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CN115583995B - Bispecific antibodies and preparation methods and applications thereof - Google Patents

Bispecific antibodies and preparation methods and applications thereof Download PDF

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Publication number
CN115583995B
CN115583995B CN202210113943.XA CN202210113943A CN115583995B CN 115583995 B CN115583995 B CN 115583995B CN 202210113943 A CN202210113943 A CN 202210113943A CN 115583995 B CN115583995 B CN 115583995B
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CN115583995A (en
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王文博
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Liling Biopharmaceutical Suzhou Co ltd
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Liling Biopharmaceutical Suzhou Co ltd
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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Abstract

The invention discloses a T cell adapter bispecific antibody targeting tumor antigens and T cell antigens and application thereof. The bispecific antibody targets CD3, CD20, CD19 or CD38. The bispecific antibody of the invention is easy to prepare, can have the affinity of a parent antibody and can generate ideal biological effect, thereby laying a new material foundation for the view of anti-tumor drugs.

Description

Bispecific antibody and preparation method and application thereof
Technical Field
The present invention relates to the field of biotechnology. More particularly, the present invention relates to bispecific antibodies, particularly bispecific antibodies that function as T cell adaptors, and methods of making and using the same.
Background
Bispecific antibodies are genetically engineered antibodies that have been engineered by artificial genes. Bispecific antibodies can target two different antibody binding epitopes simultaneously, which can be from different antigens or from the same antigen. Many studies have now shown that bispecific antibodies have great therapeutic potential in the treatment of tumors, autoimmune diseases, and viral infections. The major advantage of bispecific antibodies over monoclonal antibodies is that they mediate the steric effect of two recognition epitopes and the synergistic effect of dual targeting, resulting in biological effects that are not achieved by the combined use of both antibodies.
Bispecific antibodies are relatively high in technical threshold and development costs because of their complexity unlike monoclonal antibodies. In the process of developing the bispecific antibody, the problems of target selection, antibody engineering design, subsequent production process and the like need to be comprehensively considered. The problems of low expression level, poor stability, difficult establishment of a drug effect model and the like are frequently encountered in the process of constructing the bispecific antibody. Conventional antibodies generally consist of four chains, two heavy chains and two light chains. If conventional antibodies are used to construct bispecific antibodies, light and heavy chain mismatch problems can occur. Furthermore, it is not known whether the bispecific antibody constructed is capable of having the affinity of the parent antibody and producing the desired biological effect.
Thus, there is an urgent need in the art for bispecific antibodies that are easy to prepare, and that are capable of having the affinity of the parent antibody and that produce the desired biological effect.
Disclosure of Invention
The present invention aims to provide a bispecific antibody which is not only easy to prepare, but also has the affinity of the original antibody and simultaneously produces the ideal biological effect.
In a first aspect, the invention provides a bispecific antibody consisting of an antibody that specifically binds a tumor antigen and an antibody that specifically binds a T cell antigen.
In a preferred embodiment, the bispecific antibody is a T cell adaptor-type bispecific antibody.
In specific embodiments, the tumor antigen is CD20, CD19 or CD38 and the T cell antigen is CD3.
In a preferred embodiment, the light chain of the antibody that specifically binds to a tumor antigen and the antibody that specifically binds to a T cell antigen has a sequence similarity of 90% or more, preferably 95% or more, more preferably 98% or more.
In a specific embodiment, the bispecific antibody comprises a heavy chain derived from an antibody that specifically binds a tumor antigen and an antibody that specifically binds a T cell antigen, respectively, and a light chain derived from an antibody that specifically binds a tumor antigen and an antibody that specifically binds a T cell antigen, respectively;
Preferably, the heavy chain is derived from an antibody that specifically binds a tumor antigen and an antibody that specifically binds a T cell antigen, respectively, and the light chain is derived from an antibody that specifically binds a tumor antigen or an antibody that specifically binds a T cell antigen.
In a preferred embodiment, the 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 further linked via an optional linker to the CH1 region of the Fab fragment of the antibody that specifically binds to a tumor antigen or the CH1 region of the Fab fragment of the antibody that specifically binds to a T cell antigen;
preferably, the heavy chain variable region of said antibody specifically binding to a T cell antigen derived from said Fab fragment of said antibody is further linked via an optional linker to the CH1 region of said Fab fragment of said antibody specifically binding to a tumor antigen.
In a preferred embodiment, the linker is (GGGS) 4.
In specific embodiments, the Fab fragment of the antibody is interchanged with the Fab fragment of the antibody that specifically binds to a tumor antigen or the Fab fragment of the antibody that specifically binds to a T cell antigen.
In a specific embodiment, the light chain CDR1-3 of the antibody specifically binding to CD20 is shown as SEQ ID NO. 40-42, and the heavy chain CDR1-3 is shown as SEQ ID NO. 37-39;
the light chain CDR1-3 of the antibody specifically binding to CD19 is shown as SEQ ID NO. 52-54, and the heavy chain CDR1-3 is shown as SEQ ID NO. 49-51;
the light chain CDR1-3 of the antibody specifically binding to CD38 is shown as SEQ ID NO. 64-66, and the heavy chain CDR1-3 is shown as SEQ ID NO. 61-63;
The light chain CDR1-3 of the antibody specifically binding to CD3 is shown as SEQ ID NO 46-48 or SEQ ID NO 58-60, and the heavy chain CDR1-3 is shown as SEQ ID NO 43-45 or SEQ ID NO 55-57;
Preferably, the light chain CDR1-3 of the antibody specifically binding to CD3 is shown as SEQ ID NO 46-48, the heavy chain CDR1-3 is shown as SEQ ID NO 43-45, or the light chain CDR1-3 of the antibody specifically binding to CD3 is shown as SEQ ID NO 58-60, and the heavy chain CDR1-3 is shown as SEQ ID NO 55-57.
In a preferred embodiment, the light chain variable region of the antibody that specifically binds CD20 is shown as SEQ ID NO. 23 and the heavy chain variable region is shown as SEQ ID NO. 24;
the light chain variable region of the antibody specifically binding to CD19 is shown as SEQ ID NO. 29 or 30, and the heavy chain variable region is shown as SEQ ID NO. 27 or 28;
Preferably, the light chain variable region of the antibody that specifically binds CD19 is shown as SEQ ID NO. 29, the heavy chain variable region is shown as SEQ ID NO. 27, or the light chain variable region of the antibody that specifically binds CD19 is shown as SEQ ID NO. 30, the heavy chain variable region is shown as SEQ ID NO. 28;
the light chain variable region of the antibody specifically binding to CD38 is shown as SEQ ID NO. 36, and the heavy chain variable region is shown as SEQ ID NO. 35;
The light chain variable region of the antibody specifically binding to CD3 is shown as SEQ ID NO. 26, 33 or 34, and the heavy chain variable region is shown as SEQ ID NO. 25, 31 or 32;
preferably, the light chain variable region of the antibody specifically binding to CD3 is shown as SEQ ID NO. 26, the heavy chain variable region is shown as SEQ ID NO. 25, or the light chain variable region of the antibody specifically binding to CD3 is shown as SEQ ID NO. 33, the heavy chain variable region is shown as SEQ ID NO. 31, or the light chain variable region of the antibody specifically binding to CD3 is shown as SEQ ID NO. 34, the heavy chain variable region is shown as SEQ ID NO. 32.
