CN112010973B - Anti-4-1BB antibody, composition containing same and application thereof - Google Patents

Anti-4-1BB antibody, composition containing same and application thereof Download PDF

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CN112010973B
CN112010973B CN201911029988.3A CN201911029988A CN112010973B CN 112010973 B CN112010973 B CN 112010973B CN 201911029988 A CN201911029988 A CN 201911029988A CN 112010973 B CN112010973 B CN 112010973B
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CN112010973A (en
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宋德勇
董创创
徐洪光
窦昌林
刘秀
宁振飞
仉慧敏
韩镇
宗梦琦
贾惠言
沙春洁
沈振铎
王广珺
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Shandong Boan Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

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Abstract

The present invention provides an antibody or antigen-binding fragment specifically binding to human 4-1BB, comprising a VL domain and a VH domain comprising specific complementarity determining region sequences, a composition comprising the same, and use thereof in the preparation of an antitumor drug. The antibody or antigen binding fragment exhibits a T of at least 30h in a pharmacokinetic assay 1/2 And an AUC of at least 9000.00 μ g/mL × h 0‑t And the antitumor drug containing the antibody or the antigen binding fragment has better tumor growth inhibition activity and lower hepatotoxicity.

Description

Anti-4-1BB antibody, composition containing same and application thereof
Technical Field
The invention relates to the field of biomedicine, and particularly relates to an anti-4-1BB antibody, a composition containing the same and application thereof.
Background
The 4-1BB factor belongs to the tumor necrosis factor receptor superfamily, and is a transmembrane protein with 255 amino acids. 4-1BB is expressed on NK and NKT cells, regulatory T cells, Dendritic Cells (DCs), stimulated mast cells, differentiating myeloid cells, monocytes, neutrophils, and eosinophils. The ligand of 4-1BB is 4-1BBL, a glycoprotein belonging to the TNF superfamily, and is expressed in activated Antigen Presenting Cells (APC), myeloid progenitor cells, and hematopoietic stem cells. 4-1BB is combined with a ligand to provide a costimulation signal for a 4-1BB expression cell, and the costimulation signal can promote the proliferation and activation of a T cell and inhibit the activation-induced apoptosis, thereby enhancing the immune killing function of the T cell. At the same time, the activation of monocytes and the like is induced, the secretion of cytokines is promoted, and the monocytes, dendritic cells and the like play a role in immune regulation.
In the development process of the chimeric antigen engineered T cell, 4-1BB has an important role as an intracellular signal costimulatory molecule, and is applied to cell therapy research and clinic of different targets. Currently, a number of 4-1BB related drug research projects are in progress. The reported 4-1BB antibody sequence is not ideal in the aspects of protein binding sensitivity, protein affinity and the like, and mostly has no good tumor growth inhibition activity or obvious toxic and side effects. Therefore, there is a need to develop novel 4-1BB antibody drugs with better activity and lower toxic side effects.
Disclosure of Invention
The antibody or antigen-binding fragment of the present invention refers to an antibody or antigen-binding fragment that is detached from a human or animal body, i.e., an isolated antibody or antigen-binding fragment. Such antibodies include monoclonal produced antibodies, as well as chimeric or humanized forms of antibodies.
The English abbreviations of the present invention have the following meanings, VL represents a light chain variable region, VH represents a heavy chain variable region, L-CDR represents a light chain complementarity determining region, and H-CDR represents a heavy chain complementarity determining region.
The various embodiments described throughout this disclosure with respect to the VL domain, VH domain, L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 can be performed individually or in any combination.
The term "at least 70% homologous" as used herein means that the composition and order of at least 70% of the amino acids in an amino acid sequence are identical. Similarly, "at least 75% homologous," "at least 80% homologous," "at least 85% homologous," "at least 90% homologous," "at least 93% homologous," "at least 95% homologous," "at least 98% homologous," and "at least 99% homologous" refer to amino acid sequences that are identical in composition and order for at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99% of the amino acids, and "100% homologous" refers to amino acid sequences that are identical.
According to one aspect of the invention, there is provided an antibody or antigen-binding fragment that specifically binds human 4-1BB, the antibody or antigen-binding fragment comprising:
VH domain comprising a complementarity determining region selected from the amino acid sequences shown in SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63.
In some embodiments, the antibodies or antigen-binding fragments provided herein that specifically bind human 4-1BB further comprise a VL domain comprising a complementarity determining region selected from the group consisting of the amino acid sequences set forth in SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, and SEQ ID NO 40.
In some embodiments, the VL domain of an antibody or antigen-binding fragment provided herein that specifically binds human 4-1BB contains the complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein:
L-CDR1 is selected from the amino acid sequences shown in SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 32;
the L-CDR2 is selected from the amino acid sequences shown in SEQ ID NO. 33 and SEQ ID NO. 34;
the L-CDR3 is selected from the amino acid sequences shown in SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39 and SEQ ID NO. 40.
In other embodiments, the VH domain of an antibody or antigen-binding fragment that specifically binds human 4-1BB provided by the present invention comprises the complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein:
H-CDR1 is selected from the amino acid sequences shown in SEQ ID NO 41, 42, 43, 44, 45, 46, 47 and 48;
H-CDR2 is selected from the amino acid sequences shown in SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54 and SEQ ID NO 55;
H-CDR3 is selected from the amino acid sequences shown in SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, and SEQ ID NO 63.
In some embodiments, the VL domain of the antibody or antigen-binding fragment that specifically binds human 4-1BB comprises a combination of complementarity determining regions according to any one of (1) to (8):
(1) the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 35;
(2) the amino acid sequence of L-CDR1 shown in SEQ ID NO. 30, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 34, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 36;
(3) the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 37;
(4) an amino acid sequence of L-CDR1 shown in SEQ ID NO. 31, an amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and an amino acid sequence of L-CDR3 shown in SEQ ID NO. 38;
(5) the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 39;
(6) the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 38;
(7) the amino acid sequence of L-CDR1 shown in SEQ ID NO. 30, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 34, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 40; or
(8) The amino acid sequence of L-CDR1 shown in SEQ ID NO. 32, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 39.
In other embodiments, the VH domain of the antibody or antigen-binding fragment that specifically binds human 4-1BB comprises a combination of complementarity determining regions according to any one of (1) to (8):
(1) the amino acid sequence of H-CDR1 shown in SEQ ID NO. 41, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 49, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 56;
(2) the amino acid sequence of H-CDR1 shown in SEQ ID NO. 42, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 50, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 57;
(3) the amino acid sequence of H-CDR1 shown in SEQ ID NO. 43, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 51, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 58;
(4) the amino acid sequence of H-CDR1 shown in SEQ ID NO. 44, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 52, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 59;
(5) the amino acid sequence of H-CDR1 shown in SEQ ID NO. 45, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 52, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 60;
(6) the amino acid sequence of H-CDR1 shown in SEQ ID NO. 46, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 53, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 61;
(7) the amino acid sequence of H-CDR1 shown in SEQ ID NO. 47, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 54, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 62; or
(8) The amino acid sequence of H-CDR1 shown in SEQ ID NO. 48, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 55, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 63.
In some embodiments, the antibody or antigen-binding fragment that specifically binds human 4-1BB comprises any one combination of a VL domain and a VH domain as in (1) - (10) below:
(1) a VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 35, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 41, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 49, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 56;
(2) a VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 30, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 34, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 36, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 42, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 50, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 57;
(3) a VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 37, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 43, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 51, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 58;
(4) a VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 31, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 38, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 44, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 52, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 59;
(5) a VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 35, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 45, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 52, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 60;
(6) a VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 39, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 46, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 53, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 61;
(7) a VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 38, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 44, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 52, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 59;
(8) a VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 30, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 34, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 40, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 47, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 54, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 62;
(9) a VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 32, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 39, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 48, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 55, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 63; or
(10) A VL domain comprising the amino acid sequence of L-CDR1 shown in SEQ ID NO. 29, the amino acid sequence of L-CDR2 shown in SEQ ID NO. 33, and the amino acid sequence of L-CDR3 shown in SEQ ID NO. 35, and a VH domain comprising the amino acid sequence of H-CDR1 shown in SEQ ID NO. 44, the amino acid sequence of H-CDR2 shown in SEQ ID NO. 52, and the amino acid sequence of H-CDR3 shown in SEQ ID NO. 59.
