CN112538114A - Anti-human CD38 antibody and application thereof - Google Patents
Anti-human CD38 antibody and application thereof Download PDFInfo
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Abstract
The present invention provides an antibody molecule or fragment thereof that binds to human CD38 for use in the prevention or treatment of disease. The antibody molecule is a human CD38 antibody molecule with a brand new sequence obtained by screening after a mouse is immunized and fused with myeloma cells to generate hybridoma cells, and the antibody has consistent or better functional activity with known antibodies, including in vitro ADCC (ADCC), CDC (CDC), Daudi (Daudi) apoptosis activity and the like.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to a novel anti-CD 38 antibody or a functional fragment thereof. The invention also relates to the use of said antibody or functional fragment thereof.
Background
CD38 is a type II transmembrane glycoprotein with a molecular weight of 46KD, comprising a C-terminal extracellular region (258 amino acids), a transmembrane region (21 amino acids) and an N-terminal (21 amino acids), and functions to mediate receptor-mediated adhesion and signaling, and to mediate calcium mobilization via its extracellular enzymatic activity, catalyzing the formation of cyclic ADP ribose (cADPR) and ADPR.
CD38 is uniformly highly expressed on multiple myeloma cells (MM) and is poorly expressed on normal lymphocytes and bone marrow cells as well as on some tissues of non-hematopoietic origin. CD38 belongs to transmembrane glycoprotein, has in vitro enzyme activity and simultaneously has the functions of a receptor and an adhesion molecule, so that the CD38 is an ideal target for MM treatment; the CD38 antibody is also suitable for use in combination regimens based on its unique mechanism of action, low toxicity and single agent activity. Currently, there are several studies exploring the efficacy of CD38 antibody-based therapies for newly diagnosed high-risk MM patients. In addition, the therapeutic effects of the CD38 antibody on other hematological malignancies (acute lymphoblastic leukemia, NK/T cell lymphoma, and acute myeloid leukemia) are also under investigation.
Monoclonal antibodies against CD38 are currently developed by several pharmaceutical companies and achieve anti-MM mechanisms through antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADPC), and direct inhibition of CD38 enzyme activity. Currently, CD38 antibodies in clinical trials or that have been approved include Daratumumab (Darzalex), Isatuximab, MOR-202, and TAK-079, among others.
Among the monoclonal antibody drugs currently developed against CD38, the robust Darzalex is the first globally approved CD 38-mediated, cytolytic antibody drug with broad-spectrum killing activity that can target and bind to the highly expressed transmembrane ectoenzyme CD38 molecule on the surface of multiple myeloma and various solid tumor cells, and induce rapid tumor cell death through a variety of immune-mediated mechanisms of action, including Complementary Dependent Cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP), as well as by apoptosis (apoptosis). In addition, Darzalex has also been shown to be able to target immunosuppressive cells in the tumor microenvironment to exhibit immunomodulatory activity. In addition to multiple myeloma, Darzalex also has potential to treat other types of tumors that highly express the CD38 molecule, including diffuse large B-cell lymphoma (DLBCL), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Plasma Cell Leukemia (PCL), Acute Myelogenous Leukemia (AML), Follicular Lymphoma (FL), and Mantle Cell Lymphoma (MCL), among others.
In addition, the development and use of novel anti-CD 38 antibodies is needed in the art.
Disclosure of Invention
The technical problem to be solved by the invention is to obtain a high-affinity antibody which specifically binds to CD38, particularly human CD38, by hybridoma screening and humanization technology.
In view of the above technical problems, it is an object of the present invention to provide an antibody or a fragment thereof that specifically binds to CD38, particularly human CD38, and to provide uses thereof based on the antibody or the fragment thereof. "fragments" of the antibody molecules of the invention encompass various functional fragments of antibodies, e.g., antigen-binding portions thereof, e.g., Fab, F (ab')2Or a scFv fragment.
Specifically, the present invention provides the following technical solutions.
In one aspect, the invention provides an antibody molecule or fragment thereof that binds human CD38, said antibody molecule or fragment thereof comprising a heavy chain variable region (VH), wherein said heavy chain variable region comprises a CDR1(VH-CDR1) selected from the group consisting of SEQ ID NO 1, SEQ ID NO 7, SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 20, SEQ ID NO 26, SEQ ID NO 31, SEQ ID NO 41 and SEQ ID NO 147, a CDR2(VH-CDR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 8, SEQ ID NO 12, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 27, SEQ ID NO 32, SEQ ID NO 37 and SEQ ID NO 42, and a CDR2(VH-CDR2) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 17, SEQ ID NO 9, SEQ ID NO, 18 SEQ ID NO, 22 SEQ ID NO, 28 SEQ ID NO, 33 SEQ ID NO, 38 SEQ ID NO, 43 SEQ ID NO, 46 SEQ ID NO, 47 SEQ ID NO, 48 SEQ ID NO and the CDR3(VH-CDR3) shown in SEQ ID NO 137; and
the antibody molecule or fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain variable region selected from the group consisting of SEQ ID NOs: 4. SEQ ID NO: 14. SEQ ID NO: 23. SEQ ID NO: 29. SEQ ID NO: 34. SEQ ID NO: 39. SEQ ID NO: 44. SEQ ID NO: 49. SEQ ID NO:136 and SEQ ID NO:148 (VL-CDR1) selected from the group consisting of SEQ ID NOs: 5. SEQ ID NO:24 and SEQ ID NO:35 (VL-CDR2) selected from the group consisting of SEQ ID NOs: 6. SEQ ID NO: 10. SEQ ID NO: 15. SEQ ID NO: 19. SEQ ID NO: 25. SEQ ID NO: 30. SEQ ID NO: 36. SEQ ID NO: 40. SEQ ID NO: 45. SEQ ID NO:138 and SEQ ID NO:139 (VL-CDR 3).
According to a particular embodiment of the invention, the heavy chain variable region may comprise a combination of CDRs (VH-CDR1, VH-CDR2, VH-CDR3) selected from:
(1) VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO. 2, and VH-CDR3 shown in SEQ ID NO. 3;
(2) VH-CDR1 shown in SEQ ID NO. 7, VH-CDR2 shown in SEQ ID NO. 8, VH-CDR3 shown in SEQ ID NO. 9;
(3) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 13;
(4) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 18;
(5) VH-CDR1 shown in SEQ ID NO:20, VH-CDR2 shown in SEQ ID NO:21, VH-CDR3 shown in SEQ ID NO: 22;
(6) VH-CDR1 shown in SEQ ID NO. 26, VH-CDR2 shown in SEQ ID NO. 27, VH-CDR3 shown in SEQ ID NO. 28;
(7) VH-CDR1 shown in SEQ ID NO:31, VH-CDR2 shown in SEQ ID NO:32, VH-CDR3 shown in SEQ ID NO: 33;
(8) VH-CDR1 shown in SEQ ID NO:26, VH-CDR2 shown in SEQ ID NO:37, VH-CDR3 shown in SEQ ID NO: 38;
(9) VH-CDR1 shown in SEQ ID NO:41, VH-CDR2 shown in SEQ ID NO:42, VH-CDR3 shown in SEQ ID NO: 43;
(10) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 46;
(11) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 47;
(12) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 48;
(13) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 137;
(14) VH-CDR1 shown in SEQ ID NO:147, VH-CDR2 shown in SEQ ID NO:42, VH-CDR3 shown in SEQ ID NO: 43;
and/or, the antibody molecule or fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region comprises a combination of CDRs (VL-CDR1, VL-CDR2, VL-CDR3) selected from:
(1) VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 6;
(2) VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 10;
(3) VL-CDR1 shown in SEQ ID NO.14, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 15;
(4) VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 19;
(5) VL-CDR1 shown in SEQ ID NO. 23, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 25;
(6) VL-CDR1 shown in SEQ ID NO. 29, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 30;
(7) VL-CDR1 shown in SEQ ID NO. 34, VL-CDR2 shown in SEQ ID NO. 35, VL-CDR3 shown in SEQ ID NO. 36;
(8) VL-CDR1 shown in SEQ ID NO:39, VL-CDR2 shown in SEQ ID NO:24, VL-CDR3 shown in SEQ ID NO: 40;
(9) VL-CDR1 shown in SEQ ID NO. 44, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 45;
(10) VL-CDR1 shown in SEQ ID NO. 49, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 40;
(11) VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 138;
(12) VL-CDR1 shown in SEQ ID NO:136, VL-CDR2 shown in SEQ ID NO:35, VL-CDR3 shown in SEQ ID NO: 36;
(13) VL-CDR1 shown in SEQ ID NO:136, VL-CDR2 shown in SEQ ID NO:35, VL-CDR3 shown in SEQ ID NO: 139;
(14) VL-CDR1 shown in SEQ ID NO. 148, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 45.
Preferably, the heavy chain variable region in the antibody molecule or fragment thereof comprises an amino acid sequence selected from any one of SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68 to SEQ ID NO 88, SEQ ID NO 140 to SEQ ID NO 142 or an amino acid sequence having at least 75% identity to the amino acid sequence shown; and/or
The light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO 51, 53, 55, 57, 59, 61, 63, 65, 67, 89 to 105, 134 and 135, 143 to 146 or an amino acid sequence having at least 75% identity to the amino acid sequence shown.
According to a particular embodiment of the invention, the antibody molecule or fragment thereof comprises a CDR combination selected from:
(1) VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO. 2, and VH-CDR3 shown in SEQ ID NO. 3; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 6;
(2) VH-CDR1 shown in SEQ ID NO. 7, VH-CDR2 shown in SEQ ID NO. 8, VH-CDR3 shown in SEQ ID NO. 9; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 10;
(3) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 13; VL-CDR1 shown in SEQ ID NO.14, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 15;
(4) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 18; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 19;
(5) VH-CDR1 shown in SEQ ID NO:20, VH-CDR2 shown in SEQ ID NO:21, VH-CDR3 shown in SEQ ID NO: 22; VL-CDR1 shown in SEQ ID NO. 23, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 25;
(6) VH-CDR1 shown in SEQ ID NO. 26, VH-CDR2 shown in SEQ ID NO. 27, VH-CDR3 shown in SEQ ID NO. 28; VL-CDR1 shown in SEQ ID NO. 29, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 30;
(7) VH-CDR1 shown in SEQ ID NO:31, VH-CDR2 shown in SEQ ID NO:32, VH-CDR3 shown in SEQ ID NO: 33; VL-CDR1 shown in SEQ ID NO. 34, VL-CDR2 shown in SEQ ID NO. 35, VL-CDR3 shown in SEQ ID NO. 36;
(8) VH-CDR1 shown in SEQ ID NO:26, VH-CDR2 shown in SEQ ID NO:37, VH-CDR3 shown in SEQ ID NO: 38; VL-CDR1 shown in SEQ ID NO:39, VL-CDR2 shown in SEQ ID NO:24, VL-CDR3 shown in SEQ ID NO: 40;
(9) VH-CDR1 shown in SEQ ID NO:41, VH-CDR2 shown in SEQ ID NO:42, VH-CDR3 shown in SEQ ID NO: 43; VL-CDR1 shown in SEQ ID NO. 44, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 45;
(10) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 47; VL-CDR1 shown in SEQ ID NO.14, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 15;
(11) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 137; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 19;
(12) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 137; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 138;
(13) VH-CDR1 shown in SEQ ID NO:31, VH-CDR2 shown in SEQ ID NO:32, VH-CDR3 shown in SEQ ID NO: 33; VL-CDR1 shown in SEQ ID NO:136, VL-CDR2 shown in SEQ ID NO:35, VL-CDR3 shown in SEQ ID NO: 36;
(14) VH-CDR1 shown in SEQ ID NO:147, VH-CDR2 shown in SEQ ID NO:42, VH-CDR3 shown in SEQ ID NO: 43; VL-CDR1 shown in SEQ ID NO. 44, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 45;
preferably, the antibody molecule or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(1) an amino acid sequence as shown in SEQ ID NO. 50 or an amino acid sequence with at least 75% identity to an amino acid sequence as shown in SEQ ID NO. 50; and, an amino acid sequence as set forth in SEQ ID NO. 51 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 51;
(2) an amino acid sequence as shown in SEQ ID NO. 52 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 52; and, the amino acid sequence as set forth in SEQ ID NO 53 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 53;
(3) an amino acid sequence as shown in SEQ ID NO. 54 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 54; and, an amino acid sequence as set forth in SEQ ID NO:55 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 55;
(4) an amino acid sequence as shown in SEQ ID NO. 56 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 56; and, an amino acid sequence as set forth in SEQ ID NO:57 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 57;
(5) an amino acid sequence as set forth in SEQ ID NO. 58 or an amino acid sequence with at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 58; and, the amino acid sequence as set forth in SEQ ID NO. 59 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 59;
(6) an amino acid sequence as set forth in SEQ ID NO:60 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 60; and, the amino acid sequence as set forth in SEQ ID NO 61 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 61;
(7) an amino acid sequence as set forth in SEQ ID NO. 62 or an amino acid sequence with at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 62; and, the amino acid sequence as set forth in SEQ ID NO:63 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 63;
(8) an amino acid sequence as shown in SEQ ID NO. 64 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 64; and, the amino acid sequence as set forth in SEQ ID NO. 65 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 65;
(9) an amino acid sequence as shown in SEQ ID NO. 66 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 66; and, the amino acid sequence as set forth in SEQ ID NO:67 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 67;
(10) an amino acid sequence as shown in SEQ ID NO. 69 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 69; and, an amino acid sequence as set forth in SEQ ID NO:90 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 90;
(11) 71 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID NO 71; and, an amino acid sequence as set forth in SEQ ID NO:90 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 90;
(12) an amino acid sequence as set forth in SEQ ID NO. 74 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 74; and, the amino acid sequence as set forth in SEQ ID NO 92 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 92;
(13) an amino acid sequence as set forth in SEQ ID NO. 75 or an amino acid sequence with at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 75; and, the amino acid sequence as set forth in SEQ ID NO 94 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 94;
(14) an amino acid sequence as set forth in SEQ ID NO. 76 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 76; and, the amino acid sequence as set forth in SEQ ID NO 94 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 94;
(15) 77 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID No. 77; and, an amino acid sequence as set forth in SEQ ID NO. 95 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 95;
(16) an amino acid sequence as shown in SEQ ID NO. 83 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 83; and, an amino acid sequence as set forth in SEQ ID NO. 102 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 102;
(17) an amino acid sequence as shown in SEQ ID NO. 84 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 84; and, an amino acid sequence as set forth in SEQ ID NO. 102 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 102;
(18) an amino acid sequence as shown in SEQ ID NO. 85 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 85; and, the amino acid sequence as set forth in SEQ ID NO:134 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 134;
(19) an amino acid sequence as shown in SEQ ID NO. 88 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 88; and, an amino acid sequence as set forth in SEQ ID NO. 103 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 103;
(20) an amino acid sequence as shown in SEQ ID NO. 88 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 88; and, the amino acid sequence as set forth in SEQ ID NO 104 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 104;
(21) 140 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID No. 140; and, the amino acid sequence as set forth in SEQ ID NO. 143 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 143;
(22) the amino acid sequence shown as SEQ ID NO.141 or the amino acid sequence with at least 75 percent of identity with the amino acid sequence shown as SEQ ID NO. 141; and, the amino acid sequence shown as SEQ ID NO:144 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID NO: 144;
(23) 142 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID No. 142; and, the amino acid sequence shown as SEQ ID NO:144 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID NO: 144.
