CN114437215B

CN114437215B - Anti-human CD38 antibody and preparation method and application thereof

Info

Publication number: CN114437215B
Application number: CN202011221844.0A
Authority: CN
Inventors: 朱玲巧; 党尉; 张成海; 袁玉菁; 郭锦林; 吴易潘; 邹秋玲; 李致科
Original assignee: Shanghai Mabgeek Biotechnology Co ltd
Current assignee: Shanghai Mabgeek Biotechnology Co ltd
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2023-02-07
Anticipated expiration: 2040-11-05
Also published as: CN114437215A; WO2022095698A1

Abstract

The invention discloses an antibody capable of binding to human CD38, and a preparation method and application thereof. The antibody of the invention has anti-human CD38 antibodies with different affinities of high, medium and low, can be combined with CD38 of cynomolgus monkey, and can kill tumor cells through an Fc-dependent immune effector mechanism. Can be used for treating CD38 related diseases, such as multiple myeloma, non-Hodgkin's lymphoma, AL type amyloidosis, T cell lymphoma, and diffuse large B cell lymphoma.

Description

Anti-human CD38 antibody and preparation method and application thereof

Technical Field

The invention relates to the field of antibodies, in particular to an anti-human CD38 antibody and a preparation method and application thereof.

Background

CD38 is a 46kDa two-type transmembrane glycoprotein with an extracellular segment consisting of 258 amino acids, and the functions of CD38 include receptor-mediated adhesion and signaling and bifunctional in vitro enzymatic activity to promote intracellular calcium mobilization. Under normal conditions, CD38 is expressed at low levels on both lymphoid and myeloid cells, but in multiple myeloma, malignant cells specifically express CD38 at high levels, making CD38 an ideal myeloma target.

Multiple myeloma is a type of malignant plasmacytosis that occurs in B lymphocytes. Usually, myeloma cells proliferate clonally in the bone marrow and in the bone sponge soft tissue, causing osteolytic bone destruction, and many undesirable diseases are associated with anemia, renal failure and various damages caused by the extramedullary infiltration of myeloma cells. The incidence of chinese multiple myeloma has surpassed acute leukemia, being the second hematological malignancy after non-hodgkin lymphoma. About 1.5-2 ten thousand new cases of MM occur in China each year, the median survival time of patients is about 4-5 years, and the number of sick people in China is about 8-10 ten thousand. With the advent of aging society and the influence of various factors such as deterioration of the environment, it has been found that the incidence of multiple myeloma increases.

The CD38 monoclonal antibody was found to kill tumor cells primarily through Fc-dependent immune effector mechanisms, including complement-mediated cytotoxicity (CDC), antibody-mediated cytotoxicity (ADCC), antibody-mediated phagocytosis (ADCP), and by apoptosis (apoptosis). Furthermore, the CD38 antibody has an immunomodulatory effect: myeloma cells are regulated by immune regulation, such as reduction of CD38+ immune suppression, regulation of cells, and promotion of T cell expansion and activity.

Internationally, antibody drugs against CD38 such as cataumumab, robusta and Isatuximab, sunoft, have been marketed and MOR202, a niche organism, in clinical trials. Compared with the existing medicines and the macromolecular and small-molecular medicines in research, the CD 38-targeted antibody has very obvious curative effect advantage in multiple myeloma treatment, and single medicine and combined medicine show the curative effect advantage, so that better competitive advantage can be kept.

The main treatment method of myeloma in China is stem cell transplantation and combined medication of velcade (bortezomib) of poplar or remume (lenalidomide) of Celgene. In recent two years, the FDA approves a plurality of brand-new drug molecules for treating multiple myeloma, the curative effect is improved and the prognosis is improved with the continuous emergence of new treatment drugs, the median survival time is 7-8 years at present, multiple myeloma is gradually converted into chronic disease, and the growth of the multiple myeloma treatment market is objectively driven. The advent of CD38 monoclonal antibody provides a new option for patients with multiple myeloma, particularly patients with relapsed and refractory disease. Research in the field of CD38 antibodies is expected to make more breakthroughs and benefit more patients.

Disclosure of Invention

The inventors of the present invention have conducted a number of experiments to obtain a group of monoclonal antibodies that specifically bind to cell surface CD38, and these antibodies can bind to cynomolgus monkey CD38 at the same time. A series of anti-human CD38 antibodies with different affinities of high, medium and low were obtained, which were able to kill tumor cells by Fc-dependent immune effector mechanisms.

