and SDS-PAGE proved these two new assemblies The monoclonal antibody has a complete structure, and its purity and expression level are close to those of the original parent antibody molecule. Subsequently, the heavy chains of ofatumumab and foralumab were assembled using a knob into hole, and then assembled with the light chains of ofatumumab or foralumab to form common light chain bispecific antibodies LL206-1 and LL206-2. The results of SEC-HPLC and SDS-PAGE proved that LL206-1 and LL206-2 had complete structures and high purity, but the expression level of LL206-2 was low. The above results prove that the CD3×CD20 shared light chain bispecific antibody of the present invention has correct structure and high purity.

Table 1. Expression level and purity of CD3×CD20 bispecific antibody

The expression level and purity of the CD3×CD19 bispecific antibody are shown in Table 2. 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, the results of SEC-HPLC and SDS-PAGE prove these two new assemblies The monoclonal antibody has a complete structure, and its purity and expression level are close to those of the original parent antibody molecule. Subsequently, the heavy chains of FMC and UCHT were assembled using a 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. The results of SEC-HPLC and SDS-PAGE proved that the structures of LL207-1 and LL207-2 were complete and had high expression levels, but SEC-HPLC slightly decreased. Therefore, these results prove that the CD3×CD19 shared light chain bispecific antibody of the present invention has the correct structure and high expression level.

Table 2. Expression level and purity of CD3×CD19 bispecific antibody

The expression level and purity of the CD3×CD38 bispecific antibody are shown in Table 3. For the monoclonal antibody LL208-BM2 assembled from the light chain of Foralumab and the heavy chain of Daratumumab, the results of SEC-HPLC and SDS-PAGE proved that the two newly assembled monoclonal antibodies were structurally complete, and their purity and expression were comparable to those of the original parental antibody molecule near. Therefore, the heavy chains of Daratumumab and Foralumab are assembled using a knob into hole, and the light chain of Foralumab can be assembled with it to form a CD3×CD38 shared light chain bispecific antibody.

Table 3. Expression level and purity of CD3×CD38 bispecific antibody

It can be seen from the above results that the bispecific antibodies targeting CD3 and CD20, CD3 and CD19, and CD3 and CD38 we constructed have good expression and purity, and can be used for further screening and evaluation of biological functions.

Example 4. Detection of target binding activity of bispecific antibodies

The activity of different antibodies binding to targets at the cellular level was detected by flow cytometry, and the binding ability of bispecific antibodies to protein antigens expressed on the cell surface was evaluated. The basic steps are as follows:

1. Prepare cells: Take appropriate amount of Raji (Shanghai Biochemical Cell Bank) and T cells (Shanghai Miaotong Biology), respectively adjust the cell density to 2E6/ml, and distribute them into 1.5ml EP tubes, each tube is 50ul (approx. 1E5cells);

2. Antibody dilution: Determine the initial concentration of the antibody according to the experimental requirements (LL-206-BM1, LL206-BM2, LL-206-BM3, LL206-BM4, LL207-BM1, LL207-BM2, LL-206-BM3, LL206 - The initial concentration of BM4 is 76.8ug/ml, the initial concentration of LL206-1, LL206-2, LL207-1, LL207-2 is 153.6ug/ml), the original antibody is adjusted to the initial concentration with PBS, 2 A total of 8 concentration gradients were set up (the concentration of LL-206-BM1, LL206-BM2, LL-206-BM3, LL206-BM4, LL207-BM1, LL207-BM2, LL-206-BM3, LL206-BM4 The gradient is 4.8ug/ml, 2.4ug/ml, 1.2ug/ml, 0.6ug/ml, 0.3ug/ml, 0.15ug/ml, 0.075ug/ml, 0.0375ug/ml, LL206-1, LL206-2, The concentration gradient of 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, 0.075ug/ml );

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

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

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, mix by pipetting, and protect from light at 4°C Incubate for 30 minutes;

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

7. Test on the machine: add 200ul wash buffer to each tube to resuspend, transfer to the flow tube, and test on the machine.

