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CN110615841B - Anti-human CD47 monoclonal antibody and application thereof - Google Patents

Anti-human CD47 monoclonal antibody and application thereof Download PDF

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CN110615841B
CN110615841B CN201910534489.3A CN201910534489A CN110615841B CN 110615841 B CN110615841 B CN 110615841B CN 201910534489 A CN201910534489 A CN 201910534489A CN 110615841 B CN110615841 B CN 110615841B
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崔文俊
马琳
郭树华
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Reyoung Suzhou Biology Science & Technology Co ltd
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Abstract

The invention relates to the field of biomedicine, in particular to an anti-human CD47 monoclonal antibody which comprises three heavy chain complementary determining regions (a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3) and three light chain complementary determining regions (a light chain CDR1, a light chain CDR2 and a light chain CDR3), has good specificity and high affinity and stability, and also provides application of the antibody, a nucleic acid molecule, a vector, a host cell, a conjugate and a composition in preparation of a medicine or a detection reagent for treating diseases.

Description

Anti-human CD47 monoclonal antibody and application thereof
Technical Field
The invention relates to the field of biological medicines, in particular to an anti-human CD47 monoclonal antibody and application thereof.
Background
CD47, also known as integrin, ovarian cancer antigen OA3, Rh related antigen and Mel6, is a member of the immunoglobulin superfamily that participates in a variety of cellular processes, 305 amino acids in length, with an extracellular amino-terminal IgV-like extracellular domain, 5 transmembrane segments that are highly hydrophobically extended and 1 short, selectively spliced, carboxy-terminal cytoplasmic tail, where the IgV-like domain is its major ligand binding site. The ligand comprises: signal-regulatory protein alpha chain (SIRP α), Thrombospondin (TSP), and Integrins (Integrins). CD47 is widely expressed on cell membrane surfaces and is present on various cells in all tissues, especially leukocytes, such as polymorphonuclear leukocytes (PMNs), Dendritic Cells (DCs), T cells, erythrocytes, placenta, platelets, normal Hematopoietic Stem Cells (HSCs), and other normal cells. Meanwhile, CD47 is highly expressed in most tumor cells of human body, and it can be used as a standard for tumor diagnosis and prognosis judgment. Over-expression occurs in various solid tumor cells or tumor stem cells (such as leukemia stem cells, leukemia cells) such as acute and chronic myelogenous leukemia, acute lymphocytic leukemia, non-hodgkin lymphoma, bladder cancer, ovarian cancer, breast cancer, rectal cancer, prostate cancer, renal cancer, multiple myeloma, etc., and the high expression is related to the poor clinical prognosis. It is shown that CD47 on the surface of tumor cells transmits an inhibitory signal of don 'T me' to macrophages, DC cells and other immune cells by binding to sirpa ligand on the surface of these cells, thereby evading phagocytosis of these cells and further evading killing of tumor cells by T cells caused by presentation of tumor antigens after phagocytosis.
Research shows that the interaction of CD47 on the surface of tumor cells and SIRP alpha on the surface of macrophages is blocked, so that the phagocytosis of the tumor cells by the macrophages and DC cells can be promoted, the presentation of tumor antigens is further increased, and the acquired immune response is activated. anti-CD 47 antibody or anti-SIRP alpha antibody can also promote phagocytosis of tumor cells by macrophages. Secondly, the anti-CD 47 monoclonal antibody can also eliminate tumor cells through Fc receptor mediated cytotoxicity; in addition, anti-CD 47 mab may also eliminate tumor cells by directly inducing apoptosis.
Recently, attention has been focused on the integrin-associated molecule CD47(IAP), which forms a complex with α v β 3 integrin, and the medical use of antibodies to the integrin-associated molecule CD47 has also been studied.
WO97/32601 attempted to produce a monoclonal antibody against a splenic stromal cell strain with the aim of developing a specific antibody capable of identifying splenic stromal cells, which describes obtaining a novel monoclonal antibody recognizing mouse CD47 as an antigen. On the other hand, WO97/32601 discloses that the monoclonal antibody has the property of inducing apoptosis in bone marrow cells.
WO99/12973 describes monoclonal antibodies produced against human CD47 (hereinafter referred to as human CD 47; amino acid and nucleotide sequences described in J.cell biol., 123, 485-496, 1993; Journal of Cell Science, 108, 3419-3425, 1995) which have the property of inducing apoptosis in nucleated blood cells (bone marrow cells and lymphocytes) having human CD47, i.e., monoclonal MABL-1 and MABL-2 antibodies, and hybridomas producing these antibodies, which are MABL-1(FERM BP-6100) and MABL-2 (FERM-610BP 1).
WO02/33072 and WO02/33073 disclose single chain Fv with single chain Fv regions possessing the property of inducing apoptosis in nucleated blood cells with human CD47, resulting from the generation of monoclonal antibodies against human CD 47.
The humanized antibody is the final direction of the development of the therapeutic antibody, the clinical application of the human antibody has good patient compliance, the immunogenicity of the antibody can be reduced to the maximum extent, the half-life of the drug in vivo can be prolonged, the effects of mediated immunoregulation, ADCC, CDC and the like can be realized by virtue of the Fc segment of the human immunoglobulin, and the biological effect of the antibody can be further enhanced. Currently, several anti-CD 47 therapeutic antibodies have entered clinical research (NCT02096770, NCT02216409, NCT02367196, NCT02447354, NCT 02488811).
Disclosure of Invention
The invention provides an anti-human CD47 monoclonal antibody with good specificity, higher affinity and stability.
The first purpose of the invention is to provide an amino acid sequence of an anti-human CD47 monoclonal antibody, which is specifically as follows:
an anti-human CD47 monoclonal antibody, having three heavy chain complementarity determining regions (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3) and three light chain complementarity determining regions (light chain CDR1, light chain CDR2, light chain CDR3),
heavy chain CDR1 is selected from: 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181 or 187 or a similar sequence having more than 50% homology to one of them; or a sequence that binds to the same epitope as the above sequence;
heavy chain CDR2 is selected from: 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182 or 188 or a similar sequence having more than 50% homology to one of them; or a sequence that binds to the same epitope as the above sequence;
heavy chain CDR3 is selected from: 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183 or 189 or a similar sequence having more than 50% homology to one of them; or a sequence that binds to the same epitope as the above sequence;
the light chain CDR1 is selected from: 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184 or 190 or similar sequences with more than 50% homology to one of them; or a sequence that binds to the same epitope as the above sequence;
the light chain CDR2 is selected from: 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185 or 191 or a similar sequence having more than 50% homology to one of them; or a sequence that binds to the same epitope as the above sequence;
the light chain CDR3 is selected from: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186 or 192 or similar sequences having more than 50% homology thereto; or a sequence that binds to the same epitope as the above sequence.
Preferably, the antibody further comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises the heavy chain CDR1, CDR2, and CDR3 described above; the light chain variable region comprises the light chain CDR1, CDR2, and CDR3 described above.