In a first aspect, the present application provides an antibody or antigen-binding portion thereof that specifically binds human CD38, comprising a heavy chain variable region comprising an HCDR3 sequence, optionally further comprising an HCDR1 and/or HCDR2 sequence. In some embodiments, the HCDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs:3,6,9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 and 48. In some embodiments, the HCDR2 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2,5,8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, and 47. In some embodiments, the HCDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1,4,7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, and 46.

In some embodiments, the heavy chain variable region comprises a heavy chain variable region substantially identical to a light chain variable region selected from SEQ ID Nos:97 99, 101, 103, 105, 107, 109 and 111, or the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:97, 99, 101, 103, 105, 107, 109 and 111.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds human CD38 further comprises a light chain variable region, wherein the light chain variable region comprises an LCDR1, LCDR2, and/or LCDR3 sequence. In certain embodiments, the LCDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91 and 94. In some embodiments, the LCDR1 sequence further comprises an amino acid sequence selected from SEQ ID No. 115. In certain embodiments, the LCDR2 sequence comprises an amino acid sequence selected from SEQ ID NOs:50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92 and 95. In some embodiments, the LCDR2 sequence further comprises an amino acid sequence selected from SEQ ID NO: 116. In certain embodiments, the LCDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs:51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93 and 96. In some embodiments, the LCDR3 sequence further comprises an amino acid sequence selected from SEQ ID NO: 117.

In some embodiments, the light chain variable region comprises an amino acid sequence having at least 80% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs:98, 100, 102, 104, 106, 108, 110, and 112; or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:98, 100, 102, 104, 106, 108, 110 and 112.

In alternative embodiments, the antigen binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab') 2 fragment, a Fv fragment, a scFv fragment, a Fd fragment, and a single domain antibody.

In some embodiments, the antibody that specifically binds to human CD38 of the first aspect is a murine monoclonal antibody.

In some embodiments, the antibody that specifically binds to human CD38 of the first aspect is a humanized antibody.

In a second aspect, the present application provides an expression vector comprising a nucleotide molecule encoding an amino acid sequence as described above.

In some embodiments, the expression vector is pTT5, pUC57, pDR1, pcDNA3.1 (+), pDFHF or pCHO1.0, and the like.

In a third aspect, the present application provides a host cell comprising an expression vector as described above. In some embodiments, the host cell is HEK293, COS, CHO, NS0, sf9, sf21, DH5 α, BL21 (DE 3), or TG1, etc.

In a fourth aspect, the present application provides a method of making an antibody or antigen-binding portion thereof of the first aspect that specifically binds human CD38, the method comprising the steps of:

a) Culturing said host cell under expression conditions such that said host cell of the third aspect is capable of producing said antibody, or antigen-binding portion thereof, thereby expressing said antibody, or antigen-binding portion thereof; and

b) Isolating and purifying the antibody or antigen-binding portion thereof expressed by a).

In a fifth aspect, the present application provides a pharmaceutical composition comprising an anti-human CD38 antibody or antigen-binding portion thereof according to the first aspect and a pharmaceutically acceptable carrier.

In some embodiments, the composition is for use in treating a human CD 38-associated disease.

In some embodiments, the CD 38-associated disease comprises multiple myeloma, non-hodgkin's lymphoma, AL-type amyloidosis, T-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, plasma cell leukemia, acute myelogenous leukemia, non-small cell lung cancer, pancreatic cancer, colorectal cancer, and triple negative breast cancer, among others.

In other aspects, the present application provides a method for preventing or treating a human CD 38-associated disease, comprising administering to an individual in need thereof an antibody or antigen-binding portion thereof according to the first aspect, or a pharmaceutical composition according to the fifth aspect.

The anti-human CD38 antibody or the antigen binding part thereof can be specifically bound with human CD38, has high, medium and low different affinities, and can kill tumor cells through an Fc dependent immune effector mechanism. The anti-human CD38 antibody or an antigen-binding portion thereof of the present invention can be used for preventing or treating CD 38-associated diseases such as multiple myeloma, non-hodgkin's lymphoma, AL-type amyloidosis, T-cell lymphoma, and diffuse large B-cell lymphoma.

Drawings

FIG. 1 shows the results of the binding of humanized CD38 monoclonal antibody to Daudi cells

FIG. 2 is the result of experiment for inducing apoptosis of Daudi lymphoma cells by humanized CD38 antibody

FIG. 3 is a graph showing the results of experiments on anti-Fc cross-linking induced apoptosis of Daudi lymphoma cells by humanized CD38 antibody

Detailed Description

The present application provides novel anti-CD 38 antibodies, or antigen-binding portions thereof, that specifically bind to human CD38. In a preferred embodiment, the antibodies