In some more specific embodiments, the VL domain has an amino acid sequence that is at least 70% homologous to any of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, or SEQ ID NO 27. In other embodiments, the light chain amino acid sequence of an antibody or antigen-binding fragment that specifically binds human 4-1BB provided herein is at least 70% homologous to any one of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, or SEQ ID NO 27.
In some more specific embodiments, the VH domain has an amino acid sequence that is at least 70% homologous to any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, or SEQ ID NO 28. In other embodiments, the heavy chain amino acid sequence of an antibody or antigen-binding fragment provided herein that specifically binds human 4-1BB is at least 70% homologous to any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, or SEQ ID NO 28.
In some more specific embodiments, the antibody or antigen-binding fragment that specifically binds human 4-1BB comprises any one combination of a VL domain and a VH domain as described in (1) - (14) below:
(1) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 1 and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 2;
(2) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 3, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 4;
(3) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 5 and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 6;
(4) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 7, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 8;
(5) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 9, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 10;
(6) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 11, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 12;
(7) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 13, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 14;
(8) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 15 and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 16;
(9) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID NO. 17, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 18;
(10) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 19 and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 20;
(11) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 21, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 22;
(12) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 23, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 24;
(13) a VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID No. 25, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID No. 26; or
(14) A VL domain which is at least 90% homologous to the amino acid sequence shown in SEQ ID NO. 27, and a VH domain which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 28.
In some embodiments, the above-described combination of VL and VH domains may represent a combination of light chain and heavy chain sequences, i.e., the antibody or antigen-binding fragment that specifically binds human 4-1BB comprises any one of the following combinations of light chain and heavy chain sequences (1) - (14):
(1) a light chain sequence at least 70% homologous to the amino acid sequence shown in SEQ ID No. 1, and a heavy chain sequence at least 70% homologous to the amino acid sequence shown in SEQ ID No. 2;
(2) a light chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 3, and a heavy chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 4;
(3) a light chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 5, and a heavy chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 6;
(4) a light chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 7, and a heavy chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 8;
(5) a light chain sequence which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 9, and a heavy chain sequence which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 10;
(6) a light chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 11, and a heavy chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 12;
(7) a light chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 13, and a heavy chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 14;
(8) a light chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 15, and a heavy chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 16;
(9) a light chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 17, and a heavy chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 18;
(10) a light chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 19, and a heavy chain sequence that is at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 20;
(11) a light chain sequence which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 21, and a heavy chain sequence which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 22;
(12) a light chain sequence which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 23, and a heavy chain sequence which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 24;
(13) a light chain sequence at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 25, and a heavy chain sequence at least 70% homologous to the amino acid sequence set forth in SEQ ID NO. 26; or
(14) A light chain sequence which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 27, and a heavy chain sequence which is at least 70% homologous to the amino acid sequence shown in SEQ ID NO. 28.
The above-described "at least 70% homologous" of the present invention includes at least 75% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 93% homologous, at least 95% homologous, at least 98% homologous, at least 99% homologous, and 100% homologous. Sequences that are at least 70% homologous, at least 75% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 93% homologous, at least 95% homologous, at least 98% homologous, at least 99% homologous, and 100% homologous, as described herein, are sequences that are substantially identical in biological specificity and biological activity.
In some embodiments, the antibody or antigen-binding fragment that specifically binds human 4-1BB comprises any one combination of a VL domain and a VH domain as in (1) - (14) below:
(1) a VL domain of the amino acid sequence shown in SEQ ID NO. 1 and a VH domain of the amino acid sequence shown in SEQ ID NO. 2;
(2) a VL domain of the amino acid sequence shown in SEQ ID NO. 3 and a VH domain of the amino acid sequence shown in SEQ ID NO. 4;
(3) a VL domain of the amino acid sequence shown in SEQ ID NO. 5 and a VH domain of the amino acid sequence shown in SEQ ID NO. 6;
(4) a VL domain of the amino acid sequence shown in SEQ ID NO. 7 and a VH domain of the amino acid sequence shown in SEQ ID NO. 8;
(5) a VL domain of the amino acid sequence shown in SEQ ID NO. 9 and a VH domain of the amino acid sequence shown in SEQ ID NO. 10;
(6) a VL domain of the amino acid sequence shown in SEQ ID NO. 11 and a VH domain of the amino acid sequence shown in SEQ ID NO. 12;
(7) a VL domain of the amino acid sequence shown in SEQ ID NO. 13 and a VH domain of the amino acid sequence shown in SEQ ID NO. 14;
(8) a VL domain of the amino acid sequence shown in SEQ ID NO. 15 and a VH domain of the amino acid sequence shown in SEQ ID NO. 16;
(9) a VL domain of the amino acid sequence shown in SEQ ID NO. 17 and a VH domain of the amino acid sequence shown in SEQ ID NO. 18;
(10) a VL domain of the amino acid sequence shown in SEQ ID NO. 19 and a VH domain of the amino acid sequence shown in SEQ ID NO. 20;
(11) a VL domain of the amino acid sequence shown as SEQ ID NO. 21 and a VH domain of the amino acid sequence shown as SEQ ID NO. 22;
(12) a VL domain of the amino acid sequence shown in SEQ ID NO. 23 and a VH domain of the amino acid sequence shown in SEQ ID NO. 24;
(13) a VL domain of the amino acid sequence shown in SEQ ID NO. 25 and a VH domain of the amino acid sequence shown in SEQ ID NO. 26; or
(14) The VL domain of the amino acid sequence shown in SEQ ID NO. 27 and the VH domain of the amino acid sequence shown in SEQ ID NO. 28.
The antibodies or antigen-binding fragments provided by the invention specifically bind to human 4-1BB have slower in vivo metabolic rates and higher terminal elimination half-lives (T) 1/2 ) And has a larger area under the drug time curve (AUC). At one endIn some embodiments, the antibody or antigen binding fragment exhibits a T of at least 30h in a pharmacokinetic assay 1/2 And an AUC of at least 9000.00 μ g/mL × h 0-t . In some embodiments, the antibody or antigen binding fragment exhibits a T of at least 35h, at least 38h, at least 39h, at least 40h, at least 45h, at least 50h, at least 55h, at least 60h, at least 65h, or at least 70h in a pharmacokinetic assay 1/2 . In some embodiments, the antibody or antigen binding fragment exhibits a T in the pharmacokinetic assay of 35 to 70h, preferably 50 to 70h, more preferably 55 to 70h, and most preferably 60 to 70h 1/2 . In some embodiments, the antibody or antigen binding fragment exhibits an AUC of at least 9500.00 μ g/mL xh, at least 10000.00 μ g/mL xh, at least 10500.00 μ g/mL xh, at least 11000.00 μ g/mL xh, at least 12000.00 μ g/mL hh, at least 13000.00 μ g/mL hh, at least 14000.00 μ g/mL hh, at least 15000.00 μ g/mL hh, or at least 16000.00 μ g/mL hh in a pharmacokinetic assay 0-t . In some embodiments, the antibody or antigen binding fragment exhibits an AUC of (9500.00-16000.00) μ g/mL _ hr, preferably (10000.00-16000.00) μ g/mL _ hr, more preferably (11000.00-16000.00) μ g/mL _ hr, and most preferably (15000.00-16000.00) μ g/mL _ hr, in a pharmacokinetic assay 0-t 。
According to another aspect of the present invention, there is also provided a composition comprising an antibody or antigen-binding fragment as described above, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvent, dispersant, additive, plasticizer and medicinal auxiliary material.