It can be seen from Figure 2 that LL206-BM1, LL207-BM1 and the light chain-swapped antibodies LL206-BM3 and LL207-BM3 can all bind Raji cells very well, and the binding ability of LL206-BM1 and LL206-BM3 to Raji cells is close , LL207-BM1 and LL207-BM3 have similar binding abilities to Raji cells, indicating that swapping light chains has little effect on the binding of CD20 and CD19 targets.

It can be seen from Figure 3 that the bispecific antibody LL206-1 can bind Raji cells very well, and the affinity is about 1/3 of that of the parent monoclonal antibody LL206-BM1, and the EC50 are: 3.50 and 1.12 (ug/ml) respectively . However, the binding ability of LL206-2 to Raji cells was weak. It shows that different light chain combinations seriously affect the affinity of the double antibody to CD20. Both bispecific antibodies LL207-1 and LL207-2 can bind Raji cells well, with EC50 of 2.23 and 2.41 (ug/ml), respectively, which is about half of that of the parental monoclonal antibody LL207-BM1. It shows that for the binding of LL207-1 and LL207-2 to CD19, there is little difference in the light chains of the two antibodies.

It can be seen from Figure 2 that in terms of binding T cells, LL206-BM2, LL207-BM2 and the light chain-swapped antibodies LL206-BM4, LL207-BM4 can all bind T cells well.

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

Taken together, the bispecific antibodies LL206-1 and LL207-2 of the present invention have the advantages of better affinity for both targets and T cells, and have more potential for subsequent development.

Example 5. Detection of bispecific antibody-mediated cytokine secretion by T cells

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

The basic steps to detect bispecific antibody-mediated T cell secretion of cytokines are as follows:

1. Cell co-culture. Dilute the antibodies LL206-BM2, LL206-1, LL206-2, LL207-BM2, LL207-1, LL207-2 to 4 concentration gradients with 1640 complete medium: 9.6ug/ml, 2.4ug/ml, 0.6ug/ml ml, 0.15ug/ml spare. Adjust the concentration of T cells to 5×10 ⁵ /ml, and then seed T cells into 96-well plates at 100ul per well, and temporarily place them at 37°C for incubation. Adjust the concentration of Raji cells to 5×10 ⁵ /ml with diluted antibodies in 4 concentration gradients, and then plant Raji cells in 100ul per well into a 96-well plate containing T cells. The final ratio of T cells to tumor cells was 1:1. The final concentration of each antibody is: 4.8ug/ml, 1.2ug/ml, 0.3ug/ml, 0.075ug/ml. Mix well and centrifuge at 500 rpm for 3 minutes. Place the cells in a 37°C incubator for 24 hours;

2. Cytokine secretion detection. After incubation, the supernatant was taken and transferred to a new round-bottom 96-well plate, and 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 carried out according to the procedures provided by the kit.

The results showed that both bispecific antibodies LL206-1 and LL206-2 could well mediate T cells to kill Raji tumor cells and secrete a large amount of cytokines (Figure 4). Pure maternal antibodies do not have this function. Taking 0.3ug/ml as an example, in the LL206-1, LL206-2, LL207-1 and LL207-2 groups, the secretion of IFN-g was 2130, 450, 568 and 223 (pg/ml), respectively, and the negative control group IFN-g was not secreted (Fig. 4A). This result shows that LL206-1 has better efficacy, and LL206-2 needs a higher dose to achieve the same effect as LL206-1. LL207-1 and LL207-2 can also mediate T cells to kill Raji tumor cells, but the effect is weaker than that of LL206-1, and a larger dose is required to achieve a better drug effect.

Example 6. Bispecific antibody mediates T cell killing of tumor cells

1. Cell co-culture. The Raji cell line labeled with luciferase was prepared by transfecting Raji cells with luciferase-containing lentivirus, labeled as: Raji.luc. Spread Raji.luc in a 96-well flat-bottom opaque white plate at a cell concentration of 1×10 ⁵ /ml, 50ul/well, and place it at 37°C for incubation. Antibodies LL206-BM2, LL206-1, LL206-2, LL207-BM2, LL207-1, LL207-2 were diluted with culture medium to 4 concentration gradients: 9.6ug/ml, 2.4ug/ml, 0.6ug/ml, 0.15ug/ml. Adjust the concentration of T cells to 1×10 ⁵ /ml with diluted antibodies in 4 concentration gradients, and then plant T cells in 50ul per well into 96-well flat-bottomed opaque white plates containing Raji.luc cells. Mix well and centrifuge at 500 rpm for 3 minutes. The cells were incubated in a 37°C incubator for 24 hours.