The antibody molecule or fragment thereof of the present invention may be in any form of monoclonal antibody, single chain antibody, diabody, single domain antibody, nanobody, fully or partially humanized antibody or chimeric antibody, or the antibody molecule or fragment thereof may be a half-antibody or antigen-binding fragment of a half-antibody, such as scFv, BsFv, dsFv, (dsFv)2、Fab、Fab'、F(ab')2Or Fv; as for the fragment of the antibody, an antigen-binding fragment of the antibody is particularly preferable.
Preferably, the antibody molecule or fragment thereof further comprises a human or murine constant region, preferably a human or murine light chain constant region (CL) and/or heavy chain constant region (CH);
more preferably, the antibody molecule or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of the kappa or lambda type.
According to a particular embodiment of the invention, the antibody molecule is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type;
preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO 106 or an amino acid sequence having at least 75% identity to said amino acid sequence;
preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:107 or an amino acid sequence having at least 75% identity to said amino acid sequence.
"at least 75% identity" as set forth herein is any percentage identity greater than or equal to 75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity.
Based on the antibody molecule or fragment thereof of the invention, the invention also provides a conjugate or fusion protein comprising the antibody or fragment thereof of the invention. The conjugate or fusion protein may comprise other moieties, such as cell surface receptors, small molecule compounds such as amino acids and sugars, small molecule polymers or any other moiety that modifies the antibody of the invention, or even active proteins or polypeptides, that are chemically or physically bound to the antibody or fragment thereof of the invention.
In another aspect, the invention also provides a nucleic acid molecule encoding a heavy chain CDR, a light chain variable region, a heavy chain or a light chain in any of the antibodies or fragments thereof of the invention.
In yet another aspect, the invention provides a vector comprising a nucleic acid molecule of the invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector and the like. The vectors or nucleic acid molecules of the invention may be used to transform or transfect host cells or in any way into host cells for the purpose of preserving or expressing antibodies, etc.
Thus, in a further aspect, the present invention provides a host cell comprising a nucleic acid molecule and/or vector of the invention, or transformed or transfected with a nucleic acid molecule and/or vector of the invention. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
Based on the present disclosure, the antibody molecules or fragments thereof as well as the corresponding conjugates or fusion proteins, nucleic acid molecules, vectors and/or host cells provided by the present invention may be obtained by using any conventional technical methods known in the art. The antibody molecule or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector and/or host cell may be comprised in a pharmaceutical composition, more particularly in a pharmaceutical preparation, for use for various purposes according to the actual need.
Thus, in a further aspect, the invention also provides a pharmaceutical composition comprising an antibody molecule or fragment, conjugate or fusion protein thereof, nucleic acid molecule, vector and/or host cell according to the invention, and optionally a pharmaceutically acceptable excipient.
In a further aspect, the present invention provides the use of an antibody molecule or fragment, conjugate or fusion protein thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition as described above in the manufacture of a medicament for the treatment of a disease associated with the expression of CD38 or mediated by CD 38;
preferably, the disease is a hematological malignancy, preferably multiple myeloma or non-hodgkin's lymphoma. .
In a further aspect, the invention provides a kit comprising an antibody molecule or fragment, conjugate or fusion protein thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition of the invention.
In the invention, a mouse antibody is obtained by utilizing a hybridoma technology, and a candidate mouse antibody is obtained by antibody activity analysis (ELISA binding, epitope competition with a control antibody, affinity kinetics); then, cloning a sequence encoding a candidate mouse antibody light and heavy chain variable region to the upstream of a sequence encoding a human antibody light and heavy chain constant region, carrying out mammalian cell expression to prepare a chimeric antibody, and then verifying the Complementary Dependent Cytotoxicity (CDC) of the chimeric antibody on effector cells, the antibody dependent cell mediated cytotoxicity (ADCC) and the apoptosis activity of Duadi-luc by antibody activity analysis; then selecting a humanized template according to a Germline database, carrying out humanized design on an antibody sequence, and repeatedly verifying the Complementary Dependent Cytotoxicity (CDC) of the humanized modified antibody on effector cells, the antibody dependent cell mediated cytotoxicity (ADCC) and the in vitro cytology experiment verification of the apoptosis activity of Duadi-luc and the like by the obtained humanized antibody through antibody activity analysis; and then carrying out in-vitro physicochemical property analysis on the humanized antibody, and finally obtaining an anti-CD 38 humanized brand-new antibody sequence with the affinity and specificity equal to or better than those of a control antibody.
Experiments prove that the antibody molecule provided by the invention has an antigen binding recognition epitope close to or consistent with that of a known antibody, and simultaneously has the same or better functional activities including in-vitro ADCC (ADCC), CDC (CDC), Daudi (apoptosis inducing agent), and the like with the known antibody.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1 shows the flow cytometry identification of CD38 expressing cell lines, wherein fig. 1A: cell line CHO blank; FIG. 1B: cell line 1-T-12; FIG. 1C: cell line 1-T-14.
FIG. 2 shows the results of the first (FIG. 2A) and second (FIG. 2B) rounds of fusion screening of hybridoma cells.
FIG. 3 shows the binding activity of the antibody of the present invention to the cell surface-expressed antigen CD 38.
Figure 4 shows antibody-dependent cytotoxicity of antibodies of the invention, wherein figure 4A: 8B7 humanized antibody; FIG. 4B: a 20M19 humanized antibody; FIG. 4C: a 32a7 humanized antibody; FIG. 4D: 5E19 humanized antibody.
Fig. 5 shows the results of epitope analysis of the antibody of the present invention, wherein fig. 5A: relative to Daratumumab; FIG. 5B: relative to Mor 202; FIG. 5C: relative to Isatuximab.
Figure 6 shows the serum stability results for antibodies of the invention, wherein figure 6A: daratumumab; FIG. 6B: 8B7-H3L 3; FIG. 6C: 17H13-H2L 2; FIG. 6D: 20M19-H1L 1.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified. Wherein:
the sequences of the heavy chain constant region of the IgG1 subclass of the human monoclonal antibody are shown in SEQ ID NO. 106 and SEQ ID NO. 126, and the sequences of the light chain constant region of the kappa subclass of the human monoclonal antibody are shown in SEQ ID NO. 107 and SEQ ID NO. 127.
Recombinant human CD38 protein, purchased from ACRO biosystems, Cat No: CD 8-H5224.
Example 1Synthesis and expression of control antibodies
The light chain variable region and heavy chain variable region genes of the fully synthesized Daratumumab, Isatuximab and Mor202 antibodies are respectively cloned into eukaryotic expression vectors loaded with the upstream of the human-kappa light chain constant region and human IgG1 heavy chain constant region coding genes, and Daratumumab, Isatuximab and Mor202 light chain and heavy chain expression plasmids are respectively obtained. Transferring the obtained plasmid into escherichia coli for amplification, separating to obtain a large amount of plasmids containing Daratumumab, Isatuximab or Mor202 antibody light chains and heavy chains, mixing the two plasmids with PEI, and then co-transfecting into HEK293 cells.
The cells were transfected for 5-6 days, and the culture supernatant was taken and purified by a Mabselect affinity column to obtain Daratumumab, Isatuximab and Mor202 antibody recombinant proteins.
Daratumumab: the sequences are shown in SEQ ID NO 128 to SEQ ID NO 129.
Isatuximab: the sequences are shown in SEQ ID NO:130 to SEQ ID NO: 131.
Mor 202: the sequences are shown in SEQ ID NO:132 to SEQ ID NO: 133.
Example 2Preparation of CD38 antigen-expressing cell line
A CHO cell line 1-T-14 with high human CD38 expression and a CHO cell line 1-T-12 with low human CD38 expression were prepared.
Cloning the reading frame of CD38 gene from a vector (Cat: HG10818-UT, Beijing Yinqiao, Qian Shenzhou) containing CD38 cDNA by a PCR method, and cloning the reading frame of CD38 gene (the correct sequence is verified by sequencing) into a vector containing Glutamine Synthetase (GS) by an enzyme digestion method for screeningIn a stable expression vector of the gene, CHO-K1 cells were suspension-cultured by electrotransfection (Nuclear effector IIb, Lonza), and the transfected cells were placed in CD CHO AGTTM medium (Gibco, Cat:12490-2Standing for 2-3 weeks, MSX pressure screening, prescreening under the lens, scale-up culturing, and finally selecting 2 clones (1-T-12 and 1-T-14) by flow cytometry (FACS), scale-up culturing and freezing.
The flow cytometry results of the two cell lines and the negative control CHO cell line (CHO-blank) are shown in FIG. 1, and the Mean Fluorescence Intensity (MFI) values are shown in Table 1.
TABLE 1 expression (FACS) of CD38 antigen-expressing cell lines
| Cell line | MFI |
| CHO blank | 391.3 |
| 1-T-12 | 66006.4 |
| 1-T-14 | 133008 |
Example 3Screening and identification of hybridoma cells
1. Immunization of mice
The method comprises the following steps of adopting two immunization methods of a Freund adjuvant and a water-soluble adjuvant for a Balb/c mouse aged 8-10 weeks, wherein the Freund adjuvant performs intraperitoneal immune injection twice on the 0 th day and the 14 th day, the water-soluble adjuvant performs intramuscular immune injection twice on the 0 th day and the 21 st day on the Balb/c mouse aged 8-10 weeks, and the immune antigen is as follows: human CD38 recombinant protein, recombinantly expressed in HEK293 cells by Acro biosytem Biotech, Inc. The first immunization dose was 50. mu.g and the second immunization dose was 25. mu.g. Taking mouse serum before immunization as a negative control in detection, taking blood from tail vein of Freund's adjuvant 28 days after primary immunization and water-soluble adjuvant 35 days after primary immunization, and detecting serum titer by an ELISA method by using a 96-hole enzyme label plate coated with recombinant human CD38 protein; the mice with serum titer meeting the fusion requirement are boosted, and 25 mu g of antigen is diluted into 500 mu l by D-PBS and injected into the abdominal cavity. Splenocytes from mice with high serum titers at day 18-38 after the primary immunization were taken for the next cell fusion.