According to a further aspect of the present invention there is also provided the use of an antibody or antigen-binding fragment which specifically binds human 4-1BB as described above, wherein said antibody or antigen-binding fragment exhibits an equilibrium dissociation constant KD of no more than 7.000E-7 in the binding to human 4-1BB protein in vitro, for inhibiting the growth of tumor cells, and for the preparation of an anti-tumor medicament. In some embodiments, the antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 6.000E-7 in binding to human 4-1BB protein in vitro. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 5.000E-7 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 4.000E-7 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 3.000E-7 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 2.000E-7 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 1.000E-7 in binding to human 4-1BB protein in vitro. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 9.000E-8 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 8.000E-8 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 7.000E-8 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 6.000E-8 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 5.000E-8 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 4.000E-8 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 3.000E-8 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 2.000E-8 in vitro binding to human 4-1BB protein. The antibody or antigen-binding fragment exhibits an equilibrium dissociation constant, KD, of no greater than 1.000E-8 in vitro binding to human 4-1BB protein.
According to another aspect of the present invention, there is also provided the use of an antibody or antigen-binding fragment that specifically binds human 4-1BB as described above for inhibiting the growth of tumor cells and for the preparation of an anti-tumor medicament, wherein the anti-tumor medicament comprises the antibody or antigen-binding fragment and a pharmaceutically acceptable carrier, and the tumor volume is 1/2 or less of that of a blank control group 20 days after administration of 3mg/kg of the anti-tumor medicament to a B-h4-1BB humanized mouse that is inoculated with colon cancer cells. In some embodiments, the tumor volume of a B-h4-1BB humanized mouse vaccinated with colon cancer cells after 20 days administration of 3mg/kg of the anti-tumor drug is 1/3 or less of that of a blank control group. In some embodiments, the B-h4-1BB humanized mouse inoculated with colon cancer cells has a tumor volume of 1/4 or less of that of a blank control group 20 days after administration of 3mg/kg of the anti-tumor agent. In some embodiments, the tumor volume of a B-h4-1BB humanized mouse vaccinated with colon cancer cells after 20 days administration of 3mg/kg of the anti-tumor drug is 1/5 or less of that of a blank control group. In some embodiments, the tumor volume of a B-h4-1BB humanized mouse vaccinated with colon cancer cells after 20 days administration of 3mg/kg of the anti-tumor drug is 1/10 or less of that of a blank control group. In some embodiments, the tumor volume of a B-h4-1BB humanized mouse vaccinated with colon cancer cells 24 days after administration of 3mg/kg of the anti-tumor drug is 1/5 or less of that of a blank control group. In some embodiments, the tumor volume of a B-h4-1BB humanized mouse vaccinated with colon cancer cells 24 days after administration of 3mg/kg of the anti-tumor drug is 1/10 or less of that of a blank control group. In some embodiments, the tumor volume of a B-h4-1BB humanized mouse vaccinated with colon cancer cells 24 days after administration of 3mg/kg of the anti-tumor drug is 1/20 or less of that of a blank control group. In some embodiments, a B-h4-1BB humanized mouse inoculated with colon cancer cells administered 0.75mg/kg, 1.5mg/kg of the anti-tumor drug at 21 days has a lower tumor volume than a control group administered 3mg/kg of Utomillumab.
In the above embodiments, the administration is by injection, preferably intraperitoneal injection, with a frequency of once every three days.
In some embodiments, administration of the antineoplastic drug does not cause significant fluctuations in the ALT and AST markers, and thus has lower hepatotoxicity. In some embodiments, in vivo administration of an antibody or antigen-binding fragment that specifically binds human 4-1BB as provided herein above does not cause significant fluctuations in the ALT and AST metrics. By "does not cause significant fluctuation in the ALT and AST indicators" as used herein is meant that the subject's level of ALT and AST is maintained at a level that fluctuates no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10% up and down relative to the level prior to administration. In some embodiments, the antibodies or antigen-binding fragments of the invention cause the concentration levels of ALT and AST in a subject to fluctuate by no more than ± 30%, preferably no more than ± 20%, more preferably no more than ± 10% within 1-30 days of in vivo administration. In some embodiments, the antibodies or antigen-binding fragments of the invention cause the concentration levels of ALT and AST in a subject to fluctuate by no more than ± 30%, preferably no more than ± 20%, more preferably no more than ± 10% within 1-30 days of in vivo administration. In some embodiments, the antibodies or antigen-binding fragments of the invention cause the concentration levels of ALT and AST in a subject to fluctuate by no more than ± 30%, preferably no more than ± 20%, more preferably no more than ± 10% within 1-15 days of in vivo administration. In some embodiments, the antibodies or antigen-binding fragments of the invention cause the concentration levels of ALT and AST in a subject to fluctuate by no more than ± 30%, preferably no more than ± 20%, more preferably no more than ± 10% within 1-10 days of in vivo administration. In some embodiments, the antibodies or antigen-binding fragments of the invention cause the concentration levels of ALT and AST in a subject to fluctuate by no more than ± 30%, preferably no more than ± 20%, more preferably no more than ± 10% within 1-8 days of in vivo administration. In some embodiments, the antibodies or antigen-binding fragments of the invention cause the concentration levels of ALT and AST in the subject to fluctuate by no more than ± 30%, preferably no more than ± 20%, more preferably no more than ± 10% within 1-5 days of in vivo administration.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
Figure 1 shows the transgenic mouse serum titers of the immunization protocol in example 1, wherein 1A shows the transgenic mouse serum titers after quadruplicate immunization and 1B shows the transgenic mouse serum titers after sextuple immunization;
FIGS. 2A-2C show the binding sensitivity of ELSIA detection anti-4-1BB antibody to 4-1BB protein in example 3;
FIGS. 3A-3D show the flow cytometry detection of the binding sensitivity of anti-4-1BB antibody to GS-H2/4-1BB functional cells in example 3;
FIGS. 4A-4C show that GS-H2/4-1BB functional cell line detects the activity of anti-4-1BB antibody in example 3;
FIGS. 5A-5C show the detection of anti-4-1BB antibody activity in the T cell activation assay of example 3;
FIGS. 6A-6H show Biacore's detection of the affinity of anti-4-1BB antibody to 4-1BB protein in example 3, wherein FIG. 6A represents 4BQ8-CA505-IgG2, FIG. 6B represents 4BQ16-CA630-IgG2, FIG. 6C represents 4BQ8-BA702-IgG2, FIG. 6D represents 4BQ10-BA1093-IgG2, FIG. 6E represents 4BQ33-CA1401-IgG2, FIG. 6F represents 4BQ37-CA1488-IgG2, FIG. 6G represents Utomillumab, and FIG. 6H represents Urelumab;
FIG. 7 shows the pharmacodynamic test (tumor volume change after administration) of anti-4-1BB antibody in B-h4-1BB humanized mouse MC38 colon cancer animal model in example 4;
FIG. 8 shows the pharmacodynamic experiment (tumor volume after 20 days) of anti-4-1BB antibody in B-h4-1BB humanized mouse MC38 colon cancer animal model in example 4;
FIG. 9 shows the pharmacodynamic test (tumor volume change after administration) of anti-4-1BB antibody in B-h4-1BB humanized mouse MC38 colon cancer animal model in example 4;
FIG. 10 shows the pharmacodynamic experiment (tumor volume after 24 days) of anti-4-1BB antibody in B-h4-1BB humanized mouse MC38 colon cancer animal model in example 4;
FIG. 11 shows the pharmacodynamic experiment (tumor volume change after administration) of anti-4-1BB antibody in B-h4-1BB humanized mouse MC38 colon cancer animal model in example 4;
FIG. 12 shows the pharmacokinetic experiment (one) of anti-4-1BB antibody in cynomolgus monkey in example 5;
FIG. 13 shows the pharmacokinetic experiment of anti-4-1BB antibody in cynomolgus monkey in example 5 (II);
FIGS. 14A-14B show the profile of the anti-4-1BB antibody causing the level of ALT and AST in cynomolgus monkeys in example 6.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The features mentioned in connection with the different embodiments described throughout the present application can be implemented in combination with each other.
EXAMPLE 1 Generation of anti-4-1BB monoclonal antibodies
1.1 immunization protocol
The transgenic Boan-hMab of the boansheng full-human antibody is immunized by emulsifying protein 4-1BB (Yiqiao Shenzhou, 10041-H08H) and Freund's adjuvant. The first immunization uses Freund's complete adjuvant, the subsequent immunization uses Freund's incomplete adjuvant, and 10 mice and 5 mice are immunized respectively in two batches. 8 and 4 mice with higher serum titers were selected for booster immunizations, and after 3 to 4 days, the mice were sacrificed and the spleens were removed for subsequent experiments. The serum titers of the mice are shown in FIG. 1.