2. Kill detection. After the cells were co-cultured for 24 hours, the remaining luciferase activity (relative light unit, RLU) of the target cells was measured to detect the killing ability of the T cells on the target cells in the presence of different specific antibodies. The specific steps are:

The co-cultured cells were centrifuged at 800rpm for 5min, the supernatant was sucked off, and 100ul of D-luciferin substrate (Thermo Fisher Scientific: 88293) was added to mix and develop color for 5min in the dark, and the fluorescence intensity was detected in the chemiluminescence mode on a microplate reader. Since luciferase is only expressed in target cells, the remaining luciferase activity in a well is directly related to the number of live target cells in the well. As a control, maximal luciferase activity was obtained by adding medium to target cells in the absence of effector cells and antibodies.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 appended claims of the present application.

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<120> Bispecific antibody and its preparation method and application

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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 peptides

<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 Lys

450

<210> 6

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptides

<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 peptides

<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 peptides

<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 peptides

<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

Ser Pro Gly Lys

450

<210> 10

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptides

<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 peptides

<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 peptides

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

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser 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 peptides

<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 peptides

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

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser 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 peptides

<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 peptides

<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 peptides

<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 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 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 peptides

<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 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 peptides

<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 peptides

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

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser 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 peptides

<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 peptides

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

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser 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 peptides

<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 peptides

<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 peptides

<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 peptides

<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 peptides

<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 peptides

<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 peptides

<400> 29

Asp Ile Gln Met Thr Gln Thr Thr Ser 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 peptides

<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 peptides

<400> 33

Asp Ile Gln Met Thr Gln Thr Thr Ser 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 peptides

<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 peptides

<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 peptides

<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 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 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 (14)