In a preferred embodiment, the light chain of the antibody that specifically binds CD20 is shown as SEQ ID NO.2 and the heavy chain is shown as SEQ ID NO. 1;
The light chain of the antibody specifically binding to CD19 is shown as SEQ ID NO. 6, and the heavy chain is shown as SEQ ID NO. 5;
The light chain of the antibody specifically binding to CD38 is shown as SEQ ID NO. 10, and the heavy chain is shown as SEQ ID NO. 9;
The light chain of the antibody specifically binding to CD3 is shown as SEQ ID NO. 4 or 8, and the heavy chain is shown as SEQ ID NO. 3 or 7;
preferably, the light chain of the antibody specifically binding to CD3 is shown as SEQ ID NO. 4, the heavy chain is shown as SEQ ID NO. 3, or the light chain of the antibody specifically binding to CD3 is shown as SEQ ID NO. 8, and the heavy chain is shown as SEQ ID NO. 7.
In a second aspect, the invention provides a bispecific antibody, characterized in that the bispecific antibody consists of heavy chain 1, heavy chain 2 and light chain, the bispecific antibody being selected from the group consisting of the following antibodies:
in a third aspect, the present invention provides an immunoconjugate characterized in that it comprises:
(a) The bispecific antibody of the first or second aspect, and
(B) A coupling moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.
In a fourth aspect, the present invention provides the use of a bispecific antibody according to the first or second aspect or an immunoconjugate according to the third aspect for the preparation of an anti-tumour medicament.
In a preferred embodiment, the tumor is a tumor that specifically expresses CD20, CD19 or CD 38.
In a preferred embodiment, the tumor is a lymphoma, leukemia, multiple myeloma, or the like.
In a fifth aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody according to the first or second aspect or an immunoconjugate according to the third aspect and a pharmaceutically acceptable excipient.
In a preferred embodiment, the pharmaceutical composition further comprises other drugs for treating tumors, such as cytotoxic drugs.
In a preferred embodiment, the other drug for treating tumor comprises paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, pembrolizumab.
In a preferred embodiment, the additional agent is used to block the interaction of PD-1 and PD-L1, while blocking the interaction of the additional target with its receptor or ligand.
In a preferred embodiment, the pharmaceutical composition is in the form of an injection.
In a preferred embodiment, the pharmaceutical composition is used for preparing a medicament for preventing and treating tumors.
In a fifth aspect, the invention provides an isolated polynucleotide, characterized in that said polynucleotide encodes a bispecific antibody according to the first or second aspect.
In a sixth aspect, the present invention provides a vector comprising a polynucleotide according to the first or second aspect.
In a seventh aspect, the present invention provides a host cell comprising the vector of the sixth aspect, or having integrated into its genome the polynucleotide of the fifth aspect;
Or the host cell expresses the bispecific antibody of the first or second aspect.
In an eighth aspect, the present invention provides a method of producing a bispecific antibody according to the first or second aspect, comprising the steps of:
(a) Culturing the host cell of the seventh aspect under suitable conditions to obtain a culture comprising said bispecific antibody, and
(B) Purifying and/or isolating the culture obtained in step (a) to obtain said bispecific antibody.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows a schematic diagram of the structure of a bispecific antibody of the present invention (HC: HEAVY CHAIN; LC: LIGHT CHAIN), wherein the structures of LL206-1, LL207-1 and LL208-1 are shown in FIG. 1A, the structures of LL206-3, LL207-3 and LL208-3 are shown in FIG. 1B, the structures of LL206-5, LL207-5 and LL208-5 are shown in FIG. 1C, the structures of LL206-2, LL207-2 and LL208-2 are shown in FIG. 1D, the structures of LL206-4, LL207-4 and LL208-4 are shown in FIG. 1E, and the structures of LL206-6, LL207-6 and LL208-6 are shown in FIG. 1F;
FIG. 2 shows the results of affinity assays of the monoclonal antibodies of the invention for the interchangeable light chains on Raji cells and T cells;
FIG. 3 shows the results of affinity assays of bispecific antibodies of the invention on Raji cells and T cells;
FIG. 4 shows that bispecific antibodies of the invention can mediate T cell recognition of tumor cells and release of cytokines.
Detailed Description
In a great deal of research and screening on bispecific antibodies of T cell adaptors, the present inventors have unexpectedly found that a specific monoclonal antibody directed against a specific tumor cell target and a T cell target can be used as a parent antibody in the bispecific antibodies, and the bispecific antibodies thus obtained are not only easy to prepare, but also have affinity of the parent antibody and produce an ideal biological effect, thereby being capable of being used as a brand-new antitumor drug. The present invention has been completed on the basis of this finding.
T cell adapter (T CELL ENGAGER)
"T cell adaptor (T CELL ENGAGER)" as used herein has the meaning conventionally understood by those skilled in the art. The T cell adapter (T CELL ENGAGER) can activate endogenous T cells through bispecific binding with target points and T cells on the surface of tumor cells, so as to lead the tumor cells to be cracked, thereby achieving the purpose of treating tumors. Micromet, germany (later purchased by Amgen) is one of the earliest companies developing the BiTE (Bispecific T CELL ENGAGER) platform. BiTE is prepared by ligating two scFv (Single-chain variable fragment) with a linker peptide. Thus, these molecules are bivalent, monovalent for each antigen, with typical molecular weights of 50-60kDa. In 1998, micromet developed Blinatumomab based on the BiTE platform, after which Blinatumomab successfully completed phase I, II and III clinical trials, becoming the first FDA approved BiTE class of drugs. Blinatumomab can target both CD19 and CD3 proteins simultaneously. CD19 is expressed on the surface of acute lymphoblastic leukemia and lymphoma tumor cells as a target for tumor cells. CD3 comprises multiple subunits (d, g and e) and is an important surface marker for T cells. The CD3 subunit may bind to the TCR non-covalently to form a TCR-CD3 complex. CD3 antibodies typically recognize the epsilon chain of the CD3 complex, and transmit an activation signal generated upon binding of the TCR to an antigen into the cell, inducing T cell activation, and thus killing the tumor cell. T cell adaptors have been shown to be useful in the treatment of tumors, and subsequent T cell adaptors against other targets, such as CD20, BCMA and HER2, have been in the clinical stage of research.
Bispecific antibodies of the invention
Herein, "the antibody of the present invention" has the same meaning as "the bispecific antibody of the present invention".
Based on the teachings of the present invention, one skilled in the art will appreciate that the present invention is a T cell adaptor class bispecific antibody, i.e., a bispecific antibody that targets both tumor antigen and T cell antigen.
In a specific embodiment, the bispecific antibodies of the invention consist of an antibody that specifically binds a tumor antigen, such as CD20, CD19 or CD38, and an antibody that specifically binds a T cell antigen, such as CD 3.