1.2 phage library creation
Taking spleen cells of an immune mouse, adding Trizol (Thermo Scientific, catalog number 15596-026), adding 1/5 volumes of chloroform after full lysis, fully mixing, standing at room temperature for 20min, centrifuging at 4 ℃ 12000rpm for 20min, taking an upper layer aqueous solution, adding isopropanol with the same volume, standing at room temperature for 20min, centrifuging at 4 ℃ 12000rpm for 20min, discarding a supernatant aqueous solution, adding 75% ethanol for washing twice, centrifuging at 4 ℃ 12000rpm for 5min, discarding an aqueous solution, keeping a precipitate, air-drying at room temperature, adding DEPC water for re-suspending the precipitate to obtain RNA, and performing reverse transcription on the RNA into cDNA by using a Roche Kit Transcriptor First and StrcDNAsthesis Kit (Roche Applied Science, catalog number 4897030001) according to the specification. Phage library establishment procedures reference Carlos F.Barbas III, Phage displayy: the method described in A laboratory manual was performed by obtaining the variable regions of the heavy and light chains from cDNA by PCR, obtaining ScFv by overlap extension PCR of the variable regions of the heavy and light chains, digesting ScFv with SfiI (NEB, catalog # R0123L) at 50 ℃ for 5 hours, ligating the digested ScFv (single chain Fv) with plasmid pCOMB3x, transfecting the ligation product into E.coli TG1 competent cells, incubating at 37 ℃ for 1 hour, adding 75mL of 2YT medium (supplemented with ampicillin and glucose), incubating at 37 ℃ for 1.5 hours at 200rpm, taking 2mL of the cells for OD600 determination, adding the prepared phase at a ratio of 1:20, standing at 37 ℃ for 30min, centrifuging at 4000rpm, taking the bacterial pellet, resuspending at 30 rpm after incubation with 100mL of the medium supplemented with ampicillin and kanamycin, then at 200rpm, taking supernatant for 10 days, resuspending at 12000rpm, adding 4% PEG8000 and 3% NaCl, ice-cooling for 1h, centrifuging at 4 deg.C 12000rpm for 30min, adding 2YT culture medium into the precipitate, resuspending, adding 7% DMSO, and freezing. Phage library 4-1BB Q33, library volume 1.4X 10, established with mouse accession number 4-1BB Q33 8 (ii) a Phage library 4-1BB Q37, library volume 1.76X 10, established with mouse accession number 4-1BB Q37 8 。
1.3 phage library screening
1. The plates were screened with 4-1BB-His protein (10041-H08H, Okayama, supra) coated at 1. mu.g/well, left overnight at 4 ℃, blocked with 3% skimmed milk for 1H the following day, and phage library (2X 10) 12 ) Incubate for 2h, wash 4-10 times, and elute 4-1 BB-specifically bound phage using an Elution Buffer (pH 2.2).
2. Magnetic bead screening, in which 4-1BB-Fc protein (10041-H02H, Chinese, supra) is biotinylated according to conventional procedures (molar ratio of the input 4-1BB protein to biotin 1:2), and then bound to Thermo magnetic beads (Invitrogen Dynabeads M-280 Streptavidin, 00355871), and incubated with a phage library, and 4-10 times of washing are performed, and then 4-1 BB-specifically bound phage are eluted by an Elution Buffer (pH 2.2). The clones obtained by screening and the sources are shown in Table 1.
TABLE 1 screening to obtain anti-4-1BB antibody source table
Cloning Source bank Mouse of origin Elutriation method
4BQ8-CA505-IgG2 PLQ8 4-1BBQ08 Plate screening
4BQ16-CA630-IgG2 PLQ16 4-1BBQ16 Plate screening
4BQ8-BA702-IgG2 PLQ8 4-1BBQ08 Magnetic bead screening
4BQ9-BA912-IgG2 PLQ19 4-1BBQ19 Magnetic bead screening
4BQ13-BA959-IgG2 PLQ19 4-1BBQ19 Magnetic bead screening
4BQ10-BA1093-IgG2 PLQ22 4-1BBQ22 Magnetic bead screening
4BQ11-BA1109-IgG2 PLQ29 4-1BBQ29 Magnetic bead screening
4BQ11-BA1142-IgG2 PLQ30 4-1BBQ30 Magnetic bead screening
4BQ11-BA1145-IgG2 PLQ30 4-1BBQ30 Magnetic bead screening
4BQ33-CA1401-IgG2 PLQ33 4-1BBQ33 Plate screening
4BQ33-CA1412-IgG2 PLQ33 4-1BBQ33 Plate screening
4BQ34-CA1455-IgG2 PLQ34 4-1BBQ34 Plate screening
4BQ37-CA1488-IgG2 PLQ37 4-1BBQ37 Plate screening
4BQ37-CA1491-IgG2 PLQ37 4-1BBQ37 Plate screening
Example 2 molecular construction and production of candidate antibodies
Clones 4BQ8-CA505/BA702, 4BQ16-CA630, 4BQ9-BA912, 4BQ13-BA959, 4BQ10-BA1093, 4BQ11-BA1109/BA1142/BA1145, 4BQ33-CA1401/CA1412, 4BQ34-CA1455, 4BQ37-CA1488/CA1491 were sent to Invitrogen biotechnology Co., Ltd for sequencing, the CA1401 clone had the heavy chain belonging to the germline gene IGHV3-30 family and the light chain belonging to the germline gene IGKV1-5 family by sequence analysis. The amino acid sequence of each clone is shown in Table 2 below.
TABLE 2 screening of cloned amino acid sequences
Figure BDA0002249859860000151
Figure BDA0002249859860000161
Figure BDA0002249859860000171
Figure BDA0002249859860000181
Fusion of the antibody VH Gene to the IgG2 heavy chain constant region sequence SEQ ID NO 64After the VL gene was fused with the Kappa light chain constant region SEQ ID NO:65, the resulting fusion was confirmed by a conventional molecular biology technique: variable region amplification: (
Figure BDA0002249859860000182
DNA Polymerase manufacturer: TAKARA cargo number: R010A batch No.: # N5901BA), signal peptide overlap extension with variable region, homologous recombination (Clonexpress II One Step Cloning Kit manufacturer: vazyme cat # s: batch number C112-01: 7E151F7) into the vector pCDNA3.4(Life Technology), transfected into HEK293 cells at 37 ℃ \ 8% CO 2 Culture in a shaker at 125rpm, purifying the transient expression supernatant after 6-7 days by ProteinA affinity chromatography to obtain 4-1BB antibody, and determining the antibody concentration by UV280 binding extinction coefficient.
64(IgG2 heavy chain constant region sequence)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-
65(Kappa light chain constant region sequence)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Control antibody production: the amino acid sequence of the Hurrill 4-1BB antibody Utomillumab is determined by IMGT data and patent CN201180054004, the amino acid sequence is inserted into a vector pCDNA3.4 after full gene synthesis and is expressed by HEK293 cells, and the produced antibody is named as Utomillumab. The Urelumab antibody was expressed by the same expression method as another positive control.
Utomillumab heavy chain
EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-(SEQ ID NO:66)
Utomillumab light chain
SYELTQPPSVSVSPGQTASITCSGDNIGDQYAHWYQQKPGQSPVLVIYQDKNRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCATYTGFGSLAVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS-(SEQ ID NO:67)
Urelumab heavy chain
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK-(SEQ ID NO:68)
Urelumab light chain
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:69)
Example 3 characterization of candidate antibodies
3.1 Elisa detection of binding of candidate antibodies to 4-1BB protein
Antigen 4-1BB (10041-H08H, Cassia) coated at different concentrations (0.2. mu.g/mL, 0.05. mu.g/mL, 0.0125. mu.g/mL, 0.003125. mu.g/mL, and 0.0009766. mu.g/mL), 100. mu.L/well overnight at 4 ℃; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100 mu L of each candidate antibody with the concentration of 1 mu g/mL into each hole, and incubating for 1h at 37 ℃; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader. The results are shown in FIGS. 2A-2C.