Further preferably, the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 49, heavy chain CDR1 as set forth in SEQ ID NO:50 and the heavy chain CDR2 as set forth in SEQ ID NO:51, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:52, light chain CDR1 as set forth in SEQ ID NO:53 and a light chain CDR2 as set forth in SEQ ID NO:54, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 55, heavy chain CDR1 as set forth in SEQ ID NO: 56 and the heavy chain CDR2 as set forth in SEQ ID NO: heavy chain CDR3 shown in 57; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 58, light chain CDR1 as set forth in SEQ ID NO: 59 and a light chain CDR2 as set forth in SEQ ID NO: 60, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 61, heavy chain CDR1 as set forth in SEQ ID NO: 62 and the heavy chain CDR2 as set forth in SEQ ID NO: 63, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 64, light chain CDR1 as set forth in SEQ ID NO: 65 and a light chain CDR2 as set forth in SEQ ID NO: 66, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:67, heavy chain CDR1 as set forth in SEQ ID NO:68 and the heavy chain CDR2 as set forth in SEQ ID NO:69, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:70, light chain CDR1 as set forth in SEQ ID NO:71 and a light chain CDR2 as set forth in SEQ ID NO:72, the light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:73, the heavy chain CDR1 as shown in SEQ ID NO:74 and a heavy chain CDR2 as set forth in SEQ ID NO:75 heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:76, light chain CDR1 as set forth in SEQ ID NO:77 and a light chain CDR2 as set forth in SEQ ID NO:78, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 79, as set forth in SEQ ID NO: 80 and a heavy chain CDR2 as set forth in SEQ ID NO: (ii) the heavy chain CDR3 shown in 81; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 82, light chain CDR1 as set forth in SEQ ID NO: 83 and a light chain CDR2 as set forth in SEQ ID NO: 84, the light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 85, as shown in SEQ ID NO: 86 and the heavy chain CDR2 as shown in SEQ ID NO: 87, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 88, light chain CDR1 as set forth in SEQ ID NO: 89 and a light chain CDR2 as set forth in SEQ ID NO: 90, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 91, heavy chain CDR1 as set forth in SEQ ID NO: 92 and a heavy chain CDR2 as set forth in SEQ ID NO: 93 heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 94, light chain CDR1 as set forth in SEQ ID NO: 95 and a light chain CDR2 as set forth in SEQ ID NO: 96, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:97, heavy chain CDR1 as set forth in SEQ ID NO:98 and a heavy chain CDR2 as set forth in SEQ ID NO:99, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:100, light chain CDR1 as set forth in SEQ ID NO:101 and a light chain CDR2 as set forth in SEQ ID NO:102, the light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:103, heavy chain CDR1 as set forth in SEQ ID NO:104 and a heavy chain CDR2 as set forth in SEQ ID NO:105, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:106, light chain CDR1 as set forth in SEQ ID NO:107 and a light chain CDR2 as set forth in SEQ ID NO:108, the light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:109, heavy chain CDR1 as set forth in SEQ ID NO:110 and the heavy chain CDR2 as set forth in SEQ ID NO:111, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:112, light chain CDR1 as set forth in SEQ ID NO:113 and a light chain CDR2 as set forth in SEQ ID NO:114, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:115, heavy chain CDR1 as set forth in SEQ ID NO:116 and a heavy chain CDR2 as set forth in SEQ ID NO:117 heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:118, light chain CDR1 as set forth in SEQ ID NO:119 and a light chain CDR2 as set forth in SEQ ID NO:120, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:121, heavy chain CDR1 as set forth in SEQ ID NO:122 and a heavy chain CDR2 as set forth in SEQ ID NO:123 heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:124, light chain CDR1 as set forth in SEQ ID NO:125 and a light chain CDR2 as set forth in SEQ ID NO:126, light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:127, heavy chain CDR1 as set forth in SEQ ID NO:128 and a heavy chain CDR2 as set forth in SEQ ID NO:129, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:130, light chain CDR1 as set forth in SEQ ID NO:131 and a light chain CDR2 as set forth in SEQ ID NO:132, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:133, a heavy chain CDR1 as set forth in SEQ ID NO:134 and a heavy chain CDR2 as set forth in SEQ ID NO:135, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:136, light chain CDR1 as set forth in SEQ ID NO:137 and a light chain CDR2 as set forth in SEQ ID NO:138, light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:139, heavy chain CDR1 as set forth in SEQ ID NO:140 and the heavy chain CDR2 as set forth in SEQ ID NO:141, CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:142, light chain CDR1 as set forth in SEQ ID NO:143 and a light chain CDR2 as set forth in SEQ ID NO:144, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:145, heavy chain CDR1 as set forth in SEQ ID NO:146 and a heavy chain CDR2 as set forth in SEQ ID NO:147 of heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:148, light chain CDR1 as set forth in SEQ ID NO:149 and a light chain CDR2 as set forth in SEQ ID NO:150, light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:151, heavy chain CDR1 as set forth in SEQ ID NO:152 and a heavy chain CDR2 as set forth in SEQ ID NO:153, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:154, light chain CDR1 as set forth in SEQ ID NO:155 and a light chain CDR2 as set forth in SEQ ID NO:156, a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:157, heavy chain CDR1 as shown in SEQ ID NO:158 and the heavy chain CDR2 as set forth in SEQ ID NO:159, CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:160, light chain CDR1 as set forth in SEQ ID NO:161 and a light chain CDR2 as set forth in SEQ ID NO:162, light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:163, heavy chain CDR1 as set forth in SEQ ID NO:164 and the heavy chain CDR2 as shown in SEQ ID NO:165 heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:166, light chain CDR1 as set forth in SEQ ID NO:167 and the light chain CDR2 as shown in SEQ ID NO:168, light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:169, heavy chain CDR1 as set forth in SEQ ID NO:170 and the heavy chain CDR2 as set forth in SEQ ID NO:171, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:172, light chain CDR1 as set forth in SEQ ID NO:173 and a light chain CDR2 as set forth in SEQ ID NO:174, and a light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:175, heavy chain CDR1 as set forth in SEQ ID NO:176 and a heavy chain CDR2 as shown in SEQ ID NO:177 of the heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:178, light chain CDR1 as set forth in SEQ ID NO:179 and the light chain CDR2 as set forth in SEQ ID NO:180, light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:181, heavy chain CDR1 as set forth in SEQ ID NO:182 and a heavy chain CDR2 as set forth in SEQ ID NO:183 heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:184, light chain CDR1 as shown in SEQ ID NO:185 and a light chain CDR2 as set forth in SEQ ID NO:186 light chain CDR 3; or
The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:187, as set forth in SEQ ID NO:188 and a heavy chain CDR2 as set forth in SEQ ID NO:189 heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO:190, a light chain CDR1 as set forth in SEQ ID NO:191 and a light chain CDR2 as set forth in SEQ ID NO:192, light chain CDR 3.
Preferably, the heavy chain CDR1 is SEQ ID NO 49, the heavy chain CDR1 is SEQ ID NO 50, the heavy chain CDR1 is SEQ ID NO 51 and the light chain CDR1 is SEQ ID NO 52, the light chain CDR1 is SEQ ID NO 53, and the light chain CDR1 is SEQ ID NO 54; or
Heavy chain CDR1 is SEQ ID NO 55, heavy chain CDR1 is SEQ ID NO 56, heavy chain CDR1 is SEQ ID NO 57, light chain CDR1 is SEQ ID NO 58, light chain CDR1 is SEQ ID NO 59, light chain CDR1 is SEQ ID NO 60; or
Heavy chain CDR1 is SEQ ID NO 61, heavy chain CDR1 is SEQ ID NO 62, heavy chain CDR1 is SEQ ID NO 63, light chain CDR1 is SEQ ID NO 64, light chain CDR1 is SEQ ID NO 65, light chain CDR1 is SEQ ID NO 66; or
Heavy chain CDR1 is SEQ ID NO 67, heavy chain CDR1 is SEQ ID NO 68, heavy chain CDR1 is SEQ ID NO 69 and light chain CDR1 is SEQ ID NO 70, light chain CDR1 is SEQ ID NO 71, light chain CDR1 is SEQ ID NO 72; or
Heavy chain CDR1 is SEQ ID NO 73, heavy chain CDR1 is SEQ ID NO 74, heavy chain CDR1 is SEQ ID NO 75 and light chain CDR1 is SEQ ID NO 76, light chain CDR1 is SEQ ID NO 77, light chain CDR1 is SEQ ID NO 78; or
Heavy chain CDR1 is SEQ ID NO 79, heavy chain CDR1 is SEQ ID NO 80, heavy chain CDR1 is SEQ ID NO 81, light chain CDR1 is SEQ ID NO 82, light chain CDR1 is SEQ ID NO 83, light chain CDR1 is SEQ ID NO 84; or
Heavy chain CDR1 is SEQ ID NO 85, heavy chain CDR1 is SEQ ID NO 86, heavy chain CDR1 is SEQ ID NO 87, light chain CDR1 is SEQ ID NO 88, light chain CDR1 is SEQ ID NO 89, light chain CDR1 is SEQ ID NO 90; or
Heavy chain CDR1 is SEQ ID NO 91, heavy chain CDR1 is SEQ ID NO 92, heavy chain CDR1 is SEQ ID NO 93 and light chain CDR1 is SEQ ID NO 94, light chain CDR1 is SEQ ID NO 95, light chain CDR1 is SEQ ID NO 96; or
The heavy chain CDR1 is SEQ ID NO 97, the heavy chain CDR1 is SEQ ID NO 98, the heavy chain CDR1 is SEQ ID NO 99, the light chain CDR1 is SEQ ID NO 100, the light chain CDR1 is SEQ ID NO 101, and the light chain CDR1 is SEQ ID NO 102; or
Heavy chain CDR1 is SEQ ID NO 103, heavy chain CDR1 is SEQ ID NO 104, heavy chain CDR1 is SEQ ID NO 105 and light chain CDR1 is SEQ ID NO 106, light chain CDR1 is SEQ ID NO 107, light chain CDR1 is SEQ ID NO 108; or
Heavy chain CDR1 is SEQ ID NO 109, heavy chain CDR1 is SEQ ID NO 110, heavy chain CDR1 is SEQ ID NO 111 and light chain CDR1 is SEQ ID NO 112, light chain CDR1 is SEQ ID NO 113, light chain CDR1 is SEQ ID NO 114; or
Heavy chain CDR1 is SEQ ID NO 115, heavy chain CDR1 is SEQ ID NO 116, heavy chain CDR1 is SEQ ID NO 117 and light chain CDR1 is SEQ ID NO 118, light chain CDR1 is SEQ ID NO 119, light chain CDR1 is SEQ ID NO 120; or
Heavy chain CDR1 is SEQ ID NO. 121, heavy chain CDR1 is SEQ ID NO. 122, heavy chain CDR1 is SEQ ID NO. 123 and light chain CDR1 is SEQ ID NO. 124, light chain CDR1 is SEQ ID NO. 125, light chain CDR1 is SEQ ID NO. 126; or
Heavy chain CDR1 is SEQ ID NO 127, heavy chain CDR1 is SEQ ID NO 128, heavy chain CDR1 is SEQ ID NO 129 and light chain CDR1 is SEQ ID NO 130, light chain CDR1 is SEQ ID NO 131, light chain CDR1 is SEQ ID NO 132; or
Heavy chain CDR1 is SEQ ID NO. 133, heavy chain CDR1 is SEQ ID NO. 134, heavy chain CDR1 is SEQ ID NO. 135 and light chain CDR1 is SEQ ID NO. 136, light chain CDR1 is SEQ ID NO. 137, light chain CDR1 is SEQ ID NO. 138; or
Heavy chain CDR1 is SEQ ID NO 139, heavy chain CDR1 is SEQ ID NO 140, heavy chain CDR1 is SEQ ID NO 141 and light chain CDR1 is SEQ ID NO 142, light chain CDR1 is SEQ ID NO 143, light chain CDR1 is SEQ ID NO 144; or
Heavy chain CDR1 is SEQ ID NO. 145, heavy chain CDR1 is SEQ ID NO. 146, heavy chain CDR1 is SEQ ID NO. 147 and light chain CDR1 is SEQ ID NO. 148, light chain CDR1 is SEQ ID NO. 149, light chain CDR1 is SEQ ID NO. 150; or
Heavy chain CDR1 is SEQ ID NO 151, heavy chain CDR1 is SEQ ID NO 152, heavy chain CDR1 is SEQ ID NO 153 and light chain CDR1 is SEQ ID NO 154, light chain CDR1 is SEQ ID NO 155, light chain CDR1 is SEQ ID NO 156; or
Heavy chain CDR1 is SEQ ID NO:157, heavy chain CDR1 is SEQ ID NO:158, heavy chain CDR1 is SEQ ID NO:159 and light chain CDR1 is SEQ ID NO:160, light chain CDR1 is SEQ ID NO:161, light chain CDR1 is SEQ ID NO: 162; or
Heavy chain CDR1 is SEQ ID NO 163, heavy chain CDR1 is SEQ ID NO 164, heavy chain CDR1 is SEQ ID NO 165 and light chain CDR1 is SEQ ID NO 166, light chain CDR1 is SEQ ID NO 167, light chain CDR1 is SEQ ID NO 168; or
Heavy chain CDR1 is SEQ ID NO 169, heavy chain CDR1 is SEQ ID NO 170, heavy chain CDR1 is SEQ ID NO 171 and light chain CDR1 is SEQ ID NO 172, light chain CDR1 is SEQ ID NO 173, light chain CDR1 is SEQ ID NO 174; or
Heavy chain CDR1 is SEQ ID NO 175, heavy chain CDR1 is SEQ ID NO 176, heavy chain CDR1 is SEQ ID NO 177 and light chain CDR1 is SEQ ID NO 178, light chain CDR1 is SEQ ID NO 179, light chain CDR1 is SEQ ID NO 180; or
Heavy chain CDR1 is SEQ ID NO. 181, heavy chain CDR1 is SEQ ID NO. 182, heavy chain CDR1 is SEQ ID NO. 183, light chain CDR1 is SEQ ID NO. 184, light chain CDR1 is SEQ ID NO. 185, light chain CDR1 is SEQ ID NO. 186; or
Heavy chain CDR1 is SEQ ID NO. 187, heavy chain CDR1 is SEQ ID NO. 188, heavy chain CDR1 is SEQ ID NO. 189, light chain CDR1 is SEQ ID NO. 190, light chain CDR1 is SEQ ID NO. 191, and light chain CDR1 is SEQ ID NO. 192.