2. Cell fusion and hybridoma preparation
2.1 myeloma cell preparation
Culturing myeloma cells P3X63Ag8.653 required for fusion in a 500ml triangular culture flask to the cell density of 0.8-1.0E +6, and changing the culture solution to complete hybridoma culture medium for later use.
2.2 lymph node and B lymphocyte preparation
Selecting a mouse with the titer meeting the requirement, removing an eyeball to collect blood, and separating serum to be used as positive control serum during antibody detection; and the spleen of the mouse is taken aseptically, and the B lymphocyte suspension is prepared according to the conventional method.
2.3 electrofusion (Electro Cell Fusion, ECF)
Myeloma cells P3X63Ag8.653 and B lymphocytes were mixed at a ratio of 4:1 and then subjected to electrofusion. After electric shock, the cells were placed in CO at 37 deg.C2And standing in an incubator for 30 minutes, centrifuging at 1000RPM for 10 minutes at room temperature, re-suspending the cells by using 360ml of hybridoma selection medium, and then plating the cells into a 384-well plate to ensure that the plating density is 8000-20000 cells/well. And changing the liquid once after 2 to 3 days, and screening positive hybridomas on 7 to 10 days.
2.4 Positive hybridoma selection (FACS)
The fused hybridoma cells were cultured in 384-well plates, and the supernatant was analyzed by FACS for antibodies secreted from the hybridoma cells, and several clones were selected, which were capable of binding to the human CD 38-highly expressed strain 1-T-14 but not to CHO-blank cells. The selected clones were unicellularized by limiting dilution method, and only one antibody was secreted per hybridoma clone obtained after 3 rounds.
The results of the first and second rounds of fusion screening are shown in FIG. 2.
Example 4Identification of variable region sequence of murine monoclonal antibody, production of chimeric anti-human CD38 antibody, and antibody screening
After the hybridoma secreting the anti-human CD38 antibody is subjected to amplification culture, total RNA of the cell is extracted according to the steps of an RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); reverse transcribing hybridoma cell total RNA to cDNA using 5 XPrimeScript RT Master Mix (Takara); amplifying antibody light chain variable region IgVL (κ) and heavy chain variable region VH sequences using degenerate primers (ake krebber.1997) and Extaq PCR reagents (Takara); purifying the PCR amplification product by using a PCR clean-up Gel extraction kit (Macherey-Nagel Co.); connecting the amplified PCR product to a T Vector according to the specification of a pClone007 Simple Vector Kit (Scopheraceae Biotechnology limited), converting escherichia coli competent cells, amplifying the strain, extracting plasmids, and performing DNA sequencing to obtain the variable region sequence of the monoclonal antibody.
Splicing the heavy chain variable region sequence of a mouse anti-human CD38 monoclonal antibody and a publicly published heavy chain constant region sequence of a human monoclonal antibody IgG1 subclass together to construct a mammalian cell expression vector pCDNA3.4; the variable region sequence of the light chain of the mouse anti-human CD38 monoclonal antibody is spliced with the published constant region sequence of the light chain of the kappa subclass of the human monoclonal antibody to construct a mammalian cell expression vector pCDNA3.1. The constructed heavy chain vector and light chain vector of the anti-human CD38 chimeric antibody are mixed in pairs, HEK293 cells are transfected by Polyethyleneimine (PEI), cell supernatants are collected after about 7 days, and the anti-human CD38 chimeric antibody protein is obtained by purifying by using Mabselect.
The resulting chimeric antibody is referred to herein as "murine antibody nomenclature-xiIgG".
The screening of monoclonal antibodies such as an antigen ELISA binding experiment, an Octet binding experiment, an in vitro cell binding experiment, an in vitro cytology experiment (complement dependent cytotoxicity (CDC) and antibody dependent cytotoxicity (ADCC)) of the obtained antibody obtained by performing hybridoma cell culture supernatant or purification on the obtained murine antibody and the chimeric antibody (the experimental operation is shown below), the murine antibody and the corresponding chimeric antibody are verified with each other, and the combination of each experiment is integrated to screen and obtain a part of the murine antibody and the chimeric antibody thereof.
The light and heavy chain variable region (VH and VL) sequences from a portion of murine antibody are shown below, with the antigen Complementarity Determining Regions (CDRs) of the heavy and light chains underlined.
Murine antibody 2H 17:
>2H17_VH(SEQ ID NO:50)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYYCAKGMITTGYAMDYWGQGTSVTVSS
>2H17_VL(SEQ ID NO:51)
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIK
murine antibody 4J 1:
>4J1_VH(SEQ ID NO:52)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSFGIHWVRQSPGKGLEWLGVIWRGGSTDYNAAFLSRLSITKDNSKSQVFFKINSLRADDTAIYFCAKSMITTGYAMDYWGQGTSVTVSS
>4J1_VL(SEQ ID NO:53)
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYILSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIK
murine antibody 5E 19:
>5E19_VH(SEQ ID NO:54)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWLRQSPGKGLEWLGVIWRGGSTDYSAAFMSRLNITKDNSKSQVFFKMNSLQADDTAIYYCAKTTLTRVWYCDVWGAGTTVTVSS
>5E19_VL(SEQ ID NO:55)
DIQMTQSSSSFSVSLGDRVTITCKSSEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVVTYYCQQYWNTPLTFGAGTKLELK
murine antibody 8B 7:
>8B7_VH(SEQ ID NO:56)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLLSYGVHWVRQSPGKGLEWLGVIWRGGSADYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYYCAKSMITTGFTLDYWGQGTSVTVSS
>8B7_VL(SEQ ID NO:57)
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGHAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQFWNTPWTFGGGTKLEIK
murine antibody 13D 15:
>13D15_VH(SEQ ID NO:58)
QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGWINTYTGEPTNADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARKGFAYWGQGTLVTVSA
>13D15_VL(SEQ ID NO:59)
DIVLTQSPASLAVSLGQRTTISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQINEDPFTFGSGTKLEIK
murine antibody 15a 10:
>15A10_VH(SEQ ID NO:60)
QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTAEPTYADDFKGRFAFSLEASASTAYLQINNLKNEDMATYFCARKYGGYWGQGTTLTVSS
>15A10_VL(SEQ ID NO:61)
DIVLTQSPPSLAVSLGQRATISCRASESVDSYGNSFIHWYQQKPGQSPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQVNEDPLTFGAGTKLELK
murine antibody 17H 13:
>17H13_VH(SEQ ID NO:62)
QVQLQQSGAELAKPGASVKMSCKASGYTFTNYWMNWIKQRPGQGLEWIGYIIPSTDYTEYNQKFKDKATLTADKSSGTAYMQLSSLTSEDSAVYYCSRYPYRGYAMDYWGQGTSVTVSS
>17H13_VL(SEQ ID NO:63)
QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSTNPWTFGGGTKLELK
murine antibody 20M 19:
>20M19_VH(SEQ ID NO:64)
QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTVADDFKGRFAFSLDTSASTAYLQINNLKNEDTATYFCARNPGWFAYWGQGTLVTVSA
>20M19_VL(SEQ ID NO:65)
DIVLTQSPVFLAVSLGQRATISCRASESVDNYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSHEAPWTFGGGTRLEIK
murine antibody 32a 7:
>32A7_VH(SEQ ID NO:66)
QIQLVQSGPELKKPGETVKISCKASGYNLTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARYPGWFAYWGQGTLVTVSA
>32A7_VL(SEQ ID NO:67)
DIVLTQSPASLAVSLGQRATISCRTSESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPWTFGGGTKLEIK
the results show that all the murine antibodies with the light and heavy chains have better binding effect with human CD38 recombinant protein compared with other murine antibodies, the cell binding experiment effect is obvious, and the murine antibodies have stronger complement-dependent cytotoxicity and antibody-dependent cytotoxicity; moreover, the effect of the murine antibody was retained after conversion to the chimeric antibody, and the binding activity was similar to or higher than that of the control antibody Daratumumab or Isatuximab.
Example 5Humanization of anti-human CD38 murine antibody and preparation of humanized antibody
Combining the antibody coding schemes of Kabat, Chothia, the amino acid sequence regions of the 6 antigen Complementarity Determining Regions (CDRs) of the heavy and light chains of the murine antibody and the framework regions (framework regions) that support the conserved three-dimensional conformation of the antibody were determined.
Then, the sequences of known human antibodies are searched by analysis, the heavy chain variable region sequences of the human antibodies which are most similar to the murine antibodies are selected, such as IGHV1| IGHJ4 x 01, the sequences of the framework regions of the antibodies are selected as templates, the CDRs of the heavy chains of the murine antibodies are combined with the framework regions of the human antibodies, and finally, the heavy chain variable region sequences of the humanized antibodies are generated. The same procedure produces the humanized antibody light chain variable region sequence.
In the above process, it was found that the antibody obtained by directly grafting the CDR of the murine antibody to the human framework region often has a sharp decrease in binding activity, and thus it is necessary to change the individual amino acids in the framework region from those of human origin to those of murine origin. When determining the back mutation site, firstly, checking the difference of amino acids by contrasting the designed humanized antibody sequence and the original murine antibody sequence; the second is to examine whether these amino acids play an important role in supporting antibody structure or in binding to antigen. For the humanized designed sequence, it is also necessary to check whether there are some potential post-translational modification sites, such as N (asparagine) glycosylation site, N deamidation site, D (aspartic acid) isomerization site, etc.
Constructing a humanized antibody heavy chain variable region gene into a mammalian cell expression vector containing a heavy chain constant region gene of IgG1 subclass of the human monoclonal antibody; the light chain variable region gene is constructed into a mammalian cell expression vector containing the light chain constant region gene of the kappa subclass of the human monoclonal antibody. The constructed anti-human CD38 humanized antibody heavy chain carrier and light chain carrier are mixed in pair, HEK293 cells are transfected by Polyethyleneimine (PEI), cell supernatants are collected after about 7 days, and the anti-human CD38 humanized antibody protein is obtained by purifying by using Mabselect.
The humanized, engineered light and heavy chain variable region sequences are shown below, with antigenic Complementarity Determining Regions (CDRs) of the heavy and light chains underlined.