The T cell adapter bispecific antibody is a T cell adapter constructed by utilizing a common light chain antibody. The T cell adapter of the present invention has great advantages over other types of T cell adapters. Bispecific antibodies based on a common light chain mean that two bispecific antibodies combined together have a common light chain, thus avoiding the problem of light-heavy chain mismatches. In addition, such bispecific antibodies have a structure similar to that of conventional antibodies, and thus have great advantages in terms of patentability.
Thus, in a specific embodiment, the light chain of the antibody that specifically binds to a tumor antigen and the antibody that specifically binds to a T cell antigen in the T cell adaptor-type bispecific antibody of the present invention has a sequence similarity of 90% or more, preferably 95% or more, more preferably 98% or more.
The bispecific antibodies of the invention comprise heavy and light chains, the heavy chains may be derived from an antibody specifically binding to a tumor antigen and an antibody specifically binding to a T cell antigen, respectively, and the light chains may also be derived from an antibody specifically binding to a tumor antigen and an antibody specifically binding to a T cell antigen, respectively, preferably the light chains are identical, i.e. the light chains are derived from an antibody specifically binding to a tumor antigen, or from an antibody specifically binding to a T cell antigen.
The bispecific antibodies of the invention may be further engineered to add a Fab fragment. In a specific embodiment, the heavy chain variable region in the Fab fragment of the bispecific antibody of the invention is further linked via an optional linker, e.g. (GGGS) 4 to the CH1 region of the Fab fragment of the antibody that specifically binds to a tumor antigen or the CH1 region of the Fab fragment of the antibody that specifically binds to a T cell antigen.
In a preferred embodiment, the heavy chain variable region of said antibody derived from said antibody specifically binding to a T cell antigen in the Fab fragment of said antibody is further linked via an optional linker to the CH1 region of said Fab fragment of said antibody specifically binding to a tumor antigen.
In another preferred embodiment, the Fab fragment of the antibody is interchanged with the Fab fragment of the antibody that specifically binds to a tumor antigen or the Fab fragment of the antibody that specifically binds to a T cell antigen.
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 a host cell comprising the expression vector or the genome thereof into which the polynucleotide is integrated or which expresses the bispecific antibody.
Those skilled in the art will recognize the types of expression vectors that may be used in the practice of 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. The type of host cell used in the practice of the present invention is also known to those skilled in the art, and includes, but is not limited to, prokaryotic or eukaryotic cells, preferably E.coli, yeast cells or mammalian cells.
The person skilled in the art knows how to use the above-described host cells for the preparation of the bispecific antibodies of the invention, e.g. said host cells can be cultured under suitable conditions, thereby obtaining a culture comprising said bispecific antibodies. After obtaining the culture, the resulting culture may be purified and/or isolated to obtain the bispecific antibody. In a preferred embodiment, the purification is protein a affinity column purification and isolation to obtain the antibody of interest.
The invention includes not only whole antibodies, but also fragments, derivatives and analogues of said antibodies. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art in view of the teachings herein.
Bispecific antibodies of the invention also include variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. Such variants include, but are not limited to, deletions, insertions and/or substitutions of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and the addition of one or more (typically less than 20, preferably less than 10, more preferably less than 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter 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, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
In addition to nearly full length polypeptides, the invention also includes fragments of 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 the antibody of the invention.
In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to Table 1.
TABLE 1
Initial residues Representative substitution 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 Ala
His(H) Asn;Gln;Lys;Arg Arg
Ile(I) Leu;Val;Met;Ala;Phe Leu
Leu(L) Ile;Val;Met;Ala;Phe Ile
Lys(K) Arg;Gln;Asn Arg
Met(M) Leu;Phe;Ile Leu
Phe(F) Leu;Val;Ile;Ala;Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr;Phe Tyr
Tyr(Y) Trp;Phe;Thr;Ser Phe
Val(V) Ile;Leu;Met;Phe;Ala Leu
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. Polynucleotides encoding the mature polypeptides of the present invention include coding sequences encoding only the mature polypeptide, coding sequences and various additional coding sequences for the mature polypeptide, coding sequences (and optionally additional coding sequences) for the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences. The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means (1) hybridization and elution at a relatively low ionic strength and a relatively high temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃, or (2) hybridization with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃, etc., or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95%. Furthermore, 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 a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may 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. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of 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 E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, drosophila S2 or Sf9 insect cells, CHO, COS7, 293 cell animal cells, and the like.
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 prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl 2 method using procedures well known in the art. Another approach is to use MgCl 2. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above. Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product.
Accordingly, the present invention also provides an immunoconjugate comprising a bispecific antibody of the invention, and a conjugated moiety. Therapeutic agents that may be conjugated or coupled to the antibodies of the invention include, but are not limited to, 1. Radionuclides, 2. Biotoxins, 3. Cytokines such as IL-2, etc., 4. Gold nanoparticles/nanorods, 5. Viral particles, 6. Liposomes, 7. Nanomagnetic particles, 8. Drug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)), 9. Therapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc. In a preferred embodiment, the immunoconjugate is an Antibody-Drug-Conjugate (ADC).
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising the bispecific antibody or an active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the invention can be directly used for binding to PD-1 protein, thereby blocking the interaction of PD-1 and PD-L1, and can be directly used for binding to other targets, thereby blocking the binding of the other targets to the receptor or ligand thereof. Thus, the pharmaceutical composition of the present invention can be used for treating tumors. In a preferred embodiment, the tumor is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, multiple myelopathy, lymphocytic leukemia, lymphoma, colorectal cancer, breast cancer, gall bladder cancer, gastric cancer, liver cancer, kidney cancer, head and neck tumor, brain tumor, glioblastoma, lung cancer, small intestine cancer, bone cancer, prostate cancer, ovarian cancer, cervical cancer, adrenal tumor, bladder tumor, sarcoma, and the like. In addition, the pharmaceutical compositions of the present invention may also be used in combination with other therapeutic agents.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the single domain antibodies (or conjugates thereof) of the invention described above, as well as a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions.
The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Application of
As described above, the bispecific antibody of the present invention has a wide range of biological and clinical applications, and its use relates to various fields of diagnosis and treatment of diseases associated with cell therapy, basic medical research, biological research, and the like.
In a preferred embodiment, the bispecific antibodies of the invention are used for targeted tumor therapy against CD20, CD19 or CD38 targets.
The invention has the advantages that:
1. The bispecific antibody of the present invention is easy to prepare;
2. The bispecific antibody of the invention can have the affinity of a parent antibody and can produce ideal biological effects;
3. The bispecific antibody of the invention has high safety degree;
4. the bispecific antibody lays a new material foundation for the development of anti-tumor drugs.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, a laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.
Examples
Example 1 selection of bispecific antibody candidate antibody sequences
The present inventors selected three membrane protein targets CD20, CD19 and CD38 and CD3 as the sequences of the parent antibodies in the bispecific antibodies.
1. The inventor selects CD20 antibody Ofatumumab as a parent antibody, and the heavy chain variable region amino acid sequence is as follows:
EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS(SEQ ID NO:23)
Wherein HCDR1-3 is GFTFNDYAMH (SEQ ID NO: 37), TISWNSGSIGYADSVKG (SEQ ID NO: 38), DIQYGNYYYGMDV (SEQ ID NO: 39), respectively.