Preferably, the heavy chain variable region amino acid sequence is selected from the group consisting of: 1, 3, 5, 7; one of 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, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45 or SEQ ID NO 47;
the light chain variable region amino acid sequence is selected from: 2, 4, 6 and 8; 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48.
Preferably, the amino acid sequence of the heavy chain variable region has a similar sequence with at least 50% homology with the sequence a, wherein the sequence a is SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7; 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47.
Preferably, the light chain variable region amino acid sequence is a similar sequence having at least 50% homology with sequence b, which is SEQ ID NO 2, 4, 6, 8; 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48.
Preferably, the heavy chain variable region is SEQ ID NO 1 and the light chain variable region is SEQ ID NO 2; or
The heavy chain variable region is SEQ ID NO 3 and the light chain variable region is SEQ ID NO 4; or
The heavy chain variable region is SEQ ID NO. 5 and the light chain variable region is SEQ ID NO. 6; or
The heavy chain variable region is SEQ ID NO. 7 and the light chain variable region is SEQ ID NO. 8; or
The heavy chain variable region is SEQ ID NO 9 and the light chain variable region is SEQ ID NO 10; or
The heavy chain variable region is SEQ ID NO 11 and the light chain variable region is SEQ ID NO 12; or
The heavy chain variable region is SEQ ID NO 13 and the light chain variable region is SEQ ID NO 14; or
The heavy chain variable region is SEQ ID NO. 15 and the light chain variable region is SEQ ID NO. 16; or
The heavy chain variable region is SEQ ID NO 17 and the light chain variable region is SEQ ID NO 18; or
The heavy chain variable region is SEQ ID NO 19 and the light chain variable region is SEQ ID NO 20; or
The heavy chain variable region is SEQ ID NO 21 and the light chain variable region is SEQ ID NO 22; or
The heavy chain variable region is SEQ ID NO: 23 and the light chain variable region is SEQ ID NO: 24; or
The heavy chain variable region is SEQ ID NO. 25 and the light chain variable region is SEQ ID NO. 26; or
The heavy chain variable region is SEQ ID NO 27 and the light chain variable region is SEQ ID NO 28; or
The heavy chain variable region is SEQ ID NO: 29 and the light chain variable region is SEQ ID NO: 30; or
The heavy chain variable region is SEQ ID NO 31 and the light chain variable region is SEQ ID NO 32; or
The heavy chain variable region is SEQ ID NO 33 and the light chain variable region is SEQ ID NO 34; or
The heavy chain variable region is SEQ ID NO 35 and the light chain variable region is SEQ ID NO 36; or
The heavy chain variable region is SEQ ID NO: 37 and the light chain variable region is SEQ ID NO: 38; or
The heavy chain variable region is SEQ ID NO: 39 and the light chain variable region is SEQ ID NO: 40; or
The heavy chain variable region is SEQ ID NO 41 and the light chain variable region is SEQ ID NO 42; or
The heavy chain variable region is SEQ ID NO 43 and the light chain variable region is SEQ ID NO 44; or
The heavy chain variable region is SEQ ID NO 45 and the light chain variable region is SEQ ID NO 46; or
The heavy chain variable region is SEQ ID NO 47 and the light chain variable region is SEQ ID NO 48.
The above antibody also has a heavy chain amino acid sequence comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, a light chain CDR 3; heavy chain CDR1 is selected from: 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181 or 187 or a similar sequence having more than 50% homology to one of them;
heavy chain CDR2 is selected from: 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182 or 188 or a similar sequence having more than 50% homology to one of them;
heavy chain CDR3 is selected from: 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183 or 189 or a similar sequence having more than 50% homology to one of them;
the light chain CDR1 is selected from: 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184 or 190 or similar sequences with more than 50% homology to one of them;
the light chain CDR2 is selected from: 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185 or 191 or a similar sequence having more than 50% homology to one of them;
the light chain CDR3 is selected from: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186 or 192 or similar sequences having more than 50% homology to one of them.
The second objective of the invention is to provide a nucleic acid sequence of an anti-human CD47 monoclonal antibody, which is specifically as follows:
a nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain CDR and/or a light chain CDR, heavy chain CDR1 selected from the group consisting of: one of SEQ ID NO 49, SEQ ID NO 55, SEQ ID NO 61, SEQ ID NO 67, SEQ ID NO 73, SEQ ID NO 79, SEQ ID NO 85, SEQ ID NO 91, SEQ ID NO 97, SEQ ID NO 103, SEQ ID NO 109, SEQ ID NO 115, SEQ ID NO 121, SEQ ID NO 127, SEQ ID NO 133, SEQ ID NO 139, SEQ ID NO 145, SEQ ID NO 151, SEQ ID NO 157, SEQ ID NO 163, SEQ ID NO 169, SEQ ID NO 175, SEQ ID NO 181, or SEQ ID NO 187 or a sequence having more than 50% homology thereto;
heavy chain CDR2 is selected from: 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182 or 188 or a sequence having more than 50% homology thereto;
heavy chain CDR3 is selected from: 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183 or 189 or a sequence having more than 50% homology to one of them;
the light chain CDR1 is selected from: 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184 or 190 or a sequence with more than 50% homology thereto;
the light chain CDR2 is selected from: 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185 or 191 or a sequence having more than 50% homology thereto;
the light chain CDR3 is selected from: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186 or 192 or a sequence having more than 50% homology thereto.
A nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain variable region and/or a light chain variable region having the amino acid sequence: the sequence a or a sequence with at least 50 percent of homology with the sequence a, wherein the sequence a is SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7; one of 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, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45 or SEQ ID NO 47;
the amino acid sequence of the light chain variable region is as follows: sequence b or a sequence with at least 50 percent of homology with the sequence b, wherein the sequence b is SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8; 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48.
The present invention also provides a vector comprising the nucleic acid molecule of any one of the above.
The invention also provides a host cell comprising any one of the amino acid sequences, nucleic acid molecules or vectors described above.
The present invention also provides a conjugate comprising the antibody of any one of the above.
The invention also provides a composition comprising a main component and an auxiliary component, wherein the main component comprises one or more of the antibody or the nucleic acid molecule of any one of the above or the carrier, the host cell or the conjugate, and the auxiliary component is selected from a pharmaceutically acceptable carrier or excipient, and other optional bioactive substances.
Such other bioactive substances include, but are not limited to, other antibodies, fusion proteins, or drugs (e.g., antineoplastic drugs, such as radiation, chemotherapeutic drugs).
The invention also provides application of the antibody, the nucleic acid molecule, the vector, the host cell, the conjugate and the composition in preparation of a medicament or a detection reagent for treating diseases.
The disease is malignant tumor, cardiovascular disease or inflammation disease.