3.2 flow assay of binding of candidate antibodies to 4-1BB cells
To a 96-well round bottom plate, 50. mu.L of 4-1BB functional cells (Kinsery, M00609) with a cell number of 5E 4/well were added, each candidate antibody was diluted in a gradient manner with FACS buffer (sterile PBS, 0.2% BSA), and added to the 96-well round bottom plate at 50. mu.L/well, followed by incubation at 4 ℃ for 1 hour. The supernatant was discarded after centrifugation at 2000rpm for 3min, washed 2 times with FACS buffer, 100. mu.L/well fluorescent secondary antibody (Southern Biotech, 2040-09) was added to a final concentration of 1. mu.g/mL, incubated at 4 ℃ for 1h, centrifuged 3min at 2000rpm and discarded, washed 2 times with FACS buffer, resuspended in 100. mu.L/well FACS buffer and examined by flow cytometry (Ason, Novocyte 2060). The results are shown in tables 3-6 and FIGS. 3A-3D.
As shown in tables 3-6 and FIGS. 3A-3D, candidate antibodies CA1401, CA1412, CA1455 and CA1491 bind to 4-1BB functional cells at 0.66. mu.g/mL, 0.22. mu.g/mL, 0.07. mu.g/mL and 0.02. mu.g/mL, significantly more than control Utomillumab or Urelumab. 4-1BB on the surface of 4-1 BB-functional cells is much closer to the native conformation, suggesting that these candidate antibodies will have a strong ability to bind native antigen in vivo.
TABLE 3 concentration-mean fluorescence intensity data corresponding to FIG. 3A
Figure BDA0002249859860000201
Figure BDA0002249859860000211
TABLE 4 concentration-mean fluorescence intensity data for FIG. 3B
Figure BDA0002249859860000214
TABLE 5 concentration-mean fluorescence intensity data for FIG. 3C
Figure BDA0002249859860000212
TABLE 6 concentration-mean fluorescence intensity data for FIG. 3D
Figure BDA0002249859860000213
3.3 in vitro functional assays of antibodies
3.3.1 GS-H2/4-1BB functional cell line for detecting antibody cytological Activity
Adding FBS (Gibco, 10091-148) with the final concentration of 2 percent into an MEM culture medium (Gibco, 41090-036) to obtain a working culture medium, resuspending GS-H2/4-1BB cells to 1E5 cells/mL by using the working culture medium, inoculating 50 mu L/well of the cells into a 384-well cell culture plate, placing the plate at 37 ℃ and 5 percent CO 2 The incubator lasts 18-20 h. To the antibody samples, a cross-linked secondary antibody (SA 015, kyoho) was added or not, at a molar ratio of cross-linked secondary antibody to antibody sample of 1:2, and each antibody sample was diluted in working medium gradient and 50 μ L of antibody per well was added to 384-well cell culture plates. 37 ℃ and 5% CO 2 Culturing in an incubator for 24-26 h. The supernatant IL8 concentration was measured using IL8 kit (cisbio, 62IL8PEB), and the results are shown in FIGS. 4A-4C.
For the 4-1BB antibody, hepatotoxicity is a major challenge during treatment. From phase I clinical results, hepatotoxicity of Utomilumab was lower than Urelumab. In the experiment, the activity of the Utomilumab on the 4-1BB functional cells in the absence of the cross-linked secondary antibody is lower than that in the presence of the cross-linked secondary antibody, while the Urelumab shows stronger activity of the 4-1BB functional cells in the presence of the cross-linked secondary antibody, and the difference behavior is probably related to the difference of hepatotoxicity of two control drugs. Thus, for candidate antibodies, the activity on 4-1BB functional cells is lower without cross-linked secondary antibody, and there is a possibility that the resulting hepatotoxicity is also lower.
As shown in fig. 4A-4C, candidate antibodies CA505, CA630, BA702.1, BA1093, CA1401, CA1412, CA1455, CA1488 and CA1491 had lower 4-1BB functional cellular activity in the absence of cross-linked secondary antibody than in the presence of cross-linked secondary antibody, similar to the behavior of Utomilumab with low hepatotoxicity, suggesting that these candidate antibodies are also highly likely to have lower hepatotoxicity.
3.3.2T cell activation assay for detection of antibody cell Activity
Adding to 1640 medium (Gibco, 11875-119)In FBS (Gibco, 10091-148) with a final concentration of 10%, namely complete medium, OKT3 (Pepstosse, CDE-M120a) was diluted to 20. mu.g/mL with DPBS (Gibco, 14190-144), and 50. mu.L of each well was added to a 96-well cell culture plate; antibody samples were diluted in a DPBS gradient, 50 μ Ι _ of formulated antibody per well was added to a 96-well cell culture plate, and the cell culture plate was incubated overnight in a refrigerator at 4 ℃. The CD3+ T cells were revived, centrifuged at 1000rpm for 5min, the complete medium resuspended and the cell density adjusted to 1E 5/mL. The antibody sample and OKT3 were aspirated from the cell culture plate and 200. mu.L of cell suspension was added to each well at 37 ℃ with 5% CO 2 After 3-5 days of culture in an incubator, cell supernatants were collected and assayed for IL-2 concentration using an IL-2 assay kit (Biolegend, 431805) and IFN-. gamma.concentration using an IFN-. gamma.assay kit (Biolegend, 430105). The results are shown in FIGS. 5A-5C.
As shown in FIGS. 5A-5C, candidate antibodies CA1401, BA1093 and BA702 stimulated T cells to secrete cytokine IFN-. gamma.better than the control antibody under the same concentration conditions, indicating that the cytological activity of the candidate antibodies was better than that of the control antibody.
3.4 SPR detection of the affinity of the antibody to the 4-1BB protein
Antibody binding kinetics were measured using a biacore x100 instrument based on surface plasmon resonance (SRP) technology. The anti-Human IgG antibody amino groups were coupled to a CM5 biosensor chip by the GE anti Human IgG FC amino coupling kit (GE, cat # BR-1008-39) to obtain approximately 1000 Response Units (RU). For kinetic measurements, 4-1BB protein (10041-H08H, see Chinesen, supra) was serially diluted 2-fold in HBS-EP +1 × (GE, BR-1006-69) buffer, starting at 50nM, diluted 2-fold in 4 concentration gradients, and set at 0 concentration. Antibody: 1 mu g/ml, sample introduction time of 60s, flow rate of 5 mu L/min, and stability of 5 s; 4-1BB protein: binding for 60s, flow rate 30 uL/min, and dissociation for 300 s; regeneration: with 3M MgCl 2 buffer was regenerated for 30s, Startup 2 times. The association constant (ka) and dissociation constant (KD) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2), with the equilibrium dissociation constant (KD) calculated as the ratio KD/ka. The affinity data for each antibody is shown in Table 7, and the affinity measurement curves are shown in FIGS. 6A-6H.
TABLE 7 BiAcore detection of candidate antibody binding kinetics
Antibody ID Ka(1/Ms) Kd(1/s) KD(M)
4BQ8-CA505-IgG2 4.19E+05 0.01841 4.39E-08
4BQ16-CA630-IgG2 1.76E+05 2.57E-04 1.46E-09
4BQ8-BA702-IgG2 9.00E+05 0.02342 2.60E-08
4BQ10-BA1093-IgG2 2.42E+04 4.23E-04 1.75E-08
4BQ33-CA1401-IgG2 4.11E+05 0.02306 5.61E-08
4BQ37-CA1488-IgG2 3.79E+04 3.80E-04 1.01E-08
Utomilumab 2.57E+06 0.01778 6.92E-09
Urelumab 5.16E+05 0.002513 4.87E-09
Example 4 pharmacodynamic Studies of anti-4-1BB antibody in B-h4-1BB humanized mouse MC38 Colon cancer subcutaneous graft tumor model
A mouse colon cancer MC38 cell is inoculated to a B-h4-1BB humanized mouse of a Beijing Baioeiosi chart to establish a mouse colon cancer animal model for the function research of an anti-4-1BB antibody. Resuspended MC38 cells in PBS at 5X 10 5 The cells were inoculated subcutaneously on the right side of B-h4-1BB humanized mouse at a concentration of 0.1mL, at a volume of 0.1 mL. When the mean tumor volume reached about 114mm 3 At this time, appropriate mice were selected for group entry based on tumor volume and body weight of the mice. The drug was distributed evenly to each experimental group of 6 individuals each, and the administration was started on the day of the group. The route of administration was i.p., the frequency of administration was Q3D (once every three days), and the number of administrations was 6. Tumor volume and body weight were measured 2 times per week after grouping and mouse body weight and tumor volume were recorded. The results of the inhibition of tumor volume growth in mice by the antibodies are shown in tables 8-10 and FIGS. 7-11. Under the same dosage condition, the 4BQ33-CA1401-IgG2 has the most obvious effect of inhibiting the tumor growth and is superior to the Utomilumab (FIGS. 9-10). In addition, compared with the dose of Utomillumab 3mg/kg, 4BQ33-CA1401-IgG2 also showed better tumor suppression effects than Utomillumab at 0.75mg/kg and 1.5mg/kg (FIG. 11).