The amino acid sequence of the light chain variable region is as follows:
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK(SEQ ID NO:24)
Wherein LCDR1-3 is RASQSVSSYLA (SEQ ID NO: 40), DASNRAT (SEQ ID NO: 41), QQRSNWPIT (SEQ ID NO: 42), respectively.
The present inventors determined that the CD3 antibody corresponding to CD20 antibody Ofatumumab was Foralumab by screening. Two antibodies have very similar light chain sequences, and are likely to be able to retain sufficient affinity and biological activity under conditions where the same light chain is used.
The amino acid sequence of the heavy chain variable region of the CD3 antibody, foralumab, is:
QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS(SEQ ID NO:25)
wherein HCDR1-3 is GFKFSGYGMH (SEQ ID NO: 43), VIWYDGSKKYYVDSVKG (SEQ ID NO: 44), QMGYWHFDL (SEQ ID NO: 45), respectively;
The amino acid sequence of the light chain variable region of the CD3 antibody, foralumab, is:
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK(SEQ ID NO:26)
wherein LCDR1-3 is RASQSVSSYLA (SEQ ID NO: 46), DASNRAT (SEQ ID NO: 47), QQRSNWPPLT (SEQ ID NO: 48), respectively.
2. The inventors selected the CD 19-targeting antibody clone FMC63 as a candidate antibody. The heavy chain variable region amino acid sequence of CD19 antibody is:
EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:27)
Wherein HCDR1-3 is GVSLPDYGVS (SEQ ID NO: 49), VIWGSETTYYNSALKS (SEQ ID NO: 50), HYYYGGSYA (SEQ ID NO: 51), respectively
Humanized modification is carried out on the heavy chain variable region sequence of the CD19 antibody by a CDR grafting method, and the amino acid sequence after the humanization is as follows:
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 is RASQDISKYLN (SEQ ID NO: 52), HTSRLHS (SEQ ID NO: 53), QQGNTLPYT (SEQ ID NO: 54), respectively
The light chain variable region sequence of the CD19 antibody is subjected to humanization modification by a CDR grafting method, and the amino acid sequence after humanization is as follows:
DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPYTFGQGTKVEIK(SEQ ID NO:30)
The CD3 antibody corresponding to the CD19 antibody clone FMC63 was clone UCHT-1. The heavy chain variable region amino acid sequence 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 is GYSFTGYTMN (SEQ ID NO: 55), LINPYKGVSTYNQKFKD (SEQ ID NO: 56), SGYYGDSDWYFDV (SEQ ID NO: 57), respectively
The amino acid sequence of the heavy chain variable region sequence cloned by the UCHT-1 antibody after sequence humanization transformation is as follows:
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS(SEQ ID NO:32)
the light chain variable region amino acid sequence of CD3 antibody is derived from PDB 1XIW_C
>UCHT-VL
DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK(SEQ ID NO:33)
Wherein LCDR1-3 is RASQDIRNYLN (SEQ ID NO: 58), YTS LHS (SEQ ID NO: 59), QQGNTLPWT (SEQ ID NO: 60), respectively
The amino acid sequence of the UCHT-1 antibody cloned light chain variable region sequence after sequence humanization transformation is as follows:
DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIK(SEQ ID NO:34)
3. The inventors selected CD 38-targeting antibody Daratumumab as a candidate antibody. The heavy chain variable region amino acid sequence of CD38 antibody Daratumumab is:
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS(SEQ ID NO:35)
Wherein HCDR1-3 is GFTFNSFAMS (SEQ ID NO: 61), AISGSGGGTYYADSVKG (SEQ ID NO: 62), DKILWFGEPVFDY (SEQ ID NO: 63), respectively
The light chain variable region amino acid sequence of CD38 antibody Daratumumab is:
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK(SEQ ID NO:36)
Wherein LCDR1-3 is RASQSVSSYLA (SEQ ID NO: 64), DASNRAT (SEQ ID NO: 65), QQRSNWPPT (SEQ ID NO: 66), respectively
CD3 antibodies corresponding to CD38 antibody Daratumumab were selected Foralumab for combination.
Example 2 design of bispecific antibody sequences
1. Design of bispecific antibodies targeting CD20 and CD3
Monoclonal antibodies were first designed to exchange light chains for CD20 antibody Ofatumumab and CD3 antibody Foralumab to verify whether CD20 antibody and CD3 antibody remained with affinity for the target after exchange of light chains.
The monoclonal antibody selected from the combination of the heavy chain and the light chain of the CD20 antibody is named LL206-BM3, and the amino acid sequence is:
heavy chain SEQ ID NO. 1
EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain SEQ ID NO. 4
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
The monoclonal antibody selected from the combination of the heavy chain and the light chain of the CD3 antibody is named LL206-BM4, and the amino acid sequence is:
Heavy chain SEQ ID NO. 3
QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain SEQ ID NO. 2
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
The CD20 antibody Ofatumumab and CD3 antibody Foralumab were prepared simultaneously as control antibodies. The CD20 antibody Ofautumumab was designated LL206-BM1.CD3 antibody Foralumab was designated LL206-BM2.
Bispecific antibodies targeting CD20 and CD3 were constructed by the method of interchanging light chains. Bispecific antibodies targeting CD20 and CD3 were also constructed using Knobs-into-hops technology (J B Ridgway, L G Presta, P Carter.protein Eng.1996Jul;9 (7): 617-21.) "1+1 structure" and "2+1 structure".
The bispecific antibodies of the "1+1 structure" were designated LL206-1 and LL206-2, the Ofatumumab heavy chain was the knot chain and the Foralumab heavy chain was the hole chain. LL206-1 uses the light chain of Ofatumumab and LL206-2 uses the light chain of Foralumab.
The LL206-1 amino acid sequence is:
heavy chain 1:SEQ ID NO:11
EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Heavy chain 2:SEQ ID NO:12
QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK
Light chain SEQ ID NO. 2
The LL206-2 amino acid sequence 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 antigen binding regions of Ofatumumab (Antigen binding fragments; fab) are used, and linker:4 x (GGGS) is used to link the two variable regions. Wherein, the bispecific antibody of the "2+1 structure" constructed by connecting the Fab end of CD20 to the Fab end of CD3 on the basis of LL206-1 and LL206-2 is named LL206-3 and LL206-4, respectively.
Bispecific antibodies of the "2+1 structure" constructed by placing the Fab end of CD3 on the outside, i.e., linking the Fab end of CD20 to the Fc end, and linking the Fab end of CD3 to the Fab end of CD20 were designated LL206-5 and LL206-6, respectively.
The LL206-3 amino acid sequence is:
heavy chain 1:SEQ ID NO:11
Heavy chain 2:SEQ ID NO:13
EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK
Light chain SEQ ID NO. 2
The LL206-4 amino acid sequence is:
heavy chain 1:SEQ ID NO:11
Heavy chain 2:SEQ ID NO:13
Light chain SEQ ID NO. 4
The LL206-5 amino acid sequence is:
heavy chain 1:SEQ ID NO:11
Heavy chain 2:SEQ ID NO:14
QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK
Light chain SEQ ID NO. 2
The LL206-6 amino acid sequence 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
Monoclonal antibodies were first designed for the exchange of light chains for the CD19 antibody clone FMC63 and for the CD3 clone UCHT-1 to verify whether the CD20 and CD3 antibodies remained affinity for the target after exchange of light chains. Meanwhile, a light chain with better retention affinity of the two light chains is selected as a candidate light chain for constructing the bispecific antibody.