The present invention also provides a diagnostic reagent or kit comprising the antibody according to any one of the above. The diagnostic reagent or kit is used for diagnosing CD47 related diseases in vitro (such as cells or tissues) or in vivo (such as human or animal models).
The invention is further described below: in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).
In the present invention, the term "antigen-binding portion" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen to which the antibody binds, competing with the intact antibody for specific binding to the antigen. See generally, Fundamental Immunology, ch.7(Paul, w., ed., 2 nd edition, Raven Press, n.y. (1989), which is incorporated herein by reference in its entirety for all purposes.
"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences when aligned most preferably.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 shows the result of the affinity test between the human and mouse chimeric antibody and human CD 47;
FIG. 2 shows the structure of the cell binding activity test of the human-mouse chimeric antibody and the CD47 engineered cell line;
FIGS. 3A-3B show the results of the activity of macrophages induced by human murine chimeric antibodies in phagocytosis of tumor cells;
FIGS. 4A-4B show the results of the blood coagulation test of the chimeric human-mouse antibody;
FIG. 5 shows the verification of the efficacy of the humanized antibody in a mouse tumor model.
Detailed Description
The invention discloses a monoclonal antibody and application thereof, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein may be made and equivalents employed without departing from the spirit and scope of the invention.
The monoclonal antibody and the raw materials and reagents used in the application thereof provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
the experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1:
1. animal immunization
SD rats of appropriate age were immunized using a classical immunization schedule with human CD47 protein (purchased from near shore protein technologies) as the immunogen to produce antibodies against CD47 in the animals.
2. Cell fusion and hybridoma cell screening
Adjusting the state of mouse myeloma SP2/0 before fusion to ensure that the growth density is not more than 1.0 × 106The cells are terminally immunized 3 days earlier by tail vein injection, and SP2/0 cells are cultured one day earlier, and the number of plates is 2.0 × 104Individual cells/well. Fusing by electrofusion instrument to ensure the ratio of spleen cell or lymph node cell to SP2/0 cell is 10:1 to 5:1, and the number of spleen cells laid in each hole is not more than 1.0 × 105And (4) respectively. Supernatants were harvested 7 days after fusion and media changed.
The harvested supernatant was first subjected to direct ELISA binding method, flow cytometry to determine cell binding activity and ligand binding blocking assay to obtain 3 hybridoma cell lines which were positive in screening experiments.
The specific steps of the related experimental methods are as follows:
A. ELISA binding method
Coating human CD47 on a plate, adding hybridoma culture solution supernatant, incubating and washing, adding goat anti-mouse-HRP, developing, fitting a reading to obtain a reaction curve, and calculating an EC50 value.
B. Flow cytometry for determining hybridoma supernatant cell binding activity
And (3) incubating the hybridoma supernatant with a CD47 high-expression engineering cell strain and a blank control cell strain for a certain time, adding a goat anti-mouse IgG Fc Alexa647 secondary antibody after centrifugal washing, and reading a fluorescent signal by a flow cytometer after centrifugal washing.
C. Ligand binding blocking assay
The 96-well plate is coated with human CD47, after the 96-well plate is closed, a certain amount of SIRPa protein with a human Fc label incubated with the hybrid six supernatants is added for combination, the ligand bound to the antigen is detected by the Hrp secondary antibody of sheep anti-human Fc fragment of the washed 96-well plate, and the amount of the ligand bound to the antigen is reduced by adding the 96-well plate after the antibody capable of blocking the combination of the ligand and the antigen is incubated with the ligand of the Fc label.
3. Acquisition of murine antibody Gene
Total RNA of hybridoma cells was extracted by Trizol (Ambion 15596-026) kit, and then cDNA was prepared by reverse transcription of the total RNA using Takara PrimeScript 1st Strand cDNA synthesis kit, and the heavy chain and light chain variable region sequences of the antibody were amplified with degenerate primers, respectively.
4. Preparation and activity identification of human-mouse chimeric antibody
Preparing an antibody 1-antibody 24, specifically, the heavy chain variable region of the antibody 1 is SEQ ID NO:1 and the light chain variable region is SEQ ID NO: 2;
the heavy chain variable region of antibody 2 is SEQ ID NO 3 and the light chain variable region is SEQ ID NO 4;
the heavy chain variable region of antibody 3 is SEQ ID NO 5 and the light chain variable region is SEQ ID NO 6;
the heavy chain variable region of antibody 4 is SEQ ID NO 7 and the light chain variable region is SEQ ID NO 8;
the heavy chain variable region of antibody 5 is SEQ ID NO 9 and the light chain variable region is SEQ ID NO 10;
the heavy chain variable region of antibody 6 is SEQ ID NO 11 and the light chain variable region is SEQ ID NO 12;
the heavy chain variable region of antibody 7 is SEQ ID NO 13 and the light chain variable region is SEQ ID NO 14;
the heavy chain variable region of antibody 8 is SEQ ID NO 15 and the light chain variable region is SEQ ID NO 16;
the heavy chain variable region of antibody 9 is SEQ ID NO 17 and the light chain variable region is SEQ ID NO 18;
the heavy chain variable region of antibody 10 is SEQ ID NO 19 and the light chain variable region is SEQ ID NO 20;
the heavy chain variable region of antibody 11 is SEQ ID NO 21 and the light chain variable region is SEQ ID NO 22;
the heavy chain variable region of antibody 12 is SEQ ID NO. 23 and the light chain variable region is SEQ ID NO. 24;
the heavy chain variable region of antibody 13 is SEQ ID NO 25 and the light chain variable region is SEQ ID NO 26;
the heavy chain variable region of antibody 14 is SEQ ID NO 27 and the light chain variable region is SEQ ID NO 28;
the heavy chain variable region of antibody 15 is SEQ ID NO: 29 and the light chain variable region is SEQ ID NO: 30;
the heavy chain variable region of antibody 16 is SEQ ID NO 31 and the light chain variable region is SEQ ID NO 32;
the heavy chain variable region of antibody 17 is SEQ ID NO 33 and the light chain variable region is SEQ ID NO 34;
the heavy chain variable region of antibody 18 is SEQ ID NO 35 and the light chain variable region is SEQ ID NO 36;
the heavy chain variable region of antibody 19 is SEQ ID NO 37 and the light chain variable region is SEQ ID NO 38;
the heavy chain variable region of antibody 20 is SEQ ID NO 39 and the light chain variable region is SEQ ID NO 40;
the heavy chain variable region of antibody 21 is SEQ ID NO 41 and the light chain variable region is SEQ ID NO 42;
the heavy chain variable region of antibody 22 is SEQ ID NO 43 and the light chain variable region is SEQ ID NO 44;
the heavy chain variable region of antibody 23 is SEQ ID NO 45 and the light chain variable region is SEQ ID NO 46;
the heavy chain variable region of antibody 24 is SEQ ID NO 47 and the light chain variable region is SEQ ID NO 48.
A. Construction of human murine chimeric antibodies
The variable region sequence of the mouse anti-light chain is spliced with the sequence of the human IgG kappa CL region to obtain a full-length light chain sequence, and the full-length light chain sequence is constructed on a pcDNA3.1 vector containing a nitrogen-terminal signal peptide through full-gene synthesis to obtain a light chain expression vector.
The variable region sequence of the mouse anti-heavy chain is spliced with the sequences of CH1, CH2 and CH3 regions of human IgG 4S 228P to obtain a full-length heavy chain sequence, and the full-length heavy chain sequence is constructed to a heavy chain expression vector on a pcDNA3.1 vector containing a nitrogen terminal signal peptide through whole-gene synthesis.
B. Expression and purification of human murine chimeric antibodies
The light and heavy chain expression vectors were transiently transfected into HEK293F cells by co-transfection, and the transfected 293F cells were cultured for 7 days and the culture supernatant was collected. The chimeric antibody in the supernatant was then purified by protein a affinity chromatography column.
SPR measurement of the binding Capacity of the human murine chimeric antibody to the CD47 protein
The method is carried out at 25 ℃, the human-mouse chimeric antibody flows through the surface of the Protein A chip at a certain flow rate, and after the antibody is combined with the surface of the chip, the human CD47 Protein which is diluted in a gradient manner flows through the surface of the chip on which the antibody is fixed at a certain flow rate. The assay buffer was blanked and one replicate concentration was set. The results of the experimental determinations were fitted using a 1:1 Binding model. The results are shown in figure 1, and show that the affinity of all 24 antibodies to human CD47 is in the range of 10 < -11 > to 10 < -10 > M, and the strong binding capacity to the antigen is shown.
D. Flow cytometry for determining binding capacity of human-mouse chimeric antibody and cell
After the purified human-mouse chimeric antibody, a CD47 high-expression engineering cell strain and a blank control cell strain are incubated for a certain time, a goat anti-human IgG Fc FITC secondary antibody is added after centrifugal washing, and a fluorescent signal is read by a flow cytometer after centrifugal washing. The binding curves of 8 antibodies and a human CD47 high-expression HEK293 cell line are obtained by detecting the binding signals of the antibodies and the human CD47 high-expression HEK293 cell line under different concentrations through a flow cytometer, and the final binding activity EC50 value is obtained through fitting, and the result is shown in figure 2, the EC50 value of the antibodies ranges from 10 ug/ml to 10 ug/ml, and the binding activity different from that of the cell line is shown.