1. A bispecific antibody comprising an antibody specifically binding to a tumor antigen and an antibody specifically binding to a T cell antigen. 2. The bispecific antibody according to claim 1, wherein the tumor antigen is CD20, CD19 or CD38, and the T cell antigen is CD3. 3. The bispecific antibody according to claim 2, wherein the bispecific antibody comprises a heavy chain and a light chain, and the heavy chain is derived from an antibody specifically binding to a tumor antigen and a specifically binding T Antibodies to cellular antigens, the light chains being derived from antibodies that specifically bind tumor antigens and antibodies that specifically bind T cell antigens, respectively; Preferably, the heavy chains are derived from antibodies specifically binding to tumor antigens and antibodies specifically binding to T cell antigens, respectively, and the light chains are derived from antibodies specifically binding to tumor antigens or antibodies specifically binding to T cell antigens. 4. The bispecific antibody according to claim 3, wherein the heavy chain variable region in the Fab fragment of the antibody is connected to the Fab fragment of the antibody specifically binding to a tumor antigen via an optional linker. The CH1 region or the CH1 region of the Fab fragment of the antibody specifically binding to the T cell antigen is further connected; Preferably, the Fab fragment of the antibody is derived from the heavy chain variable region of the antibody specifically binding to a T cell antigen via an optional linker with the CH1 region of the Fab fragment of the antibody specifically binding to a tumor antigen Connect further. 5. The bispecific antibody according to claim 4, wherein the Fab fragment of the antibody and the Fab fragment of the antibody specifically binding to a tumor antigen or the Fab of the antibody specifically binding to a T cell antigen Fragments swap places. 6. The bispecific antibody according to any one of claims 3-5, wherein the light chain CDR1-3 of the antibody specifically binding to CD20 is shown in SEQ ID NO: 40-42, respectively, The heavy chain CDR1-3 are respectively shown in SEQ ID NO:37-39; The light chain CDR1-3 of the antibody specifically binding to CD19 is respectively shown in SEQ ID NO:52-54, and the heavy chain CDR1-3 is respectively shown in SEQ ID NO:49-51; The light chain CDR1-3 of the antibody specifically binding to CD38 is respectively shown in SEQ ID NO:64-66, and the heavy chain CDR1-3 is respectively shown in SEQ ID NO:61-63; The light chain CDR1-3 of the antibody specifically binding to CD3 is shown in SEQ ID NO: 46-48 or SEQ ID NO: 58-60 respectively, and the heavy chain CDR1-3 is shown in SEQ ID NO: 43-45 or SEQ ID NO: 43-45 or SEQ ID NO: 58-60 respectively. ID NO:55-57; Preferably, the light chain CDR1-3 of the antibody specifically binding to CD3 is respectively shown in SEQ ID NO:46-48, and the heavy chain CDR1-3 is respectively shown in SEQ ID NO:43-45; or the specific The light chain CDR1-3 of the antibody binding to CD3 is respectively shown in SEQ ID NO:58-60, and the heavy chain CDR1-3 is respectively shown in SEQ ID NO:55-57. 7. A bispecific antibody, characterized in that the bispecific antibody is composed of heavy chain 1, heavy chain 2 and light chain, and the bispecific antibody is selected from the following antibodies: name heavy chain 1 heavy chain 2 light chain LL206-1 SEQ ID NO: 11 SEQ ID NO:12 SEQ ID NO:2 LL206-2 SEQ ID NO: 11 SEQ ID NO:12 SEQ ID NO:4 LL206-3 SEQ ID NO: 11 SEQ ID NO: 13 SEQ ID NO:2 LL206-4 SEQ ID NO: 11 SEQ ID NO: 13 SEQ ID NO:4 LL206-5 SEQ ID NO: 11 SEQ ID NO: 14 SEQ ID NO:2 LL206-6 SEQ ID NO: 11 SEQ ID NO: 14 SEQ ID NO:5 LL207-1 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO:8 LL207-2 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO:6 LL207-3 SEQ ID NO: 15 SEQ ID NO: 17 SEQ ID NO:8 LL207-4 SEQ ID NO: 15 SEQ ID NO: 17 SEQ ID NO:6 LL207-5 SEQ ID NO: 15 SEQ ID NO: 18 SEQ ID NO:8 LL207-6 SEQ ID NO: 15 SEQ ID NO: 18 SEQ ID NO:6 LL208-1 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO:10 LL208-2 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO:4 LL208-3 SEQ ID NO: 19 SEQ ID NO: 21 SEQ ID NO:10 LL208-4 SEQ ID NO: 19 SEQ ID NO: 21 SEQ ID NO:4 LL208-5 SEQ ID NO: 19 SEQ ID NO: 22 SEQ ID NO:10 LL208-6 SEQ ID NO: 19 SEQ ID NO: 22 SEQ ID NO:4. 8. An immunoconjugate, characterized in that, the immunoconjugate contains: (a) the bispecific antibody of any one of claims 1-7; and (b) a coupling moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme, gold nanoparticles/nanorods, nanomagnetic particles, viral coat protein or VLP, or combination. 9. Use of the bispecific antibody according to any one of claims 1-7 or the immunoconjugate according to claim 8 in the preparation of anti-tumor drugs. 10. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-7 or the immunoconjugate of claim 8 and a pharmaceutically acceptable excipient agent. 11. An isolated polynucleotide, characterized in that the polynucleotide encodes the bispecific antibody according to any one of claims 1-7. 12. A vector, characterized in that the vector contains the polynucleotide according to claim 11. 13. A host cell, characterized in that the host cell contains the vector according to claim 12, or the polynucleotide according to claim 11 is integrated in its genome; Alternatively, the host cell expresses the bispecific antibody according to any one of claims 1-7. 14. A method for producing the bispecific antibody according to any one of claims 1-7, comprising the steps of: (a) cultivating the host cell according to claim 13 under suitable conditions, thereby obtaining a culture containing the bispecific antibody; and (b) purifying and/or separating the culture obtained in step (a) to obtain the bispecific antibody.

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