The monoclonal antibody selected from the combination of the heavy chain and the light chain of the CD19 antibody is named LL207-BM3, and the heavy chain is named SEQ ID NO. 5
QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
The light chain is SEQ ID NO. 8
DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
The monoclonal antibody selected from the combination of the heavy chain and the light chain of the CD3 antibody was designated LL207-BM4,
Heavy chain is SEQ ID NO. 7
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
The light chain is SEQ ID NO. 6
DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
A CD19 antibody clone FMC63 and a CD3 antibody clone UCHT-1 were also prepared as control antibodies. The CD19 antibody clone FMC63 was designated LL207-BM1. The CD3 antibody clone UCHT-1 was designated LL207-BM2.
Bispecific antibodies targeting CD19 and CD3 were constructed by the method of interchanging light chains. Meanwhile, knobs-into-holes technology is utilized to construct bispecific antibodies targeting CD19 and CD3 of the '1+1 structure' and the '2+1 structure'.
The bispecific antibodies of the '1+1 structure' are named as LL207-1 and LL207-2, the heavy chain of the CD19 antibody clone FMC63 is a knot chain, and the heavy chain of the antibody clone UCHT-1 is a 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 LL207-1 amino acid sequence is:
Heavy chain 1:SEQ ID NO 15
QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Heavy chain 2:SEQ ID NO 16
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK
Light chain SEQ ID NO. 8
The LL207-2 amino acid sequence 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" two antigen binding regions of FMC63 clone (Antigen binding fragments; fab) were used, and linker:4 x (GGGS) was used to link the two variable regions. Wherein, the bispecific antibody of the "2+1 structure" constructed by linking the Fab end of CD19 to the Fab end of CD3 on the basis of LL207-1 and LL207-2 is named LL207-3 and LL207-4, respectively. Bispecific antibodies of the "2+1 structure" constructed by placing the Fab end of CD3 on the outside, i.e., linking the Fab end of CD19 to the Fc end, and linking the Fab end of CD3 to the Fab end of CD19 were designated LL207-5 and LL207-6, respectively.
The LL207-3 amino acid sequence is:
Heavy chain 1:SEQ ID NO 15
Heavy chain 2:SEQ ID NO:17
QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK
Light chain SEQ ID NO. 8
The LL207-4 amino acid sequence is:
Heavy chain 1:SEQ ID NO 15
Heavy chain 2:SEQ ID NO:17
Light chain SEQ ID NO. 6
The LL207-5 amino acid sequence is:
Heavy chain 1:SEQ ID NO 15
Heavy chain 2:SEQ ID NO:18
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK
Light chain SEQ ID NO. 8
The LL207-6 amino acid sequence 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
Monoclonal antibodies were first designed to interchange the light chains of CD38 antibody Daratumumab and CD3 antibody Foralumab to verify whether the CD38 antibody and CD3 antibody remained affinity for the target after interchange of the light chains. Meanwhile, a light chain with better retention affinity of the two light chains is selected as a candidate light chain for constructing the bispecific antibody.
The monoclonal antibody selected from the combination of the heavy chain and the light chain of the CD38 antibody was designated LL208-BM2,
Heavy chain is SEQ ID NO 9
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
The light chain is SEQ ID NO. 4
The monoclonal antibody selected from the combination of the heavy chain and the light chain of the CD3 antibody was designated LL208-BM3,
Heavy chain is SEQ ID NO. 3
The light chain is SEQ ID NO. 10
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
CD38 antibody Daratumumab was also prepared as a control antibody. CD38 antibody Daratumumab was designated LL208-BM1.
Bispecific antibodies targeting CD38 and CD3 were constructed by the method of interchanging light chains. Bispecific antibodies targeting CD38 and CD3 were also constructed using Knobs-into-holes technology for "1+1 structure" and "2+1 structure".
The bispecific antibodies of the "1+1 structure" were designated LL208-1 and LL208-2, the heavy chain of CD38 antibody Daratumumab was the knot chain, and the heavy chain of antibody Foralumab was the hole chain. LL208-1 uses the light chain of CD38 antibody Daratumumab and LL208-2 uses the light chain of antibody Foralumab.
The LL208-1 amino acid sequence is:
Heavy chain 1:SEQ ID NO 19
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Heavy chain 2:SEQ ID NO:20
QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK
Light chain SEQ ID NO. 10
The LL208-2 amino acid sequence 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 antigen binding regions of CD38 antibody Daratumumab (Antigen binding fragments; fab) are used, and linker:4 x (GGGS) is used to link the two variable regions. Wherein, on the basis of LL208-1 and LL208-2, bispecific antibodies of 2+1 structure constructed by linking the Fab end of CD38 to the Fab end of CD3 are designated as LL208-3 and LL208-4, respectively. Bispecific antibodies of the "2+1 structure" constructed by placing the Fab end of CD3 on the outside, i.e., linking the Fab end of CD38 to the Fc end, and linking the Fab end of CD3 to the Fab end of CD38 were designated LL208-5 and LL208-6, respectively.
The LL208-3 amino acid sequence is:
Heavy chain 1:SEQ ID NO 19
Heavy chain 2:SEQ ID NO:21
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKKVGGGSGGGSGGGSGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK
Light chain SEQ ID NO. 10
The LL208-4 amino acid sequence is:
Heavy chain 1:SEQ ID NO 19
Heavy chain 2:SEQ ID NO:21
Light chain SEQ ID NO. 4
The LL208-5 amino acid sequence is:
Heavy chain 1:SEQ ID NO 19
Heavy chain 2:SEQ ID NO:22
QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK
Light chain SEQ ID NO. 10
The LL208-6 amino acid sequence 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 table of the bispecific antibody, the related bispecific antibody gene sequence is synthesized, and the gene sequence of the bispecific antibody is constructed into an antibody expression vector by using a molecular cloning technology. After successful vector construction, the bispecific antibodies described above were expressed using the 293 protein expression system.
The basic procedure was to clone the bispecific antibody gene into the expression vector pCDNA4 (Invitrogen, cat V86220). Vectors carrying bispecific antibody gene sequences were separately transfected into HEK293 suspension cells using transient methods. The cells were placed in a cell incubator for suspension culture. After expression was completed, the cell culture supernatant was harvested and bispecific antibodies were purified using Protein a affinity chromatography columns. After the bispecific antibody purification was completed, the protein concentration and total protein amount of the different antibodies were detected using ultraviolet spectrophotometry, and then the expression amount of each diabody was calculated from the expression volume. Specifically, the absorbance A280 of the sample solution was read at a wavelength of 280nm using NanoDrop 1000, and the protein concentration of each sample was calculated by the formula C (mg/mL) =A280/. Epsilon. (epsilon. Was 1.482mL/mg cm-1).