>5E19_VH_hz0(SEQ ID NO:68)
QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRGGSTDYSAAFMSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTTLTRVWYCDVWGQGTLVTVSS
>5E19_VH_hz1(SEQ ID NO:69)
QVQLVESGGGVVQPGRSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWVAVIWRGGSTDYSAAFMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAKTTLTRVWYCDVWGQGTLVTVSS
>5E19_VH_hz2(SEQ ID NO:70)
QVQLVESGGGVVQPGRSLRLSCAVSGFSLTNYGVHWVRQAPGKGLEWVAVIWRGGSTDYSAAFMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAKTTLTRVWYFDVWGQGTLVTVSS
>5E19_VH_hz3(SEQ ID NO:71)
QVQLVESGGGVVQPGRSLRLSCAVSGFSLTNYGVHWVRQAPGKGLEWVAVIWRGGSTDYSAAFMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAKTTLTRVWYVDVWGQGTLVTVSS
>5E19_VH_hz4(SEQ ID NO:72)
QVQLVESGGGVVQPGRSLRLSCAVSGFSLTNYGVHWVRQAPGKGLEWVAVIWRGGSTDYSAAFMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAKTTLTRVWYYDVWGQGTLVTVSS
>5E19_VL_hz0(SEQ ID NO:89)
DIQMTQSPSSLSASVGDRVTITCKSSEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWNTPLTFGQGTKVEIK
>5E19_VL_hz1(SEQ ID NO:90)
DIQMTQSPSSLSASVGDRVTITCKSSEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYWNTPLTFGQGTKVEIK
>8B7_VH_hz0(SEQ ID NO:73)
QVQLVESGGGVVQPGRSLRLSCAASGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSMITTGFTLDYWGQGTLVTVSS
>8B7_VH_hz1(SEQ ID NO:74)
QVQLVESGGGVVQPGRSLRLSCAASGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAKSMITTGFTLDYWGQGTLVTVSS
>8B7_VH_hz3(SEQ ID NO:75)
QVQLVESGGGVVQPGRSLRLSCAVSGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISKDNSKSTVYLQMNSLRAEDTAVYYCAKSMITTGFTLDYWGQGTLVTVSS
>8B7_VH_hz4(SEQ ID NO:76)
EVQLVESGGGVVQPGRSLRLSCAVSGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISKDNSKSTVYLQMNSLRAEDTAVYYCAKSMITTGFTLDYWGQGTLVTVSS
>8B7_VH_hz5(SEQ ID NO:77)
EVQLVESGGGVVQPGRSLRLSCAVSGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAKSMITTGFTLDYWGQGTLVTVSS
>8B7_VH_hz6(SEQ ID NO:78)
EVQLVESGGGVVQPGRSLRLSCAVSGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAKSMITTGFTLDYWGQGTLVTVSS
>8B7_VH_hz7(SEQ ID NO:79)
EVQLVESGGGVVQPGRSLRLSCAVSGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKSMITTGFTLDYWGQGTLVTVSS
>8B7_VH_hz8(SEQ ID NO:80)
EVQLVESGGGVVQPGRSLRLSCAVSGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISKDNSKSTLYLQMNSLRAEDTAVYYCAKSMITTGFTLDYWGQGTLVTVSS
>8B7_VH_hz9(SEQ ID NO:81)
EVQLVESGGGVVQPGRSLRLSCAVSGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCAKSMITTGFTLDYWGQGTLVTVSS
>8B7_VH_hz10(SEQ ID NO:82)
EVQLVESGGGVVQPGRSLRLSCAVSGFSLLSYGVHWVRQAPGKGLEWVAVIWRGGSADYNAAFMSRFTISRDNSKSTVYLQMNSLRAEDTAVYYCAKSMITTGFTLDYWGQGTLVTVSS
>8B7_VL_hz0(SEQ ID NO:91)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQFWNTPWTFGPGTKLEIK
>8B7_VL_hz1(SEQ ID NO:92)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGKDYTFTISSLQPEDVATYYCQQFWNTPWTFGPGTKLEIK
>8B7_VL_hz2(SEQ ID NO:93)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQFWNTPWTFGQGTKVEIK
>8B7_VL_hz3(SEQ ID NO:94)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGKDYTFTISSLQPEDVATYYCQQFWNTPWTFGQGTKVEIK
>8B7_VL_hz4(SEQ ID NO:95)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGKDFTFTISSLQPEDIATYYCQQFWNTPWTFGQGTKVEIK
>8B7_VL_hz5(SEQ ID NO:96)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQFWNTPWTFGQGTKVEIK
>8B7_VL_hz6(SEQ ID NO:97)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDVATYYCQQFWNTPWTFGQGTKVEIK
>8B7_VL_hz7(SEQ ID NO:98)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGKDYTFTISSLQPEDIATYYCQQFWNTPWTFGQGTKVEIK
>8B7_VL_hz8(SEQ ID NO:99)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGKDFTFTISSLQPEDVATYYCQQFWNTPWTFGQGTKVEIK
>8B7_VL_hz9(SEQ ID NO:100)
DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDVATYYCQQFWNTPWTFGQGTKVEIK
>17H13_VH_hz0(SEQ ID NO:83)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPGQGLEWMGYIIPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYPYRGYAMDYWGQGTLVTVSS
>17H13_VH_hz1(SEQ ID NO:84)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPGQGLEWMGYIIPSTDYTEYNQKFKDRVTMTKDNSTSTAYMELSSLRSEDTAVYYCSRYPYRGYAMDYWGQGTLVTVSS
>17H13_VH_hz2(SEQ ID NO:85)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPGQRLEWMGYIIPSTDYTEYNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARYPYRGYAMDYWGQGTTVTVSS
>17H13_VH_hz3(SEQ ID NO:86)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPGQRLEWMGYIIPSTDYTEYNQKFKDRVTITADKSAGTAYMELSSLRSEDTAVYYCSRYPYRGYAMDYWGQGTTVTVSS
>17H13_VL_hz0(SEQ ID NO:101)
EIVLTQSPATLSLSPGERATLSCSASSSVSYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSTNPWTFGQGTKVEIK
>17H13_VL_hz1(SEQ ID NO:102)
EIVLTQSPATLSLSPGERATLSCSASSSVSYMYWYQQKPGQAPRPLIYLTSNLASGIPARFSGSGSGTDYTLTISSLEPEDAAVYYCQQWSTNPWTFGQGTKVEIK
>17H13_VL_hz2(SEQ ID NO:134)
QIVLTQSPATLSLSPGERATLTCSASSSVSYMYWYQQKPGQAPRPWIYLTSNLASGVPARFSGSGSGTSYTLTISSLEPEDFAVYYCQQWSTNPWTFGQGTKVEIK
>17H13_VL_hz3(SEQ ID NO:135)
QIVLTQSPATLSLSPGERATLTCSASSSVSYMYWYQQKPGSSPRPWIYLTSNLASGVPARFSGSGSGTSYTLTISSLEPEDFAVYYCQQWSTNPWTFGGGTKVEIK
>20M19_VH_hz0(SEQ ID NO:87)
QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTVADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARNPGWFAYWGQGTLVTVSS
>20M19_VH_hz1(SEQ ID NO:88)
QIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTVADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARNPGWFAYWGQGTLVTVSS
>20M19_VL_hz0(SEQ ID NO:103)
DIVLTQSPASLAVSPGQRATITCRASESVDNYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQQSHEAPWTFGQGTKVEIK
>20M19_VL_hz1(SEQ ID NO:104)
DIVLTQSPASLAVSPGQRATITCRASESVDNYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPARFSGSGSRTDFTLTINPVEANDTANYYCQQSHEAPWTFGQGTKVEIK
>20M19_VL_hz2(SEQ ID NO:105)
DIVLTQSPASLAVSPGQRATITCRASESVDNYGNTFMHWYQQKPGQPPKLLIYRASNLESGVPARFSGSGSRTDFTLTINPVEAGDTANYYCQQSHEAPWTFGQGTKVEIK
>32A7_VH_hz0(SEQ ID NO:140)
QVQLVQSGSELKKPGASVKVSCKASGYNLTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARYPGWFAYWGQGTLVTVSS
>32A7_VH_hz1(SEQ ID NO:141)
QIQLVQSGSELKKPGASVKVSCKASGYNLTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARYPGWFAYWGQGTLVTVSS
>32A7_VH_hz2(SEQ ID NO:142)
QIQLVQSGSELKKPGASVKVSCKASGYALTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARYPGWFAYWGQGTLVTVSS
>32A7_VL_hz0(SEQ ID NO:143)
DIVLTQSPASLAVSPGQRATITCRTSESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQQSNEDPWTFGQGTKVEIK
>32A7_VL_hz1(SEQ ID NO:144)
DIVLTQSPASLAVSPGQRATITCRTSESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPARFSGSGSRTDFTLTINPVEANDTANYYCQQSNEDPWTFGQGTKVEIK
>32A7_VL_hz2(SEQ ID NO:145)
DIVLTQSPASLAVSPGQRATITCRTSESVDSYGNTFMHWYQQKPGQPPKLLIYRASNLESGVPARFSGSGSRTDFTLTINPVEANDTANYYCQQSNEDPWTFGQGTKVEIK
>32A7_VL_hz3(SEQ ID NO:146)
DIVLTQSPASLAVSPGQRATITCRTSESVDSYGNTFMHWYQQKPGQPPKLLIYRASNLESGVPARFSGSGSRTDFTLTINPVEAQDTANYYCQQSNEDPWTFGQGTKVEIK
The humanized antibody was named "murine antibody name-HmLn" where m and n are the humanized (hz) engineered sequences (VH _ hz and VL _ hz) numbers for VH and VL, respectively.
Example 6In vitro binding verification and in vitro cytology test of anti-human CD38 antibody and antigen
Octet binding experiment
An Octet binding experiment was performed for the antibodies of the invention and the control antibody daratumumab (dara) with recombinant human CD38 protein.
Antibody affinity was determined using an anti-human antibody capture method using a Fortebio (BLITZ pro1.1.0.28) instrument. Soaking a capture Antibody (AHC) biological probe of an anti-human antibody Fc segment in PBS for 10min during measurement; mu.l of diluted antibody samples (including chimeric, humanized and control antibodies of the invention, Daratumumab; working antibody concentration 15. mu.g/mL) were loaded onto AHC bioprobes, and then equilibrated in PBS for 100s, and further subjected to binding reaction with human CD38 protein (ACRO biosystem, Cat No. CD8-H5224) at various dilution concentrations for 600s, after which the AHC probes were transferred to PBS for dissociation for 600 s. After the experiment, blank control response values were subtracted, 1:1Langmuir binding pattern fitting was performed with software, and kinetic constants for antigen-antibody binding were calculated.
The results are shown in tables 2 to 5.
TABLE 2 in vitro binding Activity of antibodies to recombinant human CD38 protein
| Antibodies | Concentration (nM) | Response to | KD(M) | kon(1/Ms) | kdis(1/s) | RMax |
| 4J1- |
100 | 0.4598 | 3.31E-08 | 2.85E+05 | 9.44E-03 | 0.6097 |
| 4J1-H0L0 | 100 | -0.01 | - | 2.32E+05 | - | 0.0001 |
| 4J1-H0L1 | 100 | -0.0089 | - | 1.09E+06 | - | 0.0001 |
| |
100 | 0.2098 | 2.44E-07 | 4.53E+04 | 1.11E-02 | 0.7031 |
TABLE 3 in vitro binding Activity of antibodies to recombinant human CD38 protein
| Antibodies | Concentration (nM) | Response to | KD(M) | kon(1/Ms) | kdis(1/s) | RMax |
| 5E19- |
100 | 0.3799 | 2.79E-08 | 4.70E+05 | 1.31E-02 | 0.4941 |
| 5E19-H0L0 | 100 | -0.0155 | - | 8.33E+04 | - | 0.0032 |
| 5E19- |
100 | 0.0184 | - | 2.26E+05 | 5.13E-01 | 0.5128 |
| 5E19- |
100 | 0.1096 | 1.30E-06 | 6.44E+04 | 8.39E-02 | 1.6077 |
| 5E19- |
100 | 0.338 | 7.76E-08 | 3.17E+05 | 2.46E-02 | 0.6181 |
| |
100 | 0.2225 | 1.60E-07 | 7.29E+04 | 1.16E-02 | 0.5712 |
TABLE 4 in vitro binding Activity of antibodies to recombinant human CD38 protein
TABLE 5 in vitro binding Activity of antibodies to recombinant human CD38 protein
| Antibodies | Concentration (nM) | Response to | KD(M) | kon(1/Ms) | kdis(1/s) | RMax |
| 17H13- |
100 | 0.2662 | 2.29E-08 | 2.87E+05 | 6.56E-03 | 0.3266 |
| 17H13-H0L0 | 100 | -0.0063 | - | 1.96E+06 | - | 0.0001 |
| 17H13- |
100 | 0.2254 | 6.16E-08 | 1.64E+05 | 1.01E-02 | 0.3658 |
| 17H13- |
100 | 0.0053 | - | 8.49E+03 | 7.90E-01 | 8.1272 |
| 17H13- |
100 | 0.2275 | 3.94E-08 | 3.08E+05 | 1.21E-02 | 0.3203 |
| |
100 | 0.2159 | 1.43E-07 | 8.83E+04 | 1.26E-02 | 0.5239 |
The experimental results show that the partially humanized and engineered antibody retains the binding activity to the human CD38 recombinant protein.
2. In vitro cell binding assay
In vitro cell binding experiments of antibodies of the invention and control antibody daratumumab (dara) to CD38 protein were performed.
The anti-human CD38 antibody was diluted 2-fold in gradient from the starting concentration of 100nM for 16 concentration points, and 10. mu.l of antibody was added to the 384-well plate for each concentration point. The CHO cell line 1-T-14 expressing CD38 on the cell surface was taken, washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 minutes at room temperature, and resuspended at a density of about 2X106Mu.l of each cell/ml was added to the wells of 384-well plates to which the antibody had been added. After incubation for 1 hour at 4 ℃, fluorescently labeled goat anti-human IgG secondary antibody was added. After further incubation at 4 ℃ for 1 hour, the cell population was analyzed for mean fluorescence intensity by flow cytometry.
The results are shown in FIG. 3 and Table 6.
TABLE 6 binding Activity of antibodies with antigens
| Antibodies | EC50(μg/ml) |
| 8B7-H3L3 | 0.7368 |
| 5E19-H3L1 | 0.9533 |
| 20M19-H1L0 | 1.101 |
| 20M19-H1L1 | 1.221 |
| Dara | 1.279 |
| 8B7-H1L1 | 1.375 |
| Unrelated antibodies | - |
3. In vitro cytology assay
3.1 Complement Dependent Cytotoxicity (CDC)
The antibodies of the invention and a control antibody, Daratumumab (Dala), were assayed for their ability to induce Complement Dependent Cytotoxicity (CDC) against CHO cell line 1-T-14 stably expressing human CD38 using commercial human serum whole complement from Quidel.