TABLE 8 comparative analysis of mean tumor volumes between groups and Tanezumab 20 days after dosing in FIGS. 7-8 (Dunnett test)
Figure BDA0002249859860000231
Figure BDA0002249859860000241
TABLE 9 comparative analysis of mean tumor volumes between groups and vehicle 24 days after dosing in FIGS. 9-10 (Dunnett test)
P P
4BQ33-CA1401-IgG2 Vehicle <0.05 7.1823E-08
4BQ37-CA1488-IgG2 Vehicle <0.05 2.9076E-07
Utomilumab Vehicle <0.05 4.3271E-07
TABLE 10 comparative analysis of mean tumor volume between groups and vehicle 21 days after dosing in FIG. 11 (Dunnett test)
P P
Utomilumab 3mg/kg Vehicle <0.05 0.000152
4BQ33-CA1401 0.75mg/kg Vehicle <0.05 3.149E-05
4BQ33-CA1401 1.5mg/kg Vehicle <0.05 9.731E-06
4BQ33-CA1401 3mg/kg Vehicle <0.05 2.221E-05
Example 5 pharmacokinetic study of anti-4-1BB antibody in cynomolgus monkeys
Male cynomolgus monkeys collected 1.5mL of whole blood into serum thrombotubes on day 1, 3, 7, 10, 14, 17, 21, 24, and 28 cephalic or saphenous veins (no administration vein) before (0 o' clock) and 5min, 30min, 6h after administration, and were left to stand at room temperature for 30min before centrifugation. The centrifugation was carried out at room temperature and 3500rpm for 10 min. Approximately 0.5mL serum was then dispensed into prepared EP tubes. After the serum is subpackaged, the serum is immediately put into a refrigerator at the temperature of 80 ℃ below zero for freezing storage until PK analysis is carried out.
The method for detecting the blood concentration comprises the following steps: 4-1BB protein coating, and standing in a refrigerator at 5 ℃ overnight. After washing, the plate is blocked for 1h at 37.5 ℃ by PBS containing 1% BSA, and after washing, 100 mu L/well of the label, the quality control substance and the sample to be tested are added and incubated for 1h at 37.5 ℃. After washing, 100. mu.L/well of HRP-labeled anti-human IgG secondary antibody was added and incubated at 37.5 ℃ for 1 hour. After washing the plate, adding 100 mu L/hole TMB color development solution, placing for 25min at 37.5 ℃ in a dark place, and reading OD450 and OD650 on a microplate reader after terminating the reaction. The pharmacokinetic curves are shown in fig. 12 and 13. The pharmacokinetic parameters are shown in tables 11 and 12.
From the metabolic curves, the metabolic rates of 4BQ33-CA1401-IgG2 and 4BQ37-CA1488-IgG2 in cynomolgus monkeys are significantly slower than the slowest metabolic rate of Utomillumab, 4BQ33-CA1401-IgG2 (FIGS. 12 and 13). AUC of 4BQ33-CA1401-IgG2 and 4BQ37-CA1488-IgG2 0-t 11180.70 and 10791.81 μ g/mL _ h, respectively, much higher than 4025.20 μ g/mL _ h of Utomilumab (tables 11, 12), indicate that lower dosages and better tumor suppression are possible.
TABLE 11 summary of pharmacokinetic experiment parameters of anti-4-1BB antibody in cynomolgus monkey
Figure BDA0002249859860000251
TABLE 12.Anti-4-1BB antibody in cynomolgus monkey Chinese medicine pharmacokinetics experiment parameter summary (II)
Figure BDA0002249859860000252
Example 6 hepatotoxicity study of anti-4-1BB antibody in cynomolgus monkeys
About 3mL of blood was collected from the monkey's upper limb vein or other suitable site about 24, 48, 96 hours after administration and on test days 8, 13, 15, 17, 22, 29 without anticoagulation. Centrifuging the blood sample at 15-25 ℃ at 1800g for 10min, separating serum, and detecting aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) by an IFCC P-5' -P method. The results are shown in FIGS. 14A-14B.
As shown in the figure, both ALT and AST indexes of the Utomillumab are obviously increased and fallen back on days 1-8 after the administration, which indicates that the medicine has certain influence on liver cells, while the ALT and AST indexes are not obviously fluctuated (the ALT is slightly fluctuated on day 13) under the same dosage condition of 4BQ33-CA1401-IgG2, which indicates that the hepatotoxicity of the 4BQ33-CA1401-IgG2 is weaker than that of the Utomillumab.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Sequence listing
<110> Shandong Bo' an Biotechnology Ltd
<120> an anti-4-1BB antibody, a composition comprising the same and use thereof
<150> 201910459133.8
<151> 2019-05-30
<150> 201910459134.2
<151> 2019-05-30
<160> 69
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Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
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Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
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Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
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Tyr Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
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Gly Glu Val Asn His Ile Gly Gly Thr Asn Tyr Asn Pro Ser Leu Lys
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Ser Arg Val Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu
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Asn Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
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Arg Gly Gly Ile Ile Thr Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr
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Met Val Thr Val Ser Ser
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Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp
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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
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Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
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Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
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Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
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Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
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Tyr Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
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Lys
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Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
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Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Arg Asp Gly Asp Ser Asn Tyr Val Pro Gln Tyr Phe Asn Tyr Trp
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Ser Asn Asn Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
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Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
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Tyr Tyr Ser Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
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<212> PRT
<213> Homo sapiens
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Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
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Gln Asp Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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<210> 11
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<213> Homo sapiens
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Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
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Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
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Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
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Tyr Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
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Lys
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<211> 124
<212> PRT
<213> Homo sapiens
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
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Gly Arg Ile Ile Pro Ile His Gly Ile Ala Asn Tyr Ala Gln Lys Phe
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Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Thr Tyr
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Met Glu Leu Arg Ser Leu Ser Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Arg Glu Arg Ala Ile Gly Ser Gly Ile Tyr Pro Tyr Ser Phe Asn