The purity of the different antibodies was assessed by SDS-PAGE using gel electrophoresis. The method comprises the following basic steps of carrying out electrophoresis separation on a sample. And diluting the sample to about 1mg/mL, adding a proper amount of loading buffer, mixing, heating at 90 ℃ for about 3 minutes, and carrying out dyeing and decoloring by referring to an SDS-PAGE standard flow after electrophoresis is completed, wherein the loading amount is 2-10 mug. After the decoloring is finished, a gel imaging system is used for photographing and analyzing to 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 method comprises the following basic steps of diluting a sample to about 1.0mg/mL, adopting a TSKgel G3000SWXL chromatographic column, setting the column temperature to 25 ℃, setting the sample injection volume to 30-50 mu L, carrying out isocratic elution for 20min at a flow rate of 1.0mL/min, detecting at a wavelength of 280nm, and adopting a peak area normalization method to obtain the content of the monomer.
The expression level and purity information of the CD3 xCD 20 bispecific antibody are shown in Table 1.LL206-BM1 is ofatumumab and LL206-BM2 is foralumab. The results of foralumab light chain assembled mab LL206-BM3 with the heavy chain of ofatumumab or heavy chain assembled mab LL206-BM4 with the light chain of ofatumumab with foralumab, SEC-HPLC and SDS-PAGE prove that the two newly assembled mabs are structurally complete and have purity and expression levels close to those of the original parent antibody molecule. Subsequently, the heavy chains of ofatumumab and foralumab were assembled using a knob intohole, and then assembled with the light chain of ofatumumab or foralumab, respectively, into common light chain bispecific antibodies LL206-1 and LL206-2. The results of SEC-HPLC and SDS-PAGE demonstrate that LL206-1 and LL206-2 are structurally complete and of higher purity, but LL206-2 is expressed in a lower amount. The above results demonstrate that the CD3 xcd 20 common light chain bispecific antibodies of the invention have the correct structure and higher purity.
TABLE 1 expression level and purity of CD3XCD20 bispecific antibody
The information on the expression level and purity of the CD3 xCD 19 bispecific antibody is shown in Table 2. Results of light chain of UCHT and heavy chain assembled monoclonal antibody LL207-BM3 of FMC or light chain of FMC and heavy chain assembled monoclonal antibody LL206-BM4 of UCHT, SEC-HPLC and SDS-PAGE prove that the two newly assembled monoclonal antibodies are complete in structure and have purity and expression quantity similar to those of original parent antibody molecules. Subsequently, FMC was assembled with the heavy chain of UCHT using knob into hole, and then assembled with the light chain of FMC or UCHT, respectively, into common light chain bispecific antibodies LL207-1 and LL207-2. The results of SEC-HPLC and SDS-PAGE prove that LL207-1 and LL207-2 have complete structures and higher expression levels, but SEC-HPLC is slightly reduced. Therefore, these results demonstrate that the CD3 xcd 19 common light chain bispecific antibodies of the invention have the correct structure and higher expression level.
TABLE 2 expression level and purity of CD3×CD19 bispecific antibody
The information on the expression level and purity of the CD3 xCD 38 bispecific antibody is shown in Table 3. Results of LL208-BM2, SEC-HPLC and SDS-PAGE of Foralumab light chain and Daratumumab heavy chain assembled monoclonal antibodies demonstrated that the two newly assembled monoclonal antibodies were structurally intact and similar in purity and expression to the original parent antibody molecule. Thus, heavy chains Daratumumab and Foralumab were assembled using a knob into hole, with which the light chain of Foralumab could be assembled into a CD3 xcd 38 shared light chain bispecific antibody.
TABLE 3 expression level and purity of CD3XCD 38 bispecific antibody
From the above results, it can be seen that the bispecific antibodies targeting CD3 and CD20, CD3 and CD19, and CD3 and CD38, which we constructed, have better expression levels and purities, can be used for further biological function screening and evaluation.
Example 4 detection of target binding Activity of bispecific antibodies
The binding capacity of bispecific antibodies to protein antigens expressed on the cell surface was evaluated by detecting the activity of different antibodies at the cellular level binding targets by flow cytometry. The basic steps are as follows:
1. preparing cells, namely taking proper amounts of Raji (cell bank of Shanghai biochemical cells) and T cells (Shanghai Miaotong organisms), respectively adjusting the cell density to 2E6/ml, and subpackaging into 1.5ml EP tubes with 50ul (about 1E5 cells) per tube;
2. antibody dilution by determining the initial concentration of the antibody (initial concentration of LL-206-BM1, LL206-BM2, LL-206-BM3, LL206-BM4, LL207-BM1, LL207-BM2, LL-206-BM3, LL206-BM4 of 76.8ug/ml, initial concentration of LL206-1, LL206-2, LL207-1, LL207-2 of 153.6 ug/ml), adjusting the initial concentration of the original antibody to the initial concentration with PBS, 2-fold dilution, setting a total of 8 concentration gradients (concentration gradients of LL-206-BM1, LL206-BM2, LL-206-BM3, LL206-BM4, LL207-BM1, LL207-BM2, LL-206-BM3, LL206-BM4 of 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、LL207-1、LL207-2 of 9.6ug/ml, 4.8ug/ml, 2.4ug/ml, 1.2ug/ml, 0.2 ug/ml, 0.6ug/ml, 0.3ug/ml, 0.15 ug/ml);
3. The antibody incubation, namely adding 50ul diluted antibody into the cells packaged in advance, blowing and mixing uniformly, and then incubating for 30min at 4 ℃ in a dark place;
4. Antibody washing, namely adding 1ml of wash buffer (1xPBS+0.5% FBS) into the incubated antibody cell mixture, and centrifuging at 2000rpm for 5 minutes to discard the supernatant;
5. Second antibody incubation, namely diluting the second antibody anti-human IgG Fc-APC (BD Biosciences) by PBS according to a ratio of 1:200, adding 50ul into the cell sediment after supernatant removal, blowing and mixing uniformly, and then incubating for 30min at 4 ℃ in a dark place;
6. Washing the secondary antibody, namely adding 1ml of wash buffer (1xPBS+0.5% FBS) into the incubated antibody cell mixture, and centrifuging at 2000rpm for 5 minutes to discard the supernatant;
7. And (3) detecting by a machine, namely adding 200ul of wash buffer to each tube for resuspension, transferring the suspension to a flow tube, and detecting by the machine.
As can be seen from FIG. 2, LL206-BM1, LL207-BM1 and antibodies LL206-BM3, LL207-BM3, which interchange light chains, all bind well to Raji cells, LL206-BM1 and LL206-BM3 bind closely to Raji cells, LL207-BM1 and LL207-BM3 bind closely to Raji cells, indicating that interchange light chains do not significantly affect binding to CD20 and CD19 targets.
As can be seen from FIG. 3, bispecific antibody LL206-1 was able to bind Raji cells well, with affinities of approximately 1/3 of the parent monoclonal antibody LL206-BM1, EC50 of 3.50 and 1.12 (ug/ml), respectively. But LL206-2 has weak binding capacity to Raji cells. It was demonstrated that different combinations of light chains severely affected the affinity of the diabodies for CD 20. Bispecific antibodies LL207-1 and LL207-2 both bind well to Raji cells with EC50 of 2.23 and 2.41 (ug/ml), respectively, approximately half that of the parent monoclonal antibody LL207-BM 1. Indicating that the light chain of the two antibodies did not differ significantly for the binding of LL207-1 and LL207-2 to CD 19.