E. Phagocytosis experiment of human-mouse chimeric antibody induced macrophage on tumor cell
Human blood was collected and human monocytes were isolated according to CD14+ monocyte isolation instructions, and then CD14+ monocytes were resuspended in human M2 macrophage differentiation medium to 2 x106cells/mL, cell culture day 7, M2 macrophages differentiated to mature for downstream experiments. Tumor single cell suspension was collected, centrifuged and resuspended in room temperature PBS according to the CFSE cell labeling kit instructions, and CFSE solution was added to a final concentration of 3. mu.M and incubated at 37 ℃ for 10 min. 44 mL of 4 ℃ pre-cooled medium wash (RPMI 1640+10% FBS) was added and centrifuged. The washing was repeated once. Resuspend M2 macrophages and CFSE labeled tumor cells in serum-free basal medium, add 90. mu.L of 4X 10 cells per well5From individual cells (macrophages: tumor cells = 1: 1) to 96-well U-shaped cell culture plates, two duplicate wells were designed for each antibody. Add 10. mu.L of antibody to the corresponding well at a final concentration of 20. mu.g/mL and mix well. Placing at 37 ℃ and 5% CO2Incubate in incubator for 2-3 hours. Resuspend cells and transfer to corresponding flow cell tubes. FACS buffer washed cells twice. And pouring out the supernatant, adding 100 mu L of FACS buffer to resuspend the cells, adding 5 mu L of APC anti-human CD14, mixing the mixture evenly and gently, placing the mixture at 4 ℃ and incubating the mixture for 30 minutes in a dark place. Cells were washed twice with 200 μ L FACS buffer. The cells were resuspended in 200 μ L FACS buffer, and 5 μ L7-AAD was added and mixed gently and incubated at room temperature (25 ℃ C.) for 5 minutes in the dark. The samples were analyzed using a flow cytometer.
Fig. 3A shows the phagocytosis of tumor cell line expressing human CD47 by M2 macrophage at 20ug/ml in one experiment, in which the phagocytosis index of tumor cell line by M2 macrophage was less than 10% in the absence of human CD47 binding isotype control antibody, and the phagocytosis index of tumor cell line by M2 macrophage and tumor cell line by M2 macrophage were significantly increased by 11 antibodies to be tested including positive control antibody at 20 ug/ml.
FIG. 3B shows the phagocytosis of tumor cell lines expressing human CD47 by M2 macrophages at 20ug/ml in another experiment in which the phagocytosis of tumor cell lines by M2 macrophages was less than 5% in the absence of human CD47 binding isotype control antibody, while the phagocytosis of tumor cell lines by M2 macrophages was enhanced by the positive control antibody, antibody 19 and antibody 23 at 20ug/ml, which showed some enhancement of phagocytosis of tumor cell lines by M2 macrophages.
F. Determination of coagulation Effect of human murine chimeric antibody
Erythrocytes were obtained from human blood by centrifugal separation, and after the erythrocytes were resuspended in PBS, a human-mouse chimeric antibody at a certain concentration was added to the erythrocytes for co-incubation at room temperature for 2 hours, and agglutination of the erythrocytes was observed visually.
FIG. 4A shows the agglutination effect of 10 antibodies on human erythrocytes at different concentrations, and shows that the other 8 antibodies did not cause significant erythrocyte agglutination, except that antibody 3 and antibody 12 caused agglutination of erythrocytes at higher antibody concentrations.
FIG. 4B shows the results of the agglutination effect of 10 antibodies on human erythrocytes at different concentrations, represented by a curve.
5. Drug effect verification of antibody in mouse tumor model
Raji cells purchased from ATCC were genetically engineered to have Luciferase added and designated Raji-Luc cells. The cells were cultured at 37 ℃ with 5% CO2The culture medium of (2) is RPMI1640 medium containing 10% inactivated fetal bovine serum. Raji-plus resuspended in PBS after 3 to 4 days of confluencyLuc cells at 5X 105At a concentration of 0.2mL, 0.2 mL/volume was inoculated into B-NSG mice via the tail vein. Measuring tumor imaging signal values on 0 day and 3 days after inoculation by using a small animal imager after inoculation, and when the average imaging signal intensity reaches 1X106 P/S, animals were randomly grouped by tumor imaging signal value, 6 per experimental group. The administration was started on the divided day 2 times per week at 5mg/kg body weight each time. After dosing, tumor growth (detected and recorded by a small animal imager) and animal body weight were measured 2 times per week. The measurement results of each time, including live body imaging pictures, body weight, mouse clinical state and the like, are all recorded and stored. As shown in FIG. 5, the growth curve of tumor cells in B-NSG mice was obtained after inoculating Raji-Luc tumor cells and administering antibody at a dose of 5mg/kg 2 times a week. The abscissa is the time of administration and the ordinate is the signal intensity of tumor cell imaging in mice. The results show that the antibodies 1, 3, 10, 11, 15, 16, 23 and 24 can effectively inhibit the growth of tumor cells in mice, and have significant anti-tumor effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Ruiyang (Suzhou) Biotechnology Ltd
<120> anti-human CD47 monoclonal antibody and application thereof
<160> 192
<170> SIPOSequenceListing 1.0
<210> 1
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Thr Leu Ser Cys Glu Ala Ser Gly Phe Ile Phe Ser Asn Tyr
20 25 30
Asp Met Asp Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Val Thr Val Tyr Gln Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Ser Ser Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Glu Tyr Ser Ser Tyr Ile Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 2
<211> 109
<212> PRT
<213> Unknown (Unknown)
<400> 2
Asp Asp Thr Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Ile Thr Ile Ser Cys Lys Ala Ser Lys Ser Val Gly Ile Arg
20 25 30
Met His Trp Tyr Gln Gln Arg Pro Gly Gln Gln Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Gly Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Trp Asn Gly Pro Pro
85 90 95
Tyr Thr Phe Gly Pro Gly Thr Arg Leu Glu Leu Lys Arg
100 105
<210> 3
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 3
Glu Val Leu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Glu Val Ser Gly Phe Ile Phe Ser Asn Tyr
20 25 30
Glu Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Tyr Thr Ser Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Ser Asn Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 4
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 4
Ile Asp Ile Val Met Thr Gln Ser Pro Gly Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Thr Ala Ser Glu Val Ile Asn Ser Arg
20 25 30
Leu His Trp Tyr Gln Arg Arg Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Asn Leu Asn Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 5
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 5
Glu Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ala Cys Val Ala Ser Gly Phe Thr Phe Ser Leu Tyr
20 25 30
Glu Met Ala Trp Val Arg Gln Ala Pro Thr Thr Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Arg Gly Thr Ser Thr Ser Phe Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Glu Lys Ala Ile Leu Ser
65 70 75 80
Leu Gln Met Asp Ser Leu Gly Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Thr Ala Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 6
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 6
Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Ile Ser Cys Lys Ala Ser Glu Gly Val Ser Ile Arg
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Arg Leu Leu Ile
35 40 45
His Lys Ala Ser Asn Leu Pro Ser Gly Val Pro Ala Arg Phe Thr Ala
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Asn Trp Asp Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 7
<211> 118
<212> PRT
<213> Unknown (Unknown)
<400> 7
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Cys Cys
85 90 95
Ala Ser Leu Tyr Ser Tyr Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 8
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 8
Gly Asp Thr Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Ile Thr Ile Ser Cys Thr Ala Ser Asp Val Ile Asn Ser Arg
20 25 30
Leu His Trp Tyr Gln Arg Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Asn Leu Asn Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 9
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 9
Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Glu Val Ser Gly Phe Ile Phe Ser Asn Tyr
20 25 30
Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Ser Thr Asn Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Thr Asn Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Thr Ala Ser Tyr Phe Asp Asn Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 10
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 10
Ile Asp Ile Leu Met Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Thr Ala Ser Glu Ile Ile Asn Ser Arg
20 25 30
Leu His Trp Tyr Gln Arg Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Asn Leu Asn Asp Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 11
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 11
Asp Lys Arg Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Glu Val Ser Gly Phe Ile Phe Ser Asn Tyr
20 25 30
Glu Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Tyr Thr Ser Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Thr Asn Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 12
<211> 107
<212> PRT
<213> Unknown (Unknown)
<400> 12
Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Thr Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 13
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 13
Asp Val Leu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Glu Val Ser Gly Phe Ile Phe Ser Asn Tyr
20 25 30
Glu Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Thr Asn Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 14
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 14
Ile Asp Ile Val Met Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Thr Ala Ser Glu Val Ile Asn Ser Arg
20 25 30
Leu His Trp Tyr Gln Arg Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ser Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Asn Leu Asn Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 15
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 15
Asp Arg Arg Met Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Glu Val Ser Gly Phe Ile Phe Ser Asn Tyr
20 25 30