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Tyr Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
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<210> 13
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<212> PRT
<213> Homo sapiens
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Asp Ile Arg Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
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Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
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Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
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Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
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Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 14
<211> 127
<212> PRT
<213> Homo sapiens
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Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
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50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Leu Leu Gly Glu Cys Ser Gly Gly Ser Cys Tyr Tyr Gly Asp
100 105 110
Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 15
<211> 112
<212> PRT
<213> Homo sapiens
<400> 15
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 16
<211> 124
<212> PRT
<213> Homo sapiens
<400> 16
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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Val Thr Leu Ser Asn Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Pro Ile His Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Glu Arg Ala Ile Gly Ser Gly Thr Tyr Pro Tyr Ser Phe Asn
100 105 110
Tyr Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 17
<211> 112
<212> PRT
<213> Homo sapiens
<400> 17
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
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Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 18
<211> 124
<212> PRT
<213> Homo sapiens
<400> 18
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Met Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Val Thr Leu Ser Asn Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Pro Ile His Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Asp Arg Val Thr Met Thr Ala 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 Thr Glu Arg Ala Ile Gly Ser Gly Thr Tyr Pro Tyr Ser Phe Asn
100 105 110
Tyr Trp Gly Arg Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 19
<211> 107
<212> PRT
<213> Homo sapiens
<400> 19
Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 20
<211> 117
<212> PRT
<213> Homo sapiens
<400> 20
Glu Val Gln Leu Val Gln 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 Thr Phe Ser Thr Tyr
20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Val Ser Tyr Asp Gly Ser Thr Gln 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 Tyr Cys
85 90 95
Ala Arg Asp Ala Ser Lys Gly Phe Asp Ser Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 21
<211> 107
<212> PRT
<213> Homo sapiens
<400> 21
Asp Ile Arg Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 22
<211> 117
<212> PRT
<213> Homo sapiens
<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 Thr Phe Ser Thr Tyr
20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Val Ser Tyr Asp Gly Ser Thr Gln Tyr His 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 Met Tyr Tyr Cys
85 90 95
Ala Arg Asp Ala Ser Lys Gly Phe Asp Ser Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 23
<211> 113
<212> PRT
<213> Homo sapiens
<400> 23
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 24
<211> 118
<212> PRT
<213> Homo sapiens
<400> 24
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Pro Glu Trp Ile
35 40 45
Gly Glu Val Asn His Ile Gly Gly Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Ser Ile Ser Leu Asp Thr Ser Lys Asn Gln Tyr 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
Arg Gly Gly Ile Ile Thr Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 25
<211> 113
<212> PRT
<213> Homo sapiens
<400> 25
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 26
<211> 121
<212> PRT
<213> Homo sapiens
<400> 26
Glu Val Gln Leu Val Gln 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 Thr Phe Ser Ser Tyr
20 25 30
Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Phe Ile Ser Tyr Asp Glu Asn Tyr Glu 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 Tyr Cys
85 90 95
Ala Arg Gly Gly Glu Leu Gln Leu Pro Arg Pro Phe Asp Val Trp Gly
100 105 110
Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 27
<211> 113
<212> PRT
<213> Homo sapiens
<400> 27
Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro Pro Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile
100 105 110
Lys
<210> 28
<211> 124
<212> PRT
<213> Homo sapiens
<400> 28
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Met Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Val Thr Leu Ser Asn Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Pro Ile His Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Asp Arg Val Thr Met Thr Ala 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 Thr Glu Arg Ala Ile Gly Ser Gly Thr Tyr Pro Tyr Ser Phe Asn
100 105 110
Tyr Trp Gly Arg Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 29
<211> 12
<212> PRT
<213> Homo sapiens
<400> 29
Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr
1 5 10
<210> 30
<211> 6
<212> PRT
<213> Homo sapiens
<400> 30
Gln Ser Ile Ser Ser Trp
1 5
<210> 31
<211> 12
<212> PRT
<213> Homo sapiens
<400> 31
Arg Ser Ile Leu Tyr Ser Ser Asn Asn Asn Asn Tyr
1 5 10
<210> 32
<211> 12
<212> PRT
<213> Homo sapiens
<400> 32
Gln Ser Ile Leu Tyr Ser Ser Asn Asn Lys Asn Tyr
1 5 10
<210> 33
<211> 3
<212> PRT
<213> Homo sapiens
<400> 33
Trp Ala Ser
1
<210> 34
<211> 3
<212> PRT
<213> Homo sapiens
<400> 34
Lys Ala Ser
1
<210> 35
<211> 9
<212> PRT
<213> Homo sapiens
<400> 35
Gln Gln Tyr Tyr Ser Thr Pro Pro Thr
1 5
<210> 36
<211> 10
<212> PRT
<213> Homo sapiens
<400> 36
Gln Gln Tyr Tyr Ser Thr Pro Pro Trp Thr
1 5 10
<210> 37
<211> 8
<212> PRT
<213> Homo sapiens
<400> 37
Gln Gln Tyr Tyr Gly Thr Pro Thr
1 5
<210> 38
<211> 8
<212> PRT
<213> Homo sapiens
<400> 38
Gln Gln Tyr Tyr Ser Thr Pro Thr
1 5
<210> 39
<211> 9
<212> PRT
<213> Homo sapiens
<400> 39
Gln Gln Tyr Tyr Ser Thr Pro Trp Thr
1 5
<210> 40
<211> 9
<212> PRT
<213> Homo sapiens
<400> 40
Gln Gln Tyr Asn Ser Tyr Ser Leu Thr
1 5
<210> 41
<211> 8
<212> PRT
<213> Homo sapiens
<400> 41
Gly Gly Ser Phe Ser Gly Tyr Tyr
1 5
<210> 42
<211> 8
<212> PRT
<213> Homo sapiens
<400> 42
Gly Phe Thr Phe Ser Gly Tyr Val
1 5
<210> 43
<211> 8
<212> PRT
<213> Homo sapiens
<400> 43
Gly Ile Thr Phe Ser Asn Tyr Ala
1 5
<210> 44
<211> 8
<212> PRT
<213> Homo sapiens
<400> 44
Gly Val Thr Leu Ser Asn Tyr Ala
1 5
<210> 45
<211> 8
<212> PRT
<213> Homo sapiens
<400> 45
Gly Gly Thr Phe Ser His Tyr Thr
1 5
<210> 46
<211> 8
<212> PRT
<213> Homo sapiens
<400> 46
Gly Gly Ser Phe Ser Gly His Tyr
1 5
<210> 47
<211> 8
<212> PRT
<213> Homo sapiens
<400> 47
Gly Phe Thr Phe Ser Thr Tyr Val
1 5
<210> 48
<211> 8
<212> PRT
<213> Homo sapiens
<400> 48
Gly Phe Thr Phe Ser Ser Tyr Ser
1 5
<210> 49
<211> 7
<212> PRT
<213> Homo sapiens
<400> 49
Val Asn His Ile Gly Gly Thr
1 5
<210> 50
<211> 8
<212> PRT
<213> Homo sapiens
<400> 50
Ile Ser Phe Asp Gly Ser Ser Lys
1 5
<210> 51
<211> 8
<212> PRT
<213> Homo sapiens
<400> 51
Ile Ser Tyr Asp Gly Ser Lys Lys
1 5
<210> 52
<211> 8
<212> PRT
<213> Homo sapiens
<400> 52
Ile Ile Pro Ile His Gly Ile Ala
1 5
<210> 53
<211> 7
<212> PRT
<213> Homo sapiens
<400> 53
Val Ile His Ser Gly Thr Thr
1 5
<210> 54
<211> 8
<212> PRT
<213> Homo sapiens
<400> 54
Val Ser Tyr Asp Gly Ser Thr Gln
1 5
<210> 55
<211> 8
<212> PRT
<213> Homo sapiens
<400> 55
Ile Ser Tyr Asp Glu Asn Tyr Glu
1 5
<210> 56
<211> 12
<212> PRT
<213> Homo sapiens
<400> 56
Ala Arg Gly Gly Ile Ile Thr Tyr Ala Phe Asp Ile
1 5 10
<210> 57
<211> 10
<212> PRT
<213> Homo sapiens
<400> 57
Ala Arg Asp Val Ala Lys Pro Phe Asp Tyr
1 5 10
<210> 58
<211> 15
<212> PRT
<213> Homo sapiens
<400> 58
Ala Arg Asp Gly Asp Ser Asn Tyr Val Pro Gln Tyr Phe Asn Tyr
1 5 10 15
<210> 59
<211> 17
<212> PRT
<213> Homo sapiens