As can be seen from FIG. 2, both LL206-BM2, LL207-BM2 and the antibodies LL206-BM4, LL207-BM4, which interchange light chains, bind T cells well.
As can be seen from FIG. 3, LL206-BM2, LL206-1 and LL206-2 have affinities EC50 for T cells of about 1.81, 12.1 and 12.8 (ug/ml), respectively. The close affinity of LL206-1 and LL206-2 for T cells, reduced by about 6-fold compared to the parent antibody, reduced the probability of nonspecifically activating T cells to some extent, suggesting better safety for both bispecific antibodies. LL207-BM2, LL207-1 and LL207-2 have affinities EC50 for T cells of about 1.92, 4.65 and 9.32 (ug/ml), respectively. The affinity of LL207-1 for T cells remains high, reduced by about 2-fold compared to the parent antibody. LL207-2 has reduced affinity by about 5-fold compared to the parent antibody, somewhat reducing the chance of nonspecifically activating T cells, suggesting better safety.
Taken together, bispecific antibodies LL206-1 and LL207-2 of the present invention have the advantage of better affinity for both target and T cells, and are more potentially amenable to subsequent development.
Example 5 detection of bispecific antibody-mediated T cell secretion of cytokines
The bispecific antibody of the invention can combine T cells and target cells simultaneously, mediate T cells to recognize and kill target cells and secrete cytokines such as IL-2, IFN-g and the like.
The basic procedure for detecting bispecific antibody-mediated cytokine secretion by T cells is as follows:
1. And (5) co-culturing the cells. Antibodies LL206-BM2, LL206-1, LL206-2, LL207-BM2, LL207-1, LL207-2 were diluted to 4 concentration gradients of 9.6ug/ml, 2.4ug/ml, 0.6ug/ml, 0.15ug/ml with 1640 complete medium for use. T-cell concentrations were adjusted to 5X 10 5/ml and then T-cells were seeded into 96-well plates at 100ul per well and temporarily placed for incubation at 37 ℃. Raji cells were seeded into 96-well plates containing T cells at 100ul per well by adjusting the concentration of Raji cells to 5X 10 5/ml with diluted 4-concentration gradient antibody. The final T cell to tumor cell ratio was 1:1. The final concentration of each antibody was 4.8ug/ml, 1.2ug/ml, 0.3ug/ml, 0.075ug/ml. After thoroughly mixing, the mixture was centrifuged at 500rpm for 3 minutes. Cells were incubated in a 37 ℃ incubator for 24 hours;
2. Cytokine secretion assay. After completion of incubation, the supernatant was transferred to a new round bottom 96-well plate and assayed for secretion of IFN-gamma and IL-2 cytokines in T cells using ELISA kit (Thermo FISHER SCIENTIFIC; cat. No. 88-7316). Plate preparation and detection of supernatant cytokines were performed according to the protocol provided for the kit.
The results show that both bispecific antibodies LL206-1 and LL206-2 mediate T cell killing Raji tumor cells well, secreting large amounts of cytokines (FIG. 4). The pure maternal antibody does not have this function. For example, 0.3ug/ml, IFN-g was secreted in groups LL206-1, LL206-2, LL207-1 and LL207-2 as 2130, 450, 568 and 223 (pg/ml), respectively, and IFN-g was not secreted in the negative control group (FIG. 4A). The results indicate that LL206-1 is better in potency and that LL206-2 requires higher doses to achieve the equivalent effect of LL 206-1. LL207-1 and LL207-2 can also mediate T cell killing Raji tumor cells, but are less effective than LL206-1 and require greater doses to achieve better efficacy.
EXAMPLE 6 bispecific antibody mediated T cell killing of tumor cells
1. And (5) co-culturing the cells. Raji cell lines labeled with luciferase were prepared by transfecting Raji cells with lentivirus carrying luciferase, and were labeled Raji. Raji.luc was plated into 96-well flat bottom opaque plates at a cell concentration of 1×10 5/ml, 50 ul/well, and placed in 37 ℃ for incubation. Antibodies LL206-BM2, LL206-1, LL206-2, LL207-BM2, LL207-1, LL207-2 were diluted to 4 concentration gradients of 9.6ug/ml, 2.4ug/ml, 0.6ug/ml, 0.15ug/ml with medium. T-cell concentrations were adjusted to 1X 10 5/ml with diluted 4-concentration gradient antibodies, and then T-cells were seeded at 50ul per well into 96-well flat bottom opaque plate containing Raji. Luc cells. After thoroughly mixing, the mixture was centrifuged at 500rpm for 3 minutes. Cells were incubated for 24 hours at 37 ℃ in an incubator.
2. And (5) killing detection. After 24h of cell co-culture, the remaining luciferase activity (relative light units, RLU) of the target cells was measured to determine the killing capacity of T cells against the target cells in the presence of antibodies of different specificities. The method comprises the following specific steps:
the cells after co-culture were centrifuged at 800rpm for 5min, the supernatant was aspirated, 100ul of D-luciferin substrate (Thermo FISHER SCIENTIFIC: 88293) was added, mixed well and developed in dark for 5min, and the fluorescence intensity was detected in a chemiluminescent mode on a microplate reader. Since luciferase is expressed only in target cells, the remaining luciferase activity in the well is directly related to the number of viable target cells in the well. In the absence of effector cells and antibodies, the maximum luciferase activity was obtained by adding the medium to the target cells as a control.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> Rabdosia Bio-pharmaceutical Co., ltd
<120> Bispecific antibody, and preparation method and application thereof
<130> P2021-2780
<160> 66
<170> PatentIn version 3.5
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr
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Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
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Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
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Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
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Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
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Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp
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Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
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Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
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Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
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Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
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Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
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Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
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His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
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Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
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Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
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His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
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Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
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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
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Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
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Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
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Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
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Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
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Ser Pro Gly Lys
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
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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
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Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
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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
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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
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Phe Asn Arg Gly Glu Cys
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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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<400> 12
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys
355 360 365
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 13
<211> 684
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<400> 14
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser Gly Gly Gly Ser
210 215 220
Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
225 230 235 240
Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala
245 250 255
Ser Gly Phe Thr Phe Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala
260 265 270
Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly
275 280 285
Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
290 295 300
Asp Asn Ala Lys Lys Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
305 310 315 320
Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn
325 330 335
Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
355 360 365
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
450 455 460
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
465 470 475 480
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
500 505 510
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
515 520 525
Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
530 535 540
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
545 550 555 560
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
565 570 575
Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg
580 585 590
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly
595 600 605
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
625 630 635 640
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
645 650 655
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg
660 665 670
Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680
<210> 15
<211> 450
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<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 Polypeptides
<400> 17
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser Gly Gly