Glu Met Ala Trp Ile Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Thr Asn Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 16
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 16
Gly Asp Thr Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Thr Ala Ser Glu Val Ile Asn Ser Arg
20 25 30
Leu His Trp Tyr Gln Arg Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Asn Leu Asn Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 17
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 17
Glu Val Gln Leu Leu Glu Thr Gly Gly Asp Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Tyr Leu Ser Cys Val Ala Ser Gly Phe Ser Phe Ser Asn Tyr
20 25 30
Glu Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Ala
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Ile Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Thr Ala Ser Phe Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 18
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 18
Ile Asp Val Val Met Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Ile Arg
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Glu Pro Lys Pro Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Asn Leu Asp Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Leu Lys Arg
100 105
<210> 19
<211> 121
<212> PRT
<213> Unknown (Unknown)
<400> 19
Ile Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
1 5 10 15
Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Asp Tyr Ser Ile Thr Ser
20 25 30
Gly Tyr Arg Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Arg Leu Glu
35 40 45
Trp Met Ala Phe Met Asn Thr Ala Gly Asn Thr Asn Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Glu Asn Gln Phe
65 70 75 80
Phe Leu His Leu Asn Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Gly Gly Tyr Asp Ser Phe Trp Phe Phe Asp Phe Trp Gly
100 105 110
Pro Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 20
<211> 107
<212> PRT
<213> Unknown (Unknown)
<400> 20
Asp Ile Glu Leu Thr Gln Ser Pro Thr Thr Val Ala Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Leu Thr Cys Arg Ala Ser Ser Ser Ile Thr Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Asp Thr Ser Asn Arg Ala Ser Gly Val Pro Asn Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Thr Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 21
<211> 120
<212> PRT
<213> Unknown (Unknown)
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr
20 25 30
Tyr Val Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Arg Thr Tyr Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Ser Thr Gln Tyr
65 70 75 80
Leu Gln Val Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Val Thr Glu Asp Asn Glu Gly Thr Ser Pro Phe Ala Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 22
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 22
Asp Ile Val Met Thr Gln Ser Pro Thr Ser Met Ser Ile Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Asn Cys Lys Ala Ser Gln Asn Val Gly Ser Ser
20 25 30
Val Asp Trp Phe Gln Gln Lys Thr Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Arg Val Ser Asn Arg Tyr Ala Gly Ile Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Gly Asp Phe Thr Phe Thr Ile Ser Asn Met Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Met Gln Ser Tyr Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Arg Arg
100 105
<210> 23
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 23
Glu Val Gln Val Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Ile Phe Ser Ser Tyr
20 25 30
Glu Met Ala Trp Val Arg Gln Gly Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Leu Ser Pro Ser Gly Gly Phe Thr Tyr Leu Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Ala Thr Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Thr Ala Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 24
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 24
Gly Asp Thr Leu Leu Thr Gln Ser Pro Ala Leu Thr Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Gln Ala Ser Glu Ile Ile Asn Ala Arg
20 25 30
Leu His Trp Tyr Gln Gln Lys Ser Gly Gln Gln Pro Arg Leu Leu Val
35 40 45
Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Thr Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Leu Asn Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 25
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 25
Val Asp Arg Arg Met Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Asp Met Asp Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Val Thr Val Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Ser Ser Leu His
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Glu Tyr Ser Ser Tyr Val Tyr Trp Gly Gln Gly Val Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 26
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 26
Ser Ile Val Met Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly Glu
1 5 10 15
Arg Val Thr Ile Ser Cys Lys Ala Ser Lys Ser Val Ser Ile Arg Val
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile Tyr
35 40 45
Lys Val Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Glu
65 70 75 80
Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Trp Asn Gly Pro Pro Tyr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 27
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 27
Asp Arg Arg Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Glu Val Ser Gly Phe Ile Phe Ser Asn Tyr
20 25 30
Glu Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Tyr Thr Ser Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Thr Asn Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 28
<211> 107
<212> PRT
<213> Unknown (Unknown)
<400> 28
Glu Ile Val Leu Thr Gln Ser Pro Thr Ile Met Ala Ala Ser Pro Gly
1 5 10 15
Glu Gln Val Thr Leu Thr Cys Arg Ala Ser Ser Ser Leu Ile Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Asp Thr Ser Asn Arg Ala Ser Gly Val Pro Asn Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asn Ser Met Glu Thr Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Glu Trp Ser Arg Asn Pro Pro Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg Arg
100 105
<210> 29
<211> 121
<212> PRT
<213> Unknown (Unknown)
<400> 29
Glu Val Gln Ile Leu Glu Thr Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe Asn Asp Tyr
20 25 30
Phe Met Asn Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val
35 40 45
Ala Gln Ile Arg Asn Lys Asn Tyr Asn Tyr Val Thr Tyr Tyr Ala Glu
50 55 60
Ser Leu Gly Asp Arg Val Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
65 70 75 80
Val Tyr Leu Gln Val Ser Ser Leu Arg Pro Glu Asp Ser Ala Ile Tyr
85 90 95
Tyr Cys Thr Arg Tyr Asn Thr His Met Gly Ala Met Asp Ala Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 30
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 30
Val Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Glu
1 5 10 15
Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ser Pro Gln Leu Leu Ile
35 40 45
Asp Ser Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser His Tyr Ser Leu Lys Ile Ser Arg Val Gln Val
65 70 75 80
Glu Asp Leu Gly Ile Tyr Tyr Cys Leu Gln Thr Tyr Ser Ser Pro Pro
85 90 95
Thr Phe Gly Gly Gly Ser Lys Leu Glu Leu Lys Arg
100 105
<210> 31
<211> 118
<212> PRT
<213> Unknown (Unknown)
<400> 31
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ile Asn Tyr
20 25 30
Trp Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Thr Asn Thr Asp Gly Thr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Ser Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Phe Gly Leu Ser Gly Pro Phe Asp Tyr Trp Gly Gln Gly Val
100 105 110
Met Val Thr Val Ser Ser
115
<210> 32
<211> 113
<212> PRT
<213> Unknown (Unknown)
<400> 32
Asp Val Val Met Thr Gln Thr Pro Val Ala Leu Pro Val Ser Leu Gly
1 5 10 15
Gly Gln Ala Ser Phe Ser Cys Arg Ser Ser Gln Ser Leu Gly His Ser
20 25 30
Asn Gly Lys Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Arg Val Ser Ile Arg Leu Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
65 70 75 80
Ser Arg Val Glu Pro Glu Asp Leu Gly Asp Tyr Tyr Cys Leu Gln Ser
85 90 95
Thr His Phe Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<210> 33
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 33
Glu Val Gln Val Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Ser Phe Asn Asn Tyr
20 25 30
Asp Met Ala Trp Ile Arg Gln Thr Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Phe Thr Asp Phe Arg Asp Ser Val
50 55 60
Lys Gly Arg Cys Thr Val Ser Arg Asp Asn Val Gly Thr Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Thr Ala Thr Tyr Phe Asp Asn Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 34
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 34
Gly Asp Thr Leu Leu Thr Gln Ser Pro Ala Leu Thr Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Gln Ala Ser Glu Ile Ile Asn Ala Arg
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile
35 40 45
Phe Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Leu Asn Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 35
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 35
Glu Val Gln Val Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Ser Phe Asn Asn Tyr
20 25 30
Asp Met Ala Trp Ile Arg Gln Thr Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Phe Thr Asp Phe Arg Asp Ser Val
50 55 60
Lys Gly Arg Cys Thr Val Ser Arg Asp Asn Val Gly Thr Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Thr Ala Thr Tyr Phe Asp Asn Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 36
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 36
Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Glu Thr Leu Ile
35 40 45
Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr
65 70 75 80
Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 37
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 37
Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Tyr Leu Ser Cys Val Ala Ser Gly Phe Ser Phe Ser Asn Tyr
20 25 30
Glu Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Ala
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Ile Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Thr Ala Ser Phe Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 38
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 38
Ile Asp Val Val Met Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Ile Arg
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Arg Glu Pro Lys Pro Leu Ile
35 40 45
Tyr Lys Ser Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Asn Leu Asp Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Leu Lys Arg
100 105
<210> 39
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 39
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr
20 25 30
Asp Met Asp Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Thr Thr Ser Tyr Arg Asp Ser Ile
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Glu Tyr Ser Ser Tyr Val Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Ile Val Ser Ser