The cells were mixed well with the antibody and control antibody at final concentrations of 50. mu.g/ml to 7.6ng/ml, respectively, and then 6.25% human serum whole complement dissolved in cell culture medium RPMI-1640 was added to the cells and incubated at 37 ℃ for 3 hours. And then, carrying out cytotoxicity detection by using a CCK-8 kit, and finally detecting the absorbance of 450nm by using an MD (enzyme-labeled) instrument. EC50 of the samples was calculated by absorbance using a 4-parameter fit curve with softmax pro7 software.
The results are shown in Table 7.
TABLE 7 complement dependent cytotoxicity of antibodies
| Plate number | Antibodies | EC50(ng/mL) | Relative Activity (%) |
| P1 | Dara | 154.5 | 100.0 |
| P1 | 8B7-H1L1 | 177.4 | 87.1 |
| P1 | 8B7-H3L3 | 214.5 | 72.0 |
| P2 | Dara | 144.5 | 100.0 |
| P2 | 20M19-H1L0 | 234.9 | 61.5 |
| P2 | 20M19-H1L1 | 239.6 | 60.3 |
| P3 | Dara | 126.4 | 100.0 |
| P3 | 32A7-H1L1 | 303.2 | 41.7 |
| P3 | 32A7-H2L1 | 203.5 | 62.1 |
| P4 | Dara | 125.9 | 100.0 |
| P4 | 5E19-H3L1 | 192.6 | 65.4 |
| P4 | Unrelated antibodies | ～ | ～ |
3.2 Antibody Dependent Cellular Cytotoxicity (ADCC)
Antibodies of the invention and a control antibody, Daratumumab (Dara), were tested to induce antibody-dependent cellular cytotoxicity (ADCC) against CHO cell line 1-T-14 stably expressing human CD 38.
Engineered Jurkat cells stably expressing the Fc γ RIIIa-Fc ε RIa γ hybrid receptor, driven by NFAT response elements, express firefly luciferase were used as effector cells. The biological activity of the antibody in the ADCC mechanism is quantified by luciferase produced by NFAT pathway activation.
1.5E5 effector cells were mixed with the antibody of the invention and the control antibody Daratumumab at final concentrations of 33. mu.g/ml to 85pg/ml, respectively, and then 2.5E4 target cells, i.e., CHO cell line 1-T-14 stably expressing human CD38 (E: T ratio of effector cells to target cells 6:1), were added thereto and incubated at 37 ℃ for 16 hours. Thereafter, the reaction mixture was subjected to Bio-Glo reaction using a Promega kitTMThe Luciferase Assay System was used for detection, and the LUM value was finally detected by an MD microplate reader, and fold induction and EC50 calculation were performed using the following formula.
Fold induction ═ background (test well read)/(negative control well read-background)
Fold induction the samples were calculated as EC50 using a 4-parameter fit curve with prism7 software.
The results are shown in FIG. 4 and Table 8.
TABLE 8 antibody-dependent cytotoxicity of antibodies
| Plate number | Antibodies | EC50(ng/mL) | Relative Activity (%) |
| P1 | Dara | 31.76 | 100.0 |
| P1 | 8B7-H1L1 | 60.59 | 52.4 |
| P1 | 8B7-H3L3 | 37.94 | 83.7 |
| P2 | Dara | 39.01 | 100.0 |
| P2 | 20M19-H1L0 | 31.55 | 123.6 |
| P2 | 20M19-H1L1 | 41.63 | 93.7 |
| P3 | Dara | 32.59 | 100.0 |
| P3 | 32A7-H1L1 | 60.23 | 54.1 |
| P3 | 32A7-H2L1 | 56.42 | 57.8 |
| P4 | Dara | 31.61 | 100.0 |
| P4 | 5E19-H3L1 | 51.46 | 61.4 |
| P4 | Unrelated antibodies | ～ | ～ |
Example 9In vitro binding affinity and kinetic experiments for humanized antibodies against human CD38
Antibody-antigen interaction was measured using a GE BIAcore instrument S200.
Referring to the GE Biotin CAPture Kit (cat # 28-9202-34, Lot.10265137), the assay was performed by first coupling His-tagged human CD38 protein antigen to both the CAP analysis channel and the control sample channel of the sensor chip, then flowing a gradient diluted antibody sample (initial concentration 20nM, 8 concentration points at 1:3 dilution, and setting 0.741nM concentration point repeat) together in the analysis channel and the control sample channel, and determining the photoreaction value occurring after the antibody antigen binding. And finally obtaining the binding constant Kon and dissociation constant Koff and affinity constant KD of the antibody through the fitting analysis of instrument software.
The results are shown in Table 9.
TABLE 9 binding kinetics of humanized engineered antibodies
| ka(1/Ms) | kd(1/s) | KD(M) | |
| 20M19-H1L1 | 1.049E+6 | 7.136E-4 | 6.804E-10 |
| 8B7-H3L3 | 6.623E+5 | 0.001197 | 1.808E-9 |
| 8B7-H4L3 | 1.277E+6 | 0.002919 | 2.287E-9 |
Example 10Determination of apoptosis Activity of humanized antibody against human CD38
Materials: Daudi-Luc (ATCC, Cat No. CCL-213) cell
The kit comprises: alex Fluor 488Annexin V/Dead cell apoptosis kit (invitrogen, Cat: V13245, Lot:1923636)
Tool antibody: affinipuref (ab')2Fragment Goat-anti-human IgG Fcr Fragment Specific (Immo Research, Cat:109-
The experimental steps are as follows:
daudi cells were washed 1-fold with pre-chilled PBS, seeded into 96-well plates at 1E 5/well, and incubated for 30 minutes with 0.7nM anti-CD 38 antibody. Add 5. mu.g/ml sheep anti-human tool antibody to 96-well plates and incubate at 4 ℃ for 24 hours. After washing 2 times with pre-cooled PBS, the supernatant was discarded and the cells were resuspended in 100. mu.l of 1 × Annexin solution. Add 5. mu.l Alexa
488annexin V and 1. mu.L of 100. mu.g/mL PI working solution were put into a 96-well plate and incubated for 15 minutes at room temperature in the dark. After terminating the reaction with 400 μ l of 1 × annexin binding solution, the 96-well plate was placed on ice and the readings of fluorescence excitation 530nM and emission 488nM were measured using a flow cytometer.
The results are shown in Table 10.
TABLE 10 apoptotic Activity of antibodies of the invention
The "cross-linked" and "non-cross-linked" in the table are in the form of bridged Fab and unbridged Fab, respectively.
Example 11Druggability characterization of humanized antibody against human CD38
1. Antibody epitope analysis
Epitope analysis of the antibody of the present invention was performed by the Intandem method of Fortebio. Briefly, monoclonal supernatants containing murine antibodies of the invention were first used to bind to antigen cells, human CD38 high expression CHO cell line 1-T-14, and then a control antibody was added to compete with murine antibody for binding to the epitope. And detecting the binding signal of the labeled control antibody, wherein the lower the binding signal of the control antibody is, the more the murine antibody can prove to occupy the epitope recognized by the control antibody, and the antibody of the invention is close to or consistent with the antigen binding epitope of the control antibody.
The results of the competition of the antibodies of the present invention with Daratumumab, Mor202, Isatuximab are shown in FIG. 5, respectively.
Experiments prove that the binding sites of the antibody and the antigen are consistent with the epitope of the control antibody.
2. Antibody monomer ratio analysis
An experimental instrument: UPLC CLASS ACQUITYH (WATERS)
And (3) analyzing the column: TSKgel G3000SWXL 7.8 × 300 (TOSHII, Cat No 003C 03326C)
Analysis of the solution: DPBS (GIBCO, Cat No.14190-
The analysis method comprises the following steps: 50 mul of antibody with the concentration of 1mg/ml is injected into a pre-balanced chromatographic column, flows for 35min at room temperature and at the flow rate of 0.7ml/min, simultaneously detects the absorption values of a machine A280 and a machine A214, and then the monomer content and the proportion of the antibody are judged according to the peak-out time and the peak-out volume.
The results are shown in Table 11.
TABLE 11 Main Peak Retention time and monomer Rate of antibodies of the invention
3. Analysis of hydrophobic Properties of antibodies
An experimental instrument: ARC (Waters)
Analytical column for experiment: TSKgel Butyl-NPR (4.6mmX3.5cm, Cat No 14947)
Analysis of the solution: a.20mM Histidine, pH 6.0; b.20mM Histidine, 1.6M (NH)4)2SO4
The analysis method comprises the following steps: the hydrophobic nature of the antibody is analyzed according to the instructions for use of the hydrophobic chromatography column.
The results are shown in Table 12.
TABLE 12 hydrophobic Properties of antibodies of the invention
4. Antibody in vitro monkey (cyno) serum stability study
Experimental materials: the antibody to be detected, FBS, antigen human CD38 recombinant protein, anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML), and HRP-labeled goat anti-human IgG secondary antibody (Jackson, code: 109-.
An experimental instrument: 37 ℃ incubator and microplate reader.
The experimental steps are as follows:
sample preparation:
1) adjusting the concentration of the antibody to be detected to 20 mu g/ml, filtering and sterilizing, and subpackaging into 250 mu l/tube for later use;
2) adding the subpackaged antibody to be detected into the FBS in the same volume, wherein the final concentration is 50% of serum concentration and 10 mu g/ml of antibody concentration;
3) preparing 7 samples in total, sealing with a sealing film, and placing at 37 ℃ without maintaining sterility in the whole process;
4) samples are taken on days 0, 3, 7, 10, 14 and 21 and stored at 4 ℃ for testing, wherein the sample on day 21 is placed at 4 ℃ for at least one day.
The detection method comprises the following steps:
1) respectively coating the combined antigen and the anti-IgG Fab monoclonal antibody on a 96-well enzyme-linked plate by PBS (phosphate buffer solution), wherein the concentration of the combined antigen and the anti-IgG Fab monoclonal antibody is 0.2 mu g/ml and 100 mu l/well, and standing overnight at 4 ℃;
2) preparing required reagents:
blocking solution 5% BSA + PBS
Antibody dilution 5% BSA + PBS + 50% FBS
ELISA plate washing solution 0.1% Tween + PBS
3) Washing the coated enzyme-linked plate with PBS for three times, washing the coated enzyme-linked plate with PBS at 300 mu l/hole, and washing free uncoated antigen;
4) adding sealing liquid, sealing at 37 deg.C for 1h with 200 μ l/hole;
5) diluting the antibody to be detected to 2 mu g/ml by using an antibody diluent, and diluting by 3 times to obtain 8 gradients;
6) pouring off the confining liquid, respectively adding the diluted antibodies into the enzyme-linked plates of the two coating methods, and incubating for 1h at 37 ℃ in 100 mul/hole;
7) PBST plate washing 3 times;
8)1, diluting the secondary antibody by 5000 percent, adding the secondary antibody into a washed enzyme-linked plate, incubating for 40min at the temperature of 37 ℃ in a hole of 100 mul;
9) PBST plate washing 3 times;
10) TMB color development, 100 mul/hole, light shielding for 10 min;
11) addition of 50. mu.l of 2M HCl was stopped and the reading was 450 nm.
And (4) processing a result:
and (4) formulating a binding curve according to an ELISA color rendering value, observing the change of the binding curve when the antibody is placed for different time, and evaluating the stability of the binding activity of the antibody after the antibody is placed.
The results are shown in FIG. 6.
5. In vivo drug metabolism assay in antibody mice
Experimental materials: the antibody to be detected, the serum collected from the mouse at different time points, the binding antigen of the antibody to be detected, human CD38, the antibody standard product Dara, the anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML), and the goat anti-human IgG secondary antibody labeled by HRP (Jackson, code: 109-.
The experimental method comprises the following steps:
collecting serum:
1) female Balb/C mice, 3/group, respectively in the tail vein or abdominal cavity drug delivery, 200 u g to be tested antibody/;
2) collecting blood from tail vein according to experimental design time point, collecting blood sample, standing at room temperature for more than 30min, collecting serum at 4000rpm for 15min, storing at-20 deg.C, and collecting serum with volume of more than 20 μ l as far as possible to prevent evaporation of serum;
3) finally, collecting serum for the last time, and freezing and storing the serum for 24 hours at the temperature of less than-20 ℃.