210 215 220
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu Val Gln
225 230 235 240
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
245 250 255
Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg
260 265 270
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr
275 280 285
Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Ala Thr Leu
290 295 300
Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
305 310 315 320
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr
325 330 335
Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val
340 345 350
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
355 360 365
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
370 375 380
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
385 390 395 400
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
405 410 415
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
420 425 430
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
435 440 445
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
450 455 460
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
465 470 475 480
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
485 490 495
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
500 505 510
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
515 520 525
Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val
530 535 540
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
545 550 555 560
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
565 570 575
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
580 585 590
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
595 600 605
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
610 615 620
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
625 630 635 640
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
645 650 655
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
660 665 670
Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 685
<210> 18
<211> 686
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<400> 18
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser
210 215 220
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu
225 230 235 240
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu
245 250 255
Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp
260 265 270
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp
275 280 285
Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr
290 295 300
Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu Lys Leu Ser Ser
305 310 315 320
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr
325 330 335
Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
340 345 350
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
355 360 365
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
370 375 380
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
385 390 395 400
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
405 410 415
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
420 425 430
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
435 440 445
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
450 455 460
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
465 470 475 480
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
485 490 495
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
500 505 510
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
515 520 525
Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val
530 535 540
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
545 550 555 560
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
565 570 575
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
580 585 590
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
595 600 605
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
610 615 620
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
625 630 635 640
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
645 650 655
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
660 665 670
Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 685
<210> 19
<211> 452
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<400> 20
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys
355 360 365
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 21
<211> 684
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<400> 22
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Gly Gly Gly Ser Gly Gly Gly Ser
210 215 220
Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly
225 230 235 240
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val
245 250 255
Ser Gly Phe Thr Phe Asn Ser Phe Ala Met Ser Trp Val Arg Gln Ala
260 265 270
Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly
275 280 285
Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
290 295 300
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
305 310 315 320
Glu Asp Thr Ala Val Tyr Phe Cys Ala Lys Asp Lys Ile Leu Trp Phe
325 330 335
Gly Glu Pro Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
355 360 365
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
450 455 460
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
465 470 475 480
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
500 505 510
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
515 520 525
Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
530 535 540
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
545 550 555 560
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
565 570 575
Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg
580 585 590
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly
595 600 605
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
625 630 635 640
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
645 650 655
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg
660 665 670
Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680
<210> 23
<211> 122
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<400> 29
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
100 105
<210> 30
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<400> 33
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 34
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<400> 41
Asp Ala Ser Asn Arg Ala Thr
1 5
<210> 42
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<400> 47
Asp Ala Ser Asn Arg Ala Thr
1 5
<210> 48
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<400> 53
His Thr Ser Arg Leu His Ser
1 5
<210> 54
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<400> 59
Tyr Thr Ser Arg Leu His Ser
1 5
<210> 60
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<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 Polypeptides
<400> 65
Asp Ala Ser Asn Arg Ala Thr
1 5
<210> 66
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Polypeptides
<400> 66
Gln Gln Arg Ser Asn Trp Pro Pro Thr
1 5

Claims (9)

1.一种双特异性抗体，其特征在于，所述双特异性抗体由重链1、重链2和轻链构成，所述双特异性抗体选自以下抗体：1. A bispecific antibody, characterized in that the bispecific antibody is composed of a heavy chain 1, a heavy chain 2 and a light chain, and the bispecific antibody is selected from the following antibodies: 。 . 2. 一种免疫偶联物，其特征在于，所述免疫偶联物由以下构成：2. An immunoconjugate, characterized in that the immunoconjugate is composed of: (a) 如权利要求1所述的双特异性抗体；和(a) the bispecific antibody according to claim 1; and (b) 选自下组的偶联部分：可检测标记物、金纳米颗粒/纳米棒、纳米磁粒或其组合。(b) a conjugation moiety selected from the group consisting of a detectable label, gold nanoparticles/nanorods, nanomagnetic particles or a combination thereof. 3.如权利要求2所述的免疫偶联物，其特征在于，所述可检测标记物是放射性核素。3. The immunoconjugate of claim 2, wherein the detectable label is a radionuclide. 4.如权利要求1所述的双特异性抗体或权利要求2或3所述的免疫偶联物在制备抗肿瘤的药物中的用途，所述肿瘤是淋巴瘤，白血病，多发性骨髓瘤。4. Use of the bispecific antibody according to claim 1 or the immunoconjugate according to claim 2 or 3 in the preparation of an anti-tumor drug, wherein the tumor is lymphoma, leukemia, or multiple myeloma. 5.一种药物组合物，所述药物组合物中包含权利要求1所述的双特异性抗体或权利要求2或3所述的免疫偶联物以及药学上可接受的赋形剂。5. A pharmaceutical composition comprising the bispecific antibody according to claim 1 or the immunoconjugate according to claim 2 or 3 and a pharmaceutically acceptable excipient. 6.一种分离的多核苷酸，其特征在于，所述多核苷酸编码如权利要求1所述的双特异性抗体。6. An isolated polynucleotide, characterized in that the polynucleotide encodes the bispecific antibody according to claim 1. 7.一种载体，其特征在于，所述载体含有如权利要求6所述的多核苷酸。7. A vector, characterized in that the vector contains the polynucleotide according to claim 6. 8.一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求7所述的载体，或其基因组中整合有如权利要求6所述的多核苷酸；8. A host cell, characterized in that the host cell contains the vector according to claim 7, or the polynucleotide according to claim 6 is integrated into its genome; 或者，所述的宿主细胞表达如权利要求1所述的双特异性抗体。Alternatively, the host cell expresses the bispecific antibody according to claim 1. 9. 一种产生如权利要求1所述双特异性抗体的方法，其特征在于，包括步骤：9. A method for producing the bispecific antibody according to claim 1, comprising the steps of: (a) 在合适的条件下，培养如权利要求8所述的宿主细胞，从而获得含所述双特异性抗体的培养物；和(a) culturing the host cell according to claim 8 under suitable conditions to obtain a culture containing the bispecific antibody; and (b) 对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的双特异性抗体。(b) purifying and/or isolating the culture obtained in step (a) to obtain the bispecific antibody.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109071663A (en) * 2016-02-05 2018-12-21 奥里尼斯生物科学公司 Bispecific signaling agents and uses thereof
CN110540593A (en) * 2018-05-29 2019-12-06 无锡智康弘义生物科技有限公司 Novel anti-CD 3/anti-CD 20 bispecific antibodies
CN110551221A (en) * 2019-07-02 2019-12-10 广州爱思迈生物医药科技有限公司 Bispecific antibody and preparation method and application thereof

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CN103145847B (en) * 2013-02-05 2014-05-21 浙江大学 Anti-CD20 antibody-monomethyl auristatin E conjugate, preparation method and application thereof
CN104829730A (en) * 2015-04-14 2015-08-12 苏静 Bispecific antibody capable of combining immune cells to enhance tumor killing ability, preparation method and applications thereof
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Publication number Priority date Publication date Assignee Title
CN109071663A (en) * 2016-02-05 2018-12-21 奥里尼斯生物科学公司 Bispecific signaling agents and uses thereof
CN110540593A (en) * 2018-05-29 2019-12-06 无锡智康弘义生物科技有限公司 Novel anti-CD 3/anti-CD 20 bispecific antibodies
CN110551221A (en) * 2019-07-02 2019-12-10 广州爱思迈生物医药科技有限公司 Bispecific antibody and preparation method and application thereof

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