115
<210> 40
<211> 109
<212> PRT
<213> Unknown (Unknown)
<400> 40
Ile Asp Ile Leu Leu Thr Gln Ser Pro Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Lys Ala Ser Glu Asn Val Ser Pro Arg
20 25 30
Met His Trp Tyr Gln Gln Arg Pro Gly Gln Gln Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Val Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Thr
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Trp Asn Gly Pro Pro
85 90 95
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 41
<211> 120
<212> PRT
<213> Unknown (Unknown)
<400> 41
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Ser Val Thr Asp Tyr Ser Ile Thr Ser Gly
20 25 30
Tyr Arg Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Ala Phe Met Asn Thr Ala Gly Asn Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Glu Asn Gln Phe Phe
65 70 75 80
Leu His Leu Asn Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Tyr Asp Ser Leu Trp Phe Phe Asp Phe Trp Gly Pro
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 42
<211> 107
<212> PRT
<213> Unknown (Unknown)
<400> 42
Glu Ile Val Ile Thr Gln Ser Pro Thr Thr Val Ala Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Leu Thr Cys Arg Ala Ser Ser Ser Ile Thr Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Asp Thr Ser Asn Arg Ala Ser Gly Val Pro Asn Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Thr Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 43
<211> 119
<212> PRT
<213> Unknown (Unknown)
<400> 43
Glu Val Gln Leu Gln Gln Tyr Gly Ala Glu Leu Gly Lys Pro Gly Thr
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Leu Ser Gly Phe Asp Ile Trp Tyr Thr
20 25 30
Tyr Leu His Trp Val Ile Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Lys Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Lys Asn Lys Ala Thr Val Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Gln Leu Lys Ser Asp Asp Thr Ala Ile Tyr Phe Cys
85 90 95
Ala Met Asp Gly Tyr Asn Leu Gly Thr Met Asp Ala Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 44
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 44
Ile Asp Ile Val Met Thr Gln Ser Pro Ala Leu Ala Met Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Ile Ser Cys Arg Ala Ser Glu Gly Val Asn Ser Tyr
20 25 30
Met His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu Pro Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Trp Ser Asp Pro Tyr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 45
<211> 119
<212> PRT
<213> Unknown (Unknown)
<400> 45
Glu Val Gln Leu Gln Gln Tyr Gly Ala Glu Leu Gly Lys Pro Gly Thr
1 5 10 15
Ser Val Lys Leu Ser Cys Asn Leu Ser Gly Phe Asp Ile Trp Tyr Thr
20 25 30
Tyr Leu His Trp Val Ile Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Lys Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Lys Asn Lys Ala Thr Val Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Gln Leu Lys Ser Asp Asp Thr Ala Ile Tyr Phe Cys
85 90 95
Ala Met Asp Gly Tyr Asn Leu Gly Thr Met Asp Ala Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 46
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 46
Gly Asp Thr Val Leu Thr Gln Ser Pro Ala Leu Ala Met Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Ile Ser Cys Arg Ala Ser Glu Gly Val Asn Ser Tyr
20 25 30
Val His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Trp Ser Asp Pro Tyr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 47
<211> 117
<212> PRT
<213> Unknown (Unknown)
<400> 47
Gly Asp Arg Arg Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Val Thr Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr
20 25 30
Asp Met Val Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr
65 70 75 80
Leu Gln Ile Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Thr Ala Thr Ser Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Ile Ser Ser
115
<210> 48
<211> 108
<212> PRT
<213> Unknown (Unknown)
<400> 48
Gly Asp Thr Val Leu Thr Gln Ser His Asp Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Ser Cys Lys Ala Ser Glu Thr Val Ser Ala Arg
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Arg Leu Leu Ile
35 40 45
Tyr Lys Gly Ser Asn Leu Pro Ser Gly Val Pro Val Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala
65 70 75 80
Asp Asp Thr Ala Thr Tyr Phe Cys Gln Gln Asn Leu Asn Gly Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Arg Arg
100 105
<210> 49
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 49
Gly Phe Ile Phe Ser Asn Tyr Asp
1 5
<210> 50
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 50
Ser Ile Ser Pro Ser Gly Gly Val Thr
1 5
<210> 51
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 51
Ala Arg His Glu Tyr Ser Ser Tyr Ile Tyr
1 5 10
<210> 52
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 52
Lys Ser Val Gly Ile Arg
1 5
<210> 53
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 53
Lys Ile Ser Asn Leu Ala
1 5
<210> 54
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 54
Gln Gln Ser Trp Asn Gly Pro Pro Tyr Thr
1 5 10
<210> 55
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 55
Gly Phe Ile Phe Ser Asn Tyr Glu
1 5
<210> 56
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 56
Ser Ile Ser Pro Ser Gly Gly Tyr Thr
1 5
<210> 57
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 57
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr
1 5 10
<210> 58
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 58
Glu Val Ile Asn Ser Arg
1 5
<210> 59
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 59
Lys Ala Ser Asn Leu Ala
1 5
<210> 60
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 60
Gln Gln Asn Leu Asn Gly Pro Trp Thr
1 5
<210> 61
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 61
Gly Phe Thr Phe Ser Leu Tyr Glu
1 5
<210> 62
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 62
Ser Ile Ser Pro Arg Gly Thr Ser Thr
1 5
<210> 63
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 63
Ala Arg His Thr Ala Ser Tyr Phe Asp Tyr
1 5 10
<210> 64
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 64
Glu Gly Val Ser Ile Arg
1 5
<210> 65
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 65
Lys Ala Ser Asn Leu Pro
1 5
<210> 66
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 66
Gln Gln Asn Trp Asp Gly Pro Trp Thr
1 5
<210> 67
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 67
Gly Phe Asn Ile Lys Asp Thr Tyr
1 5
<210> 68
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 68
Arg Ile Asp Pro Ala Asn Gly Asn Thr
1 5
<210> 69
<211> 11
<212> PRT
<213> Unknown (Unknown)
<400> 69
Ala Ser Leu Tyr Ser Tyr Gly Ala Met Asp Tyr
1 5 10
<210> 70
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 70
Asp Val Ile Asn Ser Arg
1 5
<210> 71
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 71
Lys Ala Ser Asn Leu Ala
1 5
<210> 72
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 72
Gln Gln Asn Leu Asn Gly Pro Trp Thr
1 5
<210> 73
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 73
Gly Phe Ile Phe Ser Asn Tyr Asp
1 5
<210> 74
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 74
Ser Ile Ser Pro Ser Gly Gly Ser Thr
1 5
<210> 75
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 75
Ala Arg His Thr Ala Ser Tyr Phe Asp Asn
1 5 10
<210> 76
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 76
Glu Ile Ile Asn Ser Arg
1 5
<210> 77
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 77
Lys Ala Ser Asn Leu Ala
1 5
<210> 78
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 78
Gln Gln Asn Leu Asn Asp Pro Trp Thr
1 5
<210> 79
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 79
Gly Phe Ile Phe Ser Asn Tyr Glu
1 5
<210> 80
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 80
Ser Ile Ser Pro Ser Gly Gly Tyr Thr
1 5
<210> 81
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 81
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr
1 5 10
<210> 82
<211> 5
<212> PRT
<213> Unknown (Unknown)
<400> 82
Ser Ser Val Asn Tyr
1 5
<210> 83
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 83
Leu Thr Ser Asn Leu Ala
1 5
<210> 84
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 84
Gln Gln Trp Ser Thr Asn Pro Leu Thr
1 5
<210> 85
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 85
Gly Phe Ile Phe Ser Asn Tyr Glu
1 5
<210> 86
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 86
Ser Ile Ser Pro Ser Gly Gly Asn Thr
1 5
<210> 87
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 87
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr
1 5 10
<210> 88
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 88
Glu Val Ile Asn Ser Arg
1 5
<210> 89
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 89
Lys Ser Ser Asn Leu Ala
1 5
<210> 90
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 90
Gln Gln Asn Leu Asn Gly Pro Trp Thr
1 5
<210> 91
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 91
Gly Phe Ile Phe Ser Asn Tyr Glu
1 5
<210> 92
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 92
Ser Ile Ser Pro Ser Gly Gly Asn Thr
1 5
<210> 93
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 93
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr
1 5 10
<210> 94
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 94
Glu Val Ile Asn Ser Arg
1 5
<210> 95
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 95
Lys Ala Ser Asn Leu Ala
1 5
<210> 96
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 96
Gln Gln Asn Leu Asn Gly Pro Trp Thr
1 5
<210> 97
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 97
Gly Phe Ser Phe Ser Asn Tyr Glu
1 5
<210> 98
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 98
Ser Ile Ser Pro Ser Gly Gly Ile Thr
1 5
<210> 99
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 99
Ala Arg His Thr Ala Ser Phe Phe Asp Tyr
1 5 10
<210> 100
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 100
Glu Ser Val Ser Ile Arg
1 5
<210> 101
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 101
Lys Ala Ser Asn Leu Ala
1 5
<210> 102
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 102
Gln Gln Asn Leu Asp Gly Pro Trp Thr
1 5
<210> 103
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 103
Asp Tyr Ser Ile Thr Ser Gly Tyr Arg
1 5
<210> 104
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 104
Phe Met Asn Thr Ala Gly Asn Thr
1 5
<210> 105
<211> 13
<212> PRT
<213> Unknown (Unknown)
<400> 105
Ala Arg Gly Gly Tyr Asp Ser Phe Trp Phe Phe Asp Phe
1 5 10
<210> 106
<211> 5
<212> PRT
<213> Unknown (Unknown)
<400> 106
Ser Ser Ile Thr Tyr
1 5
<210> 107
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 107
Asp Thr Ser Asn Arg Ala
1 5
<210> 108
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 108
Gln Gln Trp Ser Thr Asn Pro Pro Thr
1 5
<210> 109