<400> 59
Ala Thr Glu Arg Ala Ile Gly Ser Gly Thr Tyr Pro Tyr Ser Phe Asn
1 5 10 15
Tyr
<210> 60
<211> 17
<212> PRT
<213> Homo sapiens
<400> 60
Ala Arg Glu Arg Ala Ile Gly Ser Gly Ile Tyr Pro Tyr Ser Phe Asn
1 5 10 15
Tyr
<210> 61
<211> 21
<212> PRT
<213> Homo sapiens
<400> 61
Ala Arg Gly Leu Leu Gly Glu Cys Ser Gly Gly Ser Cys Tyr Tyr Gly
1 5 10 15
Asp Ala Phe Asp Ile
20
<210> 62
<211> 10
<212> PRT
<213> Homo sapiens
<400> 62
Ala Arg Asp Ala Ser Lys Gly Phe Asp Ser
1 5 10
<210> 63
<211> 14
<212> PRT
<213> Homo sapiens
<400> 63
Ala Arg Gly Gly Glu Leu Gln Leu Pro Arg Pro Phe Asp Val
1 5 10
<210> 64
<211> 326
<212> PRT
<213> Homo sapiens
<400> 64
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro Gly Lys
325
<210> 65
<211> 107
<212> PRT
<213> Homo sapiens
<400> 65
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 66
<211> 442
<212> PRT
<213> Homo sapiens
<400> 66
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr
20 25 30
Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
180 185 190
Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro
210 215 220
Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
340 345 350
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 67
<211> 214
<212> PRT
<213> Homo sapiens
<400> 67
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu
85 90 95
Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys
100 105 110
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
115 120 125
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly
130 135 140
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly
145 150 155 160
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala
165 170 175
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser
180 185 190
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
195 200 205
Ala Pro Thr Glu Cys Ser
210
<210> 68
<211> 448
<212> PRT
<213> Homo sapiens
<400> 68
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 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
Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly
100 105 110
Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445
<210> 69
<211> 216
<212> PRT
<213> Homo sapiens
<400> 69
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
Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val
100 105 110
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
115 120 125
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
145 150 155 160
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205
Lys Ser Phe Asn Arg Gly Glu Cys
210 215

Claims (5)

1.An antibody or antigen-binding fragment that specifically binds human 4-1BB, comprising a VL domain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, and a VH domain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein,
the antibody or antigen binding fragment thereof comprises L-CDR1 as set forth in SEQ ID NO. 30, L-CDR2 as set forth in SEQ ID NO. 34, L-CDR3 as set forth in SEQ ID NO. 40, H-CDR1 as set forth in SEQ ID NO. 47, H-CDR2 as set forth in SEQ ID NO. 54, H-CDR3 as set forth in SEQ ID NO. 62;
the antibody or antigen binding fragment thereof comprises L-CDR1 as shown in SEQ ID NO. 32, L-CDR2 as shown in SEQ ID NO. 33, L-CDR3 as shown in SEQ ID NO. 39, H-CDR1 as shown in SEQ ID NO. 48, H-CDR2 as shown in SEQ ID NO. 55, H-CDR3 as shown in SEQ ID NO. 63;
the antibody or antigen binding fragment thereof comprises L-CDR1 as set forth in SEQ ID NO. 30, L-CDR2 as set forth in SEQ ID NO. 34, L-CDR3 as set forth in SEQ ID NO. 36, H-CDR1 as set forth in SEQ ID NO. 42, H-CDR2 as set forth in SEQ ID NO. 50, H-CDR3 as set forth in SEQ ID NO. 57;
the antibody or antigen binding fragment thereof comprises L-CDR1 as set forth in SEQ ID NO. 29, L-CDR2 as set forth in SEQ ID NO. 33, L-CDR3 as set forth in SEQ ID NO. 37, H-CDR1 as set forth in SEQ ID NO. 43, H-CDR2 as set forth in SEQ ID NO. 51, H-CDR3 as set forth in SEQ ID NO. 58;
the antibody or antigen-binding fragment thereof comprises L-CDR1 shown in SEQ ID NO. 31, L-CDR2 shown in SEQ ID NO. 33, L-CDR3 shown in SEQ ID NO. 38, H-CDR1 shown in SEQ ID NO. 44, H-CDR2 shown in SEQ ID NO. 52, and H-CDR3 shown in SEQ ID NO. 59;
the antibody or antigen binding fragment thereof comprises L-CDR1 as set forth in SEQ ID NO. 29, L-CDR2 as set forth in SEQ ID NO. 33, L-CDR3 as set forth in SEQ ID NO. 35, H-CDR1 as set forth in SEQ ID NO. 45, H-CDR2 as set forth in SEQ ID NO. 52, H-CDR3 as set forth in SEQ ID NO. 60;
the antibody or antigen binding fragment thereof comprises L-CDR1 as set forth in SEQ ID NO. 29, L-CDR2 as set forth in SEQ ID NO. 33, L-CDR3 as set forth in SEQ ID NO. 39, H-CDR1 as set forth in SEQ ID NO. 46, H-CDR2 as set forth in SEQ ID NO. 53, H-CDR3 as set forth in SEQ ID NO. 61;
the antibody or antigen binding fragment thereof comprises L-CDR1 as set forth in SEQ ID NO. 29, L-CDR2 as set forth in SEQ ID NO. 33, L-CDR3 as set forth in SEQ ID NO. 38, H-CDR1 as set forth in SEQ ID NO. 44, H-CDR2 as set forth in SEQ ID NO. 52, H-CDR3 as set forth in SEQ ID NO. 59; or,
the antibody or antigen binding fragment thereof comprises L-CDR1 as set forth in SEQ ID NO. 29, L-CDR2 as set forth in SEQ ID NO. 33, L-CDR3 as set forth in SEQ ID NO. 35, H-CDR1 as set forth in SEQ ID NO. 44, H-CDR2 as set forth in SEQ ID NO. 52, and H-CDR3 as set forth in SEQ ID NO. 59.
2.A composition comprising the antibody or antigen-binding fragment of claim 1, and a pharmaceutically acceptable carrier.
3. Use of an antibody or antigen-binding fragment that specifically binds human 4-1BB according to claim 1 in the preparation of a medicament for inhibiting tumor cell growth, wherein the antibody or antigen-binding fragment exhibits a T of at least 30h in a pharmacokinetic assay 1/2 And an AUC of at least 9000.00 μ g/mL × h 0-t 。
4. Use of the antibody or antigen-binding fragment that specifically binds human 4-1BB of claim 1 in the preparation of an anti-tumor medicament, wherein the anti-tumor medicament comprises the antibody or antigen-binding fragment and a pharmaceutically acceptable carrier, and the tumor volume is 1/2 or less of that of a blank control group 20 days after administration of 3mg/kg of the anti-tumor medicament to a B-h4-1BB humanized mouse that is seeded with colon cancer cells.
5. The use of claim 4, wherein administration of said antineoplastic agent does not cause significant fluctuation in ALT and AST markers.
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Publication number Priority date Publication date Assignee Title
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004010947A2 (en) * 2002-07-30 2004-02-05 Bristol-Myers Squibb Company Humanized antibodies against human 4-1bb
CN1867585A (en) * 2003-10-10 2006-11-22 布里斯托尔-迈尔斯·斯奎布公司 Fully human antibodies against human 4-1BB (CD137)
WO2017205745A1 (en) * 2016-05-27 2017-11-30 Abbvie Biotherapeutics Inc. Anti-4-1bb antibodies and their uses
CN109651507A (en) * 2017-10-12 2019-04-19 瑞阳(苏州)生物科技有限公司 A kind of excited type 4-1BB monoclonal antibody
CN109762066A (en) * 2017-11-09 2019-05-17 上海开拓者生物医药有限公司 4-1BB antibody and its preparation method and application

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2802994T3 (en) * 2015-09-22 2021-01-22 Dingfu Biotarget Co Ltd Fully human antibody to human CD137 and use thereof
JP7274417B2 (en) * 2016-11-23 2023-05-16 イミュノア・セラピューティクス・インコーポレイテッド 4-1BB binding protein and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004010947A2 (en) * 2002-07-30 2004-02-05 Bristol-Myers Squibb Company Humanized antibodies against human 4-1bb
CN1867585A (en) * 2003-10-10 2006-11-22 布里斯托尔-迈尔斯·斯奎布公司 Fully human antibodies against human 4-1BB (CD137)
WO2017205745A1 (en) * 2016-05-27 2017-11-30 Abbvie Biotherapeutics Inc. Anti-4-1bb antibodies and their uses
CN109651507A (en) * 2017-10-12 2019-04-19 瑞阳(苏州)生物科技有限公司 A kind of excited type 4-1BB monoclonal antibody
CN109762066A (en) * 2017-11-09 2019-05-17 上海开拓者生物医药有限公司 4-1BB antibody and its preparation method and application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity;Compte,M.等;《Nature Communications》;20181231;第4809号文章 *

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