The detection method comprises the following steps:
1) respectively coating the combined antigen and the anti-IgG Fab monoclonal antibody on a 96-well enzyme-linked plate by PBS (phosphate buffer solution), wherein the concentration of the combined antigen and the anti-IgG Fab monoclonal antibody is 0.2 mu g/ml and 100 mu l/well, and standing overnight at 4 ℃;
2) preparing required reagents:
blocking solution 5% BSA + PBS
Antibody dilutions 5% BSA + PBS + 20% blank mouse serum
ELISA plate washing solution 0.1% Tween + PBS
3) Washing the coated enzyme-linked plate with PBS for three times, wherein each hole is 300 mul;
4) adding sealing liquid, sealing at 37 deg.C for 1h with 200 μ l/hole;
5) diluting the initial serum to a proper concentration by using a confining liquid, and diluting the initial serum to a proper concentration range by using an antibody diluent containing the same serum concentration, wherein the dilution multiple of the specific concentration needs to be adjusted according to a pre-experiment, so that the final color rendering value of the detected serum is in a standard curve color rendering value range in principle;
6) the antibody dilution dilutes the antibody standard, and the dilution of the standard is still adjusted according to pre-experiments, so that the standard is fitted with a linear curve (or an S-shaped curve if appropriate software is available).
7) Pouring off the confining liquid, respectively adding the diluted antibody standard substance and the serum to be detected into the enzyme-linked plates of the two coating methods, and incubating for 1h at 37 ℃ in a way of 100 mu l/hole;
8) PBST plate washing 3 times;
9)1, diluting the secondary antibody by 5000 percent, adding the secondary antibody into a washed enzyme-linked plate, incubating for 40min at the temperature of 37 ℃ in a hole of 100 mul;
10) PBST plate washing 3 times;
11) TMB color development, 100 mul/hole, light shielding for 10 min;
12) addition of 50. mu.l of 2M HCl was stopped and the reading was 450 nm.
The results are shown in Table 13.
TABLE 13-1 in vivo drug metabolism Properties of the antibodies of the invention in mice
TABLE 13-2 in vivo drug metabolism Properties of the antibodies of the invention in mice
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Sequence listing
<110> Miwei (Shanghai) Biotech-effective Co
Shanghai Puming Biotech Co., Ltd
<120> anti-human CD38 antibody and application thereof
<130> LC19110036
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<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 9
Ser Met Ile Thr Thr Gly Tyr Ala Met Asp Tyr
1 5 10
<210> 10
<211> 9
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 10
Gln Gln Tyr Trp Ser Thr Pro Trp Thr
1 5
<210> 11
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR1
<400> 11
Gly Phe Ser Leu Thr Asn Tyr Gly Val His
1 5 10
<210> 12
<211> 16
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR2
<400> 12
Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Ser Ala Ala Phe Met Ser
1 5 10 15
<210> 13
<211> 11
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 13
Thr Thr Leu Thr Arg Val Trp Tyr Cys Asp Val
1 5 10
<210> 14
<211> 11
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
<400> 14
Lys Ser Ser Glu Asp Ile Tyr Asn Arg Leu Ala
1 5 10
<210> 15
<211> 9
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 15
Gln Gln Tyr Trp Asn Thr Pro Leu Thr
1 5
<210> 16
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR1
<400> 16
Gly Phe Ser Leu Leu Ser Tyr Gly Val His
1 5 10
<210> 17
<211> 16
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR2
<400> 17
Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met Ser
1 5 10 15
<210> 18
<211> 11
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 18
Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr
1 5 10
<210> 19
<211> 9
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 19
Gln Gln Phe Trp Asn Thr Pro Trp Thr
1 5
<210> 20
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR1
<400> 20
Gly Tyr Thr Phe Thr Asn Phe Gly Met Asn
1 5 10
<210> 21
<211> 17
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR2
<400> 21
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Asn Ala Asp Asp Phe Lys
1 5 10 15
Gly
<210> 22
<211> 5
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 22
Lys Gly Phe Ala Tyr
1 5
<210> 23
<211> 15
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
<400> 23
Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His
1 5 10 15
<210> 24
<211> 7
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR2
<400> 24
Arg Ala Ser Asn Leu Glu Ser
1 5
<210> 25
<211> 9
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 25
Gln Gln Ile Asn Glu Asp Pro Phe Thr
1 5
<210> 26
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR1
<400> 26
Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn
1 5 10
<210> 27
<211> 17
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR2
<400> 27
Trp Ile Asn Thr Tyr Thr Ala Glu Pro Thr Tyr Ala Asp Asp Phe Lys
1 5 10 15
Gly
<210> 28
<211> 5
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 28
Lys Tyr Gly Gly Tyr
1 5
<210> 29
<211> 15
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
<400> 29
Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Ile His
1 5 10 15
<210> 30
<211> 9
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 30
Gln Gln Val Asn Glu Asp Pro Leu Thr
1 5
<210> 31
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR1
<400> 31
Gly Tyr Thr Phe Thr Asn Tyr Trp Met Asn
1 5 10
<210> 32
<211> 17
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR2
<400> 32
Tyr Ile Ile Pro Ser Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210> 33
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 33
Tyr Pro Tyr Arg Gly Tyr Ala Met Asp Tyr
1 5 10
<210> 34
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
<400> 34
Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr
1 5 10
<210> 35
<211> 7
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR2
<400> 35
Leu Thr Ser Asn Leu Ala Ser
1 5
<210> 36
<211> 9
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 36
Gln Gln Trp Ser Thr Asn Pro Trp Thr
1 5
<210> 37
<211> 17
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR2
<400> 37
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Val Ala Asp Asp Phe Lys
1 5 10 15
Gly
<210> 38
<211> 7
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 38
Asn Pro Gly Trp Phe Ala Tyr
1 5
<210> 39
<211> 15
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
<400> 39
Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Asn Ser Phe Met His
1 5 10 15
<210> 40
<211> 9
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 40
Gln Gln Ser His Glu Ala Pro Trp Thr
1 5
<210> 41
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR1
<400> 41
Gly Tyr Asn Leu Thr Asn Tyr Gly Met Asn
1 5 10
<210> 42
<211> 17
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR2
<400> 42
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys
1 5 10 15
Gly
<210> 43
<211> 7
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 43
Tyr Pro Gly Trp Phe Ala Tyr
1 5
<210> 44
<211> 15
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
<400> 44
Arg Thr Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His
1 5 10 15
<210> 45
<211> 9
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 45
Gln Gln Ser Asn Glu Asp Pro Trp Thr
1 5
<210> 46
<211> 11
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 46
Thr Thr Leu Thr Arg Val Trp Tyr Phe Asp Val
1 5 10
<210> 47
<211> 11
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 47
Thr Thr Leu Thr Arg Val Trp Tyr Val Asp Val
1 5 10
<210> 48
<211> 11
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 48
Thr Thr Leu Thr Arg Val Trp Tyr Tyr Asp Val
1 5 10
<210> 49
<211> 15
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
<400> 49
Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Asn Thr Phe Met His
1 5 10 15
<210> 50
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 50
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys Gly Met Ile Thr Thr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 51
<211> 106
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 51
Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 52
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 52
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe
20 25 30
Gly Ile His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Leu
50 55 60
Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Ile Asn Ser Leu Arg Ala Asp Asp Thr Ala Ile Tyr Phe Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 53
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 53
Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Ile Leu Ser Ile Thr Ser Leu Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 54
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 54
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Leu Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Ser Ala Ala Phe Met
50 55 60
Ser Arg Leu Asn Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys Thr Thr Leu Thr Arg Val Trp Tyr Cys Asp Val Trp Gly Ala Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 55
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 55
Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr
65 70 75 80
Glu Asp Val Val Thr Tyr Tyr Cys Gln Gln Tyr Trp Asn Thr Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 56
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 56
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 57
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 57
Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly His Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 58
<211> 114
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 58
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Phe
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Asn Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Lys Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
100 105 110
Ser Ala
<210> 59
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 59
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Thr Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ile Asn
85 90 95
Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 60
<211> 114
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 60
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Ala Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Ala Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Lys Tyr Gly Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
100 105 110
Ser Ser
<210> 61
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 61
Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Val Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210> 62
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 62
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Ile Pro Ser Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Gly Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Tyr Pro Tyr Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 63
<211> 106
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 63
Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser 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 Trp Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 64
<211> 116
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 64
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Val Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Asn Pro Gly Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ala
115
<210> 65
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 65
Asp Ile Val Leu Thr Gln Ser Pro Val Phe Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser His
85 90 95
Glu Ala Pro Trp Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys
100 105 110
<210> 66
<211> 116
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 66
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asn Leu Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Tyr Pro Gly Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ala
115
<210> 67
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 67
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Thr Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 68
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 68
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Ser Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Thr Thr Leu Thr Arg Val Trp Tyr Cys Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 69
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 69
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Ser Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Thr Leu Thr Arg Val Trp Tyr Cys Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 70
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 70
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Ser Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Thr Leu Thr Arg Val Trp Tyr Phe Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 71
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 71
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Ser Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Thr Leu Thr Arg Val Trp Tyr Val Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 72
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 72
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Ser Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Thr Leu Thr Arg Val Trp Tyr Tyr Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 73
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 73
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 74
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 74
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 75
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 75
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 76
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 76
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 77
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 77
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 78
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 78
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 79
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 79
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 80
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 80
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Ser Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 81
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 81
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 82
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 82
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Leu Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Arg Gly Gly Ser Ala Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Ser Met Ile Thr Thr Gly Phe Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 83
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 83
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Ile Pro Ser Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Pro Tyr Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 84
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 84
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Ile Pro Ser Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Met Thr Lys Asp Asn Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Tyr Pro Tyr Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 85
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 85
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Tyr Ile Ile Pro Ser Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Pro Tyr Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 86
<211> 119
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 86
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Tyr Ile Ile Pro Ser Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Ala Gly Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Tyr Pro Tyr Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 87
<211> 116
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 87
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Val Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Pro Gly Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 88
<211> 116
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 88
Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Val Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Pro Gly Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 89
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 89
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Asn Thr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 90
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 90
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Asn Thr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 91
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 91
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 92
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 92
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 93
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 93
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 94
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 94
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 95
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 95
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 96
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 96
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 97
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 97
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 98
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 98
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 99
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 99
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 100
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 100
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Asn Thr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 101
<211> 106
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 101
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Thr Asn Pro Trp Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 102
<211> 106
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 102
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Leu Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Ala Ala Val Tyr Tyr Cys Gln Gln Trp Ser Thr Asn Pro Trp Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 103
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 103
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser His
85 90 95
Glu Ala Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 104
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 104
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser His
85 90 95
Glu Ala Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 105
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 105
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr
20 25 30
Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Gly Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser His
85 90 95
Glu Ala Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 106
<211> 330
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain constant region
<400> 106
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Leu Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Leu Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Leu Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 107
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain constant region
<400> 107
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 108
<211> 357
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 108
caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccata 60
acctgcacag tctctggttt ctcattaact agctatggtg tacactgggt tcgccagtct 120
ccaggaaagg gtctggagtg gctgggagtg atatggagag gtggaagcac agactacaat 180
gcagctttca tgtccagact gagcatcacc aaggacaact ccaagagcca agttttcttt 240
aaaatgaaca gtctgcaagc tgatgacact gccatatact actgtgccaa aggtatgatt 300
acgacgggct atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357
<210> 109
<211> 318
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain variable region
<400> 109
gacatccaga tgacacaatc ttcatcctcc ttttctgtat ctctaggaga cagagtcacc 60
attacttgca aggcaagtga ggacatatat aatcggttag cctggtatca gcagaaacca 120
ggaaatgctc ctaggctctt aatatctggt gcaaccagtt tggaaactgg ggttccttca 180
agattcagtg gcagtggatc tggaaaggat tacactctca gcattaccag tcttcagact 240
gaagatgttg ctacttatta ctgtcaacag tattggagta ctcccacgtt cggagggggg 300
accaagctgg aaataaaa 318
<210> 110
<211> 357
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 110
caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccata 60
acctgcacag tctctggttt ctcattaact agctttggta tacactgggt tcgccagtct 120
ccaggaaagg gtctggagtg gctgggagtg atatggagag gtggaagcac agactacaat 180
gcagctttcc tgtccagact gagcatcacc aaggacaact ccaagagcca agttttcttt 240
aaaataaaca gtcttcgagc tgatgacact gccatatact tctgtgccaa atctatgatt 300
acgaccggct atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357
<210> 111
<211> 321
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain variable region
<400> 111
gacatccaga tgacacaatc ttcatcctcc ttttctgtat ctctaggaga cagagtcacc 60
attacttgca aggcaagtga ggacatatat aatcggttag cctggtatca gcagaaacca 120
ggaaatgctc ctaggctctt aatatctggt gcaaccagtt tggaaactgg ggttccttca 180
agattcagtg gcagtggatc tggaaaggat tacattctca gcattaccag tcttcagact 240
gaagatgttg ctacttatta ctgtcaacag tattggagta ctccgtggac gttcggtgga 300
ggcaccaagc tggagatcaa a 321
<210> 112
<211> 357
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 112
caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccata 60
acctgcacag tctctggttt ctcattaact aactatggtg tacactggct tcgccagtct 120
ccaggaaagg gtctggagtg gctgggagtg atatggagag gtggaagcac agactacagt 180
gcagctttca tgtccagact gaacatcacc aaggacaact ccaagagtca agtttttttt 240
aaaatgaaca gtctgcaagc tgatgacact gccatatact actgtgccaa aactacgctt 300
acgagggtct ggtattgcga tgtctggggc gcagggacca cggtcaccgt ctcctca 357
<210> 113
<211> 321
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain variable region
<400> 113
gacatccaga tgacacaatc ttcatcctcc ttttctgtat ctctaggaga cagagtcacc 60
attacttgca agtcaagtga ggacatatat aatcggttag cctggtatca gcagaaacca 120
ggaaatgctc ctaggctctt aatatctggt gcaaccagtt tggaaactgg ggttccttca 180
agattcagtg gcagtggatc tggaaaggat tacactctca gcattaccag tcttcagact 240
gaagatgttg ttacttatta ctgtcaacag tattggaata ctccgctcac gttcggtgct 300
gggaccaagc tggagctgaa a 321
<210> 114
<211> 357
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 114
caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccata 60
acctgcacag tctctgggtt ctcattactt agttatggtg tacactgggt tcgccagtct 120
ccaggaaagg gtctggagtg gctgggagtg atatggagag gtggaagcgc agactacaat 180
gcagctttca tgtccagact gagcatcacc aaagacaact ccaagagcca agttttcttt 240
aaaatgaaca gtctgcaagc tgatgacact gccatatact actgtgccaa aagtatgatt 300
acgacggggt ttactctgga ctactggggt caaggaacct cagtcaccgt ctcctca 357
<210> 115
<211> 321
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain variable region
<400> 115
gacatccaga tgacacaatc ttcatcctcc ttttctgtat ctctaggaga cagagtcacc 60
attacttgca aggcaagtga ggacatatat aatcggttag cctggtatca gcagaaacca 120
ggacatgctc ctaggctctt aatatctggt gcaaccagtt tggaaactgg ggttccttca 180
agattcagtg gcagtggatc tggaaaggat tacactctca gcattaccag tcttcagact 240
gaagatgttg ctacttatta ctgtcaacag ttttggaata ctccgtggac gttcggtgga 300
ggcaccaagc tggaaatcaa a 321
<210> 116
<211> 342
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 116
cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60
tcctgcaagg cttctggata taccttcaca aactttggaa tgaactgggt gaagcaggct 120
ccaggaaagg gtttaaagtg gatgggctgg ataaacacct acactggaga gccaacaaat 180
gctgatgact tcaagggacg gtttgccttc tctttggaaa cctctgccag cactgcctat 240
ttgcagatca acaacctcaa aaatgaggac atggctacat atttctgtgc aagaaaaggg 300
tttgcttact ggggccaagg gactctggtc actgtctctg ca 342
<210> 117
<211> 333
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain variable region
<400> 117
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gaggaccacc 60
atatcctgca gagccagtga aagtgttgat agttatggca atagttttat gcactggtac 120
cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180
gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240
cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaattaatga ggatccattc 300
acgttcggct cggggacaaa gttggaaata aaa 333
<210> 118
<211> 342
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 118
cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60
tcctgcaagg cttctggata taccttcaca aactatggaa tgaactgggt gaagcaggct 120
ccaggaaagg gtttaaagtg gatgggctgg ataaacacct acactgcaga gccaacatat 180
gctgatgact tcaagggacg gtttgccttc tctttggaag cctctgccag cactgcctat 240
ttgcagatca acaacctcaa aaatgaggac atggctacat atttctgtgc aagaaagtat 300
ggtggctact ggggccaagg caccactctc acagtctcct ca 342
<210> 119
<211> 333
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain variable region
<400> 119
gacattgtgc tgacccaatc tccaccttct ttggctgtgt ctctagggca gagggccacc 60
atatcctgca gagccagtga aagtgttgat agttatggca atagttttat acactggtac 120
cagcagaaac caggacagtc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180
gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240
cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aagttaatga ggatccgctc 300
acgttcggtg ctgggaccaa gctggagctg aaa 333
<210> 120
<211> 357
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 120
caggtccagc ttcagcagtc tggggctgaa ctggcaaaac ctggggcctc agtgaagatg 60
tcctgcaagg cttctggcta cacctttact aactactgga tgaattggat aaaacagagg 120
cctggacagg gtctggaatg gattggatac attattccaa gcactgatta tactgagtac 180
aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccgg cacagcctac 240
atgcaactga gcagcctgac atctgaggac tctgcagtct attactgttc aaggtaccct 300
tataggggct atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357
<210> 121
<211> 318
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain variable region
<400> 121
caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 60
atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccaaca gaagccaaga 120
tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240
gatgctgcca cttattactg ccagcagtgg agtactaacc cgtggacgtt cggtggaggc 300
accaagctgg aactcaaa 318
<210> 122
<211> 348
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 122
cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60
tcctgcaagg cttctgggta taccttcaca aactatggaa tgaactgggt gaagcaggct 120
ccaggaaagg gtttaaagtg gatgggctgg ataaacacct acactggaga gccaacagtt 180
gctgatgact tcaagggacg gtttgccttc tctttggaca cctctgccag cactgcctat 240
ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagaaatccc 300
ggctggtttg cttactgggg ccaagggact ctggtcactg tctctgca 348
<210> 123
<211> 333
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain variable region
<400> 123
gacattgtgt tgacccaatc tccagttttt ttggctgtgt ctctagggca gagggccacc 60
atatcctgca gagccagtga aagtgttgat aattatggca atagttttat gcactggtac 120
cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180
gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240
cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaagtcatga ggctccgtgg 300
acgttcggtg gaggcaccag gctggaaatc aaa 333
<210> 124
<211> 348
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 124
cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60
tcctgcaagg cttctgggta taacctcaca aactatggaa tgaactgggt gaagcaggct 120
ccaggaaagg gtttaaagtg gatgggctgg ataaacacct acactggaga gccaacatat 180
gctgatgact tcaagggacg gtttgccttc tctttggaaa cctctgccag cactgcctat 240
ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagataccca 300
ggctggtttg cttactgggg ccaagggact ctggtcactg tctctgca 348
<210> 125
<211> 333
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain variable region
<400> 125
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60
atatcctgca gaaccagtga aagtgttgat agttatggca atagttttat gcactggtac 120
cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180
gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240
cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaagtaatga ggatccgtgg 300
acgttcggtg gaggcaccaa gctggaaatc aaa 333
<210> 126
<211> 990
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain constant region
<400> 126
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa 990
<210> 127
<211> 321
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> light chain constant region
<400> 127
cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60
ggaactgcct ctgttgtgtg cctgctgaat aacttctatc cccgcgaggc caaagtacag 120
tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180
agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240
aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300
agcttcaacc gcggagagtg t 321
<210> 128
<211> 122
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> Daratumumab, heavy chain variable region
<400> 128
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 129
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> Daratumumab, light chain variable region
<400> 129
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 130
<211> 120
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> Isatuximab, heavy chain variable region
<400> 130
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr
65 70 75 80
Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 131
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> Isatuximab, light chain variable region
<400> 131
Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly
1 5 10 15
Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 132
<211> 120
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> Mor202, heavy chain variable region
<400> 132
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asp Pro Ser Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Pro Leu Val Tyr Thr Gly Phe Ala Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 133
<211> 107
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> Mor202, light chain variable region
<400> 133
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg His Tyr Tyr Val
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Gly Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Thr Gly Gly Ala Ser Leu
85 90 95
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 134
<211> 106
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 134
Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 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 Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Thr Asn Pro Trp Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 135
<211> 106
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 135
Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg 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 Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Thr Asn Pro Trp Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 136
<211> 12
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
<400> 136
Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr Trp Tyr
1 5 10
<210> 137
<211> 9
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR3
<400> 137
Ser Met Ile Thr Thr Gly Phe Thr Leu
1 5
<210> 138
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 138
Gln Gln Phe Trp Asn Thr Pro Trp Thr Phe
1 5 10
<210> 139
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR3
<400> 139
Gln Gln Trp Ser Thr Asn Pro Trp Thr Phe
1 5 10
<210> 140
<211> 116
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 140
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Leu Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Pro Gly Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 141
<211> 116
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 141
Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Leu Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Pro Gly Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 142
<211> 116
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain variable region
<400> 142
Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Leu Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Pro Gly Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 143
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 143
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Thr Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 144
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 144
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Thr Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 145
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 145
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Thr Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 146
<211> 111
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 146
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Thr Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Gln Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 147
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> heavy chain, CDR1
<400> 147
Gly Tyr Ala Leu Thr Asn Tyr Gly Met Asn
1 5 10
<210> 148
<211> 15
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
<400> 148
Arg Thr Ser Glu Ser Val Asp Ser Tyr Gly Asn Thr Phe Met His
1 5 10 15
Claims (13)
1. An antibody molecule or fragment thereof that binds human CD38, said antibody molecule or fragment thereof comprising a heavy chain variable region (VH), wherein said heavy chain variable region comprises a CDR1(VH-CDR1) selected from the group consisting of SEQ ID NO 1, SEQ ID NO 7, SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 20, SEQ ID NO 26, SEQ ID NO 31, SEQ ID NO 41 and SEQ ID NO 147, a CDR2(VH-CDR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 8, SEQ ID NO 12, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 27, SEQ ID NO 32, SEQ ID NO 37 and SEQ ID NO 42, and a CDR2(VH-CDR2) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 18, SEQ ID NO 17, SEQ ID NO 27, SEQ, CDR3(VH-CDR3) shown in SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO: 137; and
the antibody molecule or fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain variable region selected from the group consisting of SEQ ID NOs: 4. SEQ ID NO: 14. SEQ ID NO: 23. SEQ ID NO: 29. SEQ ID NO: 34. SEQ ID NO: 39. SEQ ID NO: 44. SEQ ID NO: 49. SEQ ID NO:136 and SEQ ID NO:148 (VL-CDR1) selected from the group consisting of SEQ ID NOs: 5. SEQ ID NO:24 and SEQ ID NO:35 (VL-CDR2) selected from the group consisting of SEQ ID NOs: 6. SEQ ID NO: 10. SEQ ID NO: 15. SEQ ID NO: 19. SEQ ID NO: 25. SEQ ID NO: 30. SEQ ID NO: 36. SEQ ID NO: 40. SEQ ID NO: 45. SEQ ID NO:138 and SEQ ID NO:139 (VL-CDR 3).
2. The antibody molecule or fragment thereof of claim 1, wherein said heavy chain variable region comprises a combination of CDRs selected from the group consisting of:
(1) VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO. 2, and VH-CDR3 shown in SEQ ID NO. 3;
(2) VH-CDR1 shown in SEQ ID NO. 7, VH-CDR2 shown in SEQ ID NO. 8, VH-CDR3 shown in SEQ ID NO. 9;
(3) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 13;
(4) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 18;
(5) VH-CDR1 shown in SEQ ID NO:20, VH-CDR2 shown in SEQ ID NO:21, VH-CDR3 shown in SEQ ID NO: 22;
(6) VH-CDR1 shown in SEQ ID NO. 26, VH-CDR2 shown in SEQ ID NO. 27, VH-CDR3 shown in SEQ ID NO. 28;
(7) VH-CDR1 shown in SEQ ID NO:31, VH-CDR2 shown in SEQ ID NO:32, VH-CDR3 shown in SEQ ID NO: 33;
(8) VH-CDR1 shown in SEQ ID NO:26, VH-CDR2 shown in SEQ ID NO:37, VH-CDR3 shown in SEQ ID NO: 38;
(9) VH-CDR1 shown in SEQ ID NO:41, VH-CDR2 shown in SEQ ID NO:42, VH-CDR3 shown in SEQ ID NO: 43;
(10) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 46;
(11) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 47;
(12) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 48;
(13) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 137;
(14) VH-CDR1 shown in SEQ ID NO:147, VH-CDR2 shown in SEQ ID NO:42, VH-CDR3 shown in SEQ ID NO: 43;
and/or, the antibody molecule or fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region comprises a combination of CDRs selected from:
(1) VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 6;
(2) VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 10;
(3) VL-CDR1 shown in SEQ ID NO.14, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 15;
(4) VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 19;
(5) VL-CDR1 shown in SEQ ID NO. 23, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 25;
(6) VL-CDR1 shown in SEQ ID NO. 29, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 30;
(7) VL-CDR1 shown in SEQ ID NO. 34, VL-CDR2 shown in SEQ ID NO. 35, VL-CDR3 shown in SEQ ID NO. 36;
(8) VL-CDR1 shown in SEQ ID NO:39, VL-CDR2 shown in SEQ ID NO:24, VL-CDR3 shown in SEQ ID NO: 40;
(9) VL-CDR1 shown in SEQ ID NO. 44, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 45;
(10) VL-CDR1 shown in SEQ ID NO. 49, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 40;
(11) VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 138;
(12) VL-CDR1 shown in SEQ ID NO:136, VL-CDR2 shown in SEQ ID NO:35, VL-CDR3 shown in SEQ ID NO: 36;
(13) VL-CDR1 shown in SEQ ID NO:136, VL-CDR2 shown in SEQ ID NO:35, VL-CDR3 shown in SEQ ID NO: 139;
(14) VL-CDR1 shown in SEQ ID NO. 148, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 45.
3. Antibody molecule or fragment thereof according to claim 1 or2, wherein the heavy chain variable region comprises an amino acid sequence as set forth in any one of or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in any one of SEQ ID No. 50, SEQ ID No. 52, SEQ ID No. 54, SEQ ID No. 56, SEQ ID No. 58, SEQ ID No. 60, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 66, SEQ ID No. 68 to SEQ ID No. 88, SEQ ID No. 140 to SEQ ID No. 142; and/or
The light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 89 to SEQ ID NO 105, SEQ ID NO 134 and SEQ ID NO 135, SEQ ID NO 143 to SEQ ID NO 146 or an amino acid sequence having at least 75% identity to the amino acid sequence shown.
4. Antibody molecule or fragment thereof according to any one of claims 1 to 3, characterized in that it comprises a CDR combination selected from:
(1) VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO. 2, and VH-CDR3 shown in SEQ ID NO. 3; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 6;
(2) VH-CDR1 shown in SEQ ID NO. 7, VH-CDR2 shown in SEQ ID NO. 8, VH-CDR3 shown in SEQ ID NO. 9; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 10;
(3) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 13; VL-CDR1 shown in SEQ ID NO.14, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 15;
(4) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 18; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 19;
(5) VH-CDR1 shown in SEQ ID NO:20, VH-CDR2 shown in SEQ ID NO:21, VH-CDR3 shown in SEQ ID NO: 22; VL-CDR1 shown in SEQ ID NO. 23, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 25;
(6) VH-CDR1 shown in SEQ ID NO. 26, VH-CDR2 shown in SEQ ID NO. 27, VH-CDR3 shown in SEQ ID NO. 28; VL-CDR1 shown in SEQ ID NO. 29, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 30;
(7) VH-CDR1 shown in SEQ ID NO:31, VH-CDR2 shown in SEQ ID NO:32, VH-CDR3 shown in SEQ ID NO: 33; VL-CDR1 shown in SEQ ID NO. 34, VL-CDR2 shown in SEQ ID NO. 35, VL-CDR3 shown in SEQ ID NO. 36;
(8) VH-CDR1 shown in SEQ ID NO:26, VH-CDR2 shown in SEQ ID NO:37, VH-CDR3 shown in SEQ ID NO: 38; VL-CDR1 shown in SEQ ID NO:39, VL-CDR2 shown in SEQ ID NO:24, VL-CDR3 shown in SEQ ID NO: 40;
(9) VH-CDR1 shown in SEQ ID NO:41, VH-CDR2 shown in SEQ ID NO:42, VH-CDR3 shown in SEQ ID NO: 43; VL-CDR1 shown in SEQ ID NO. 44, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 45;
(10) VH-CDR1 shown in SEQ ID NO. 11, VH-CDR2 shown in SEQ ID NO. 12, VH-CDR3 shown in SEQ ID NO. 47; VL-CDR1 shown in SEQ ID NO.14, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 15;
(11) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 137; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 19;
(12) VH-CDR1 shown in SEQ ID NO 16, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 137; VL-CDR1 shown in SEQ ID NO. 4, VL-CDR2 shown in SEQ ID NO. 5, VL-CDR3 shown in SEQ ID NO. 138;
(13) VH-CDR1 shown in SEQ ID NO:31, VH-CDR2 shown in SEQ ID NO:32, VH-CDR3 shown in SEQ ID NO: 33; VL-CDR1 shown in SEQ ID NO:136, VL-CDR2 shown in SEQ ID NO:35, VL-CDR3 shown in SEQ ID NO: 36;
(14) VH-CDR1 shown in SEQ ID NO:147, VH-CDR2 shown in SEQ ID NO:42, VH-CDR3 shown in SEQ ID NO: 43; VL-CDR1 shown in SEQ ID NO. 44, VL-CDR2 shown in SEQ ID NO. 24, VL-CDR3 shown in SEQ ID NO. 45;
preferably, the antibody molecule or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(1) an amino acid sequence as shown in SEQ ID NO. 50 or an amino acid sequence with at least 75% identity to an amino acid sequence as shown in SEQ ID NO. 50; and, an amino acid sequence as set forth in SEQ ID NO. 51 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 51;
(2) an amino acid sequence as shown in SEQ ID NO. 52 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 52; and, the amino acid sequence as set forth in SEQ ID NO 53 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 53;
(3) an amino acid sequence as shown in SEQ ID NO. 54 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 54; and, an amino acid sequence as set forth in SEQ ID NO:55 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 55;
(4) an amino acid sequence as shown in SEQ ID NO. 56 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 56; and, an amino acid sequence as set forth in SEQ ID NO:57 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 57;
(5) an amino acid sequence as set forth in SEQ ID NO. 58 or an amino acid sequence with at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 58; and, the amino acid sequence as set forth in SEQ ID NO. 59 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 59;
(6) an amino acid sequence as set forth in SEQ ID NO:60 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 60; and, the amino acid sequence as set forth in SEQ ID NO 61 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 61;
(7) an amino acid sequence as set forth in SEQ ID NO. 62 or an amino acid sequence with at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 62; and, the amino acid sequence as set forth in SEQ ID NO:63 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 63;
(8) an amino acid sequence as shown in SEQ ID NO. 64 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 64; and, the amino acid sequence as set forth in SEQ ID NO. 65 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 65;
(9) an amino acid sequence as shown in SEQ ID NO. 66 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 66; and, the amino acid sequence as set forth in SEQ ID NO:67 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 67;
(10) an amino acid sequence as shown in SEQ ID NO. 69 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 69; and, an amino acid sequence as set forth in SEQ ID NO:90 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 90;
(11) 71 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID NO 71; and, an amino acid sequence as set forth in SEQ ID NO:90 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 90;
(12) an amino acid sequence as set forth in SEQ ID NO. 74 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 74; and, the amino acid sequence as set forth in SEQ ID NO 92 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 92;
(13) an amino acid sequence as set forth in SEQ ID NO. 75 or an amino acid sequence with at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 75; and, the amino acid sequence as set forth in SEQ ID NO 94 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 94;
(14) an amino acid sequence as set forth in SEQ ID NO. 76 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 76; and, the amino acid sequence as set forth in SEQ ID NO 94 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 94;
(15) 77 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID No. 77; and, an amino acid sequence as set forth in SEQ ID NO. 95 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 95;
(16) an amino acid sequence as shown in SEQ ID NO. 83 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 83; and, an amino acid sequence as set forth in SEQ ID NO. 102 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 102;
(17) an amino acid sequence as shown in SEQ ID NO. 84 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 84; and, an amino acid sequence as set forth in SEQ ID NO. 102 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 102;
(18) an amino acid sequence as shown in SEQ ID NO. 85 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 85; and, the amino acid sequence as set forth in SEQ ID NO:134 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 134;
(19) an amino acid sequence as shown in SEQ ID NO. 88 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 88; and, an amino acid sequence as set forth in SEQ ID NO. 103 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 103;
(20) an amino acid sequence as shown in SEQ ID NO. 88 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 88; and, the amino acid sequence as set forth in SEQ ID NO 104 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 104;
(21) 140 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID No. 140; and, the amino acid sequence as set forth in SEQ ID NO. 143 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 143;
(22) the amino acid sequence shown as SEQ ID NO.141 or the amino acid sequence with at least 75 percent of identity with the amino acid sequence shown as SEQ ID NO. 141; and, the amino acid sequence shown as SEQ ID NO:144 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID NO: 144;
(23) 142 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID No. 142; and, the amino acid sequence shown as SEQ ID NO:144 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID NO: 144.
5. The antibody molecule or fragment thereof of any one of claims 1 to 4, wherein said antibody molecule or fragment thereof is in any form of a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody or a chimeric antibody, or wherein said antibody molecule or fragment thereof is a half-antibody or an antigen-binding fragment of a half-antibody, such as scFv, BsFv, dsFv, (dsFv)2、Fab、Fab'、F(ab')2Or Fv;
preferably, the antibody molecule or fragment thereof further comprises a human or murine constant region, preferably a human or murine light chain constant region (CL) and/or heavy chain constant region (CH);
more preferably, the antibody molecule or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of the kappa or lambda type.
6. Antibody molecule or fragment thereof according to any one of claims 1 to 5, characterized in that said antibody molecule is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type;
preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO 106 or an amino acid sequence having at least 75% identity to said amino acid sequence;
preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:107 or an amino acid sequence having at least 75% identity to said amino acid sequence.
7. A conjugate or fusion protein comprising the antibody molecule of any one of claims 1 to 6, or a fragment thereof.
8. A nucleic acid molecule encoding the antibody molecule or fragment thereof of any one of claims 1 to 6 or encoding a heavy chain CDR, a light chain variable region, a heavy chain or a light chain comprised in said antibody molecule or fragment thereof.
9. A vector comprising the nucleic acid molecule of claim 8.
10. A host cell comprising the nucleic acid molecule of claim 8 and/or the vector of claim 9, or transformed or transfected with the nucleic acid molecule of claim 8 and/or the vector of claim 9.
11. A pharmaceutical composition comprising the antibody molecule or fragment thereof of any one of claims 1 to 6, the conjugate or fusion protein of claim 7, the nucleic acid molecule of claim 8, the vector of claim 9, or the host cell of claim 10, and optionally a pharmaceutically acceptable adjuvant.
12. Use of an antibody molecule or fragment thereof according to any one of claims 1 to 6, a conjugate or fusion protein according to claim 7, a nucleic acid molecule according to claim 8, a vector according to claim 9 or a host cell according to claim 10 in the manufacture of a medicament for the treatment of a disease associated with high expression of CD 38;
preferably, the disease is a hematological malignancy, preferably multiple myeloma or non-hodgkin's lymphoma.
13. A kit comprising the antibody molecule or fragment thereof of any one of claims 1 to 6, the conjugate or fusion protein of claim 7, the nucleic acid molecule of claim 8, the vector of claim 9, the host cell of claim 10, or the pharmaceutical composition of claim 11.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910889707.5A CN112538114A (en) | 2019-09-20 | 2019-09-20 | Anti-human CD38 antibody and application thereof |
| PCT/CN2020/116191 WO2021052465A1 (en) | 2019-09-20 | 2020-09-18 | Anti-human cd38 antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910889707.5A CN112538114A (en) | 2019-09-20 | 2019-09-20 | Anti-human CD38 antibody and application thereof |
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| Publication Number | Publication Date |
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| CN112538114A true CN112538114A (en) | 2021-03-23 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201910889707.5A Pending CN112538114A (en) | 2019-09-20 | 2019-09-20 | Anti-human CD38 antibody and application thereof |
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| Country | Link |
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| CN (1) | CN112538114A (en) |
| WO (1) | WO2021052465A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2024051057A1 (en) * | 2022-09-09 | 2024-03-14 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Multi-target chimeric antigen receptor comprising cd38 targeting |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JOP20210044A1 (en) * | 2010-12-30 | 2017-06-16 | Takeda Pharmaceuticals Co | Anti-cd38 antibodies |
| CN109053892B (en) * | 2018-09-19 | 2021-03-26 | 苏州思坦维生物技术股份有限公司 | Monoclonal antibody specifically binding to human and monkey CD38 antigens, and preparation method and application thereof |
-
2019
- 2019-09-20 CN CN201910889707.5A patent/CN112538114A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2024051057A1 (en) * | 2022-09-09 | 2024-03-14 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Multi-target chimeric antigen receptor comprising cd38 targeting |
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