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 109
Gly Phe Thr Phe Asn Asn Tyr Tyr
1 5
<210> 110
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 110
Ser Ile Ser Pro Ser Gly Gly Arg Thr
1 5
<210> 111
<211> 13
<212> PRT
<213> Unknown (Unknown)
<400> 111
Val Thr Glu Asp Asn Glu Gly Thr Ser Pro Phe Ala Tyr
1 5 10
<210> 112
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 112
Gln Asn Val Gly Ser Ser
1 5
<210> 113
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 113
Arg Val Ser Asn Arg Tyr
1 5
<210> 114
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 114
Met Gln Ser Tyr Ser Tyr Pro Leu Thr
1 5
<210> 115
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 115
Gly Phe Ile Phe Ser Ser Tyr Glu
1 5
<210> 116
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 116
Ser Leu Ser Pro Ser Gly Gly Phe Thr
1 5
<210> 117
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 117
Ala Arg His Thr Ala Ser Tyr Phe Asp Tyr
1 5 10
<210> 118
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 118
Glu Ile Ile Asn Ala Arg
1 5
<210> 119
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 119
Lys Ala Ser Asn Leu Ala
1 5
<210> 120
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 120
Gln Gln Ser Leu Asn Gly Pro Trp Thr
1 5
<210> 121
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 121
Gly Phe Thr Phe Ser Asn Tyr Asp
1 5
<210> 122
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 122
Ser Ile Ser Pro Ser Gly Gly Val Thr
1 5
<210> 123
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 123
Ala Arg His Glu Tyr Ser Ser Tyr Val Tyr
1 5 10
<210> 124
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 124
Lys Ser Val Ser Ile Arg
1 5
<210> 125
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 125
Lys Val Ser Asn Leu Ala
1 5
<210> 126
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 126
Gln Gln Ser Trp Asn Gly Pro Pro Tyr Thr
1 5 10
<210> 127
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 127
Gly Phe Ile Phe Ser Asn Tyr Glu
1 5
<210> 128
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 128
Ser Ile Ser Pro Ser Gly Gly Tyr Thr
1 5
<210> 129
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 129
Thr Arg His Thr Ala Ser Tyr Phe Asp Tyr
1 5 10
<210> 130
<211> 5
<212> PRT
<213> Unknown (Unknown)
<400> 130
Ser Ser Leu Ile Tyr
1 5
<210> 131
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 131
Asp Thr Ser Asn Arg Ala
1 5
<210> 132
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 132
Gln Glu Trp Ser Arg Asn Pro Pro Thr
1 5
<210> 133
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 133
Gly Phe Asn Phe Asn Asp Tyr Phe
1 5
<210> 134
<211> 11
<212> PRT
<213> Unknown (Unknown)
<400> 134
Gln Ile Arg Asn Lys Asn Tyr Asn Tyr Val Thr
1 5 10
<210> 135
<211> 12
<212> PRT
<213> Unknown (Unknown)
<400> 135
Thr Arg Tyr Asn Thr His Met Gly Ala Met Asp Ala
1 5 10
<210> 136
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 136
Gln Asp Ile Gly Asn Trp
1 5
<210> 137
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 137
Ser Ala Thr Ser Leu Ala
1 5
<210> 138
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 138
Leu Gln Thr Tyr Ser Ser Pro Pro Thr
1 5
<210> 139
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 139
Gly Phe Thr Phe Ile Asn Tyr Trp
1 5
<210> 140
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 140
Ser Ile Thr Asn Thr Asp Gly Thr Thr
1 5
<210> 141
<211> 11
<212> PRT
<213> Unknown (Unknown)
<400> 141
Ala Arg Phe Gly Leu Ser Gly Pro Phe Asp Tyr
1 5 10
<210> 142
<211> 11
<212> PRT
<213> Unknown (Unknown)
<400> 142
Gln Ser Leu Gly His Ser Asn Gly Lys Thr Tyr
1 5 10
<210> 143
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 143
Arg Val Ser Ile Arg Leu
1 5
<210> 144
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 144
Leu Gln Ser Thr His Phe Pro Leu Thr
1 5
<210> 145
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 145
Gly Phe Ser Phe Asn Asn Tyr Asp
1 5
<210> 146
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 146
Ser Ile Ser Pro Ser Gly Gly Phe Thr
1 5
<210> 147
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 147
Ala Arg His Thr Ala Thr Tyr Phe Asp Asn
1 5 10
<210> 148
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 148
Glu Ile Ile Asn Ala Arg
1 5
<210> 149
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 149
Lys Ala Ser Asn Leu Ala
1 5
<210> 150
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 150
Gln Gln Ser Leu Asn Gly Pro Trp Thr
1 5
<210> 151
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 151
Gly Phe Ser Phe Asn Asn Tyr Asp
1 5
<210> 152
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 152
Ser Ile Ser Pro Ser Gly Gly Phe Thr
1 5
<210> 153
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 153
Ala Arg His Thr Ala Thr Tyr Phe Asp Asn
1 5 10
<210> 154
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 154
Gln Asp Ile Asn Ser Tyr
1 5
<210> 155
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 155
Arg Ala Asn Arg Leu Val
1 5
<210> 156
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 156
Leu Gln Tyr Asp Glu Phe Pro Tyr Thr
1 5
<210> 157
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 157
Gly Phe Ser Phe Ser Asn Tyr Glu
1 5
<210> 158
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 158
Ser Ile Ser Pro Ser Gly Gly Ile Thr
1 5
<210> 159
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 159
Ala Arg His Thr Ala Ser Phe Phe Asp Tyr
1 5 10
<210> 160
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 160
Glu Ser Val Ser Ile Arg
1 5
<210> 161
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 161
Lys Ser Ser Asn Leu Ala
1 5
<210> 162
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 162
Gln Gln Asn Leu Asp Gly Pro Trp Thr
1 5
<210> 163
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 163
Gly Phe Thr Phe Ser Lys Tyr Asp
1 5
<210> 164
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 164
Ser Ile Ser Pro Ser Gly Gly Thr Thr
1 5
<210> 165
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 165
Ala Arg His Glu Tyr Ser Ser Tyr Val Tyr
1 5 10
<210> 166
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 166
Glu Asn Val Ser Pro Arg
1 5
<210> 167
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 167
Lys Val Ser Asn Leu Ala
1 5
<210> 168
<211> 10
<212> PRT
<213> Unknown (Unknown)
<400> 168
Gln Gln Ser Trp Asn Gly Pro Pro Tyr Thr
1 5 10
<210> 169
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 169
Asp Tyr Ser Ile Thr Ser Gly Tyr Arg
1 5
<210> 170
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 170
Phe Met Asn Thr Ala Gly Asn Thr
1 5
<210> 171
<211> 13
<212> PRT
<213> Unknown (Unknown)
<400> 171
Ala Arg Gly Gly Tyr Asp Ser Leu Trp Phe Phe Asp Phe
1 5 10
<210> 172
<211> 5
<212> PRT
<213> Unknown (Unknown)
<400> 172
Ser Ser Ile Thr Tyr
1 5
<210> 173
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 173
Asp Thr Ser Asn Arg Ala
1 5
<210> 174
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 174
Gln Gln Trp Ser Thr Asn Pro Pro Thr
1 5
<210> 175
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 175
Gly Phe Asp Ile Trp Tyr Thr Tyr
1 5
<210> 176
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 176
Arg Ile Asp Pro Ala Asn Gly Lys Thr
1 5
<210> 177
<211> 12
<212> PRT
<213> Unknown (Unknown)
<400> 177
Ala Met Asp Gly Tyr Asn Leu Gly Thr Met Asp Ala
1 5 10
<210> 178
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 178
Glu Gly Val Asn Ser Tyr
1 5
<210> 179
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 179
Lys Ala Ser Asn Leu Pro
1 5
<210> 180
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 180
Gln Gln Ser Trp Ser Asp Pro Tyr Thr
1 5
<210> 181
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 181
Gly Phe Asp Ile Trp Tyr Thr Tyr
1 5
<210> 182
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 182
Arg Ile Asp Pro Ala Asn Gly Lys Thr
1 5
<210> 183
<211> 12
<212> PRT
<213> Unknown (Unknown)
<400> 183
Ala Met Asp Gly Tyr Asn Leu Gly Thr Met Asp Ala
1 5 10
<210> 184
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 184
Glu Gly Val Asn Ser Tyr
1 5
<210> 185
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 185
Lys Ala Ser Asn Leu Ala
1 5
<210> 186
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 186
Gln Gln Ser Trp Ser Asp Pro Tyr Thr
1 5
<210> 187
<211> 8
<212> PRT
<213> Unknown (Unknown)
<400> 187
Gly Phe Ser Phe Ser Asn Tyr Asp
1 5
<210> 188
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 188
Ser Ile Ser Pro Ser Gly Gly Asn Thr
1 5
<210> 189
<211> 11
<212> PRT
<213> Unknown (Unknown)
<400> 189
Ala Arg His Thr Ala Thr Ser Phe Asp Tyr Trp
1 5 10
<210> 190
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 190
Glu Thr Val Ser Ala Arg
1 5
<210> 191
<211> 6
<212> PRT
<213> Unknown (Unknown)
<400> 191
Lys Gly Ser Asn Leu Pro
1 5
<210> 192
<211> 9
<212> PRT
<213> Unknown (Unknown)
<400> 192
Gln Gln Asn Leu Asn Gly Pro Trp Thr
1 5

Claims (7)

1. An anti-human CD47 monoclonal antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1 shown as SEQ ID NO: 49, heavy chain CDR2 shown as SEQ ID NO:50, and heavy chain CDR3 shown as SEQ ID NO: 51; the light chain variable region comprises the light chain CDR1 shown in SEQ ID NO:52, the light chain CDR2 shown in SEQ ID NO:53 and the light chain CDR3 shown in SEQ ID NO: 54.
2. A nucleic acid molecule, characterized in that: comprising a nucleic acid sequence capable of encoding an anti-human CD47 monoclonal antibody according to claim 1.
3. A carrier, characterized by: comprising the nucleic acid molecule of claim 2.
4. A host cell, characterized in that: the host cell comprising the nucleic acid molecule of claim 2 or comprising the vector of claim 3.
5. A conjugate, characterized by: comprising the antibody of claim 1.
6. A composition characterized by: comprising a main component comprising one or more of the antibody of claim 1 or the nucleic acid molecule of claim 2 or the vector of claim 3, the host cell of claim 4 or the conjugate of claim 5 and an accessory component selected from a pharmaceutically acceptable carrier or excipient and optionally other biologically active substances.
7. A kit, characterized in that: comprising the antibody of claim 1.
CN201910534489.3A 2018-06-20 2019-06-20 Anti-human CD47 monoclonal antibody and application thereof Active CN110615841B (en)

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CN111087470B (en) * 2020-01-19 2022-05-10 中国人民解放军第四军医大学 Anti-human CD47 monoclonal antibody 7G4mAb and application thereof
CN116693684A (en) * 2023-04-19 2023-09-05 浙江正熙生物技术有限公司 Anti-human CD8 antibody and preparation method and application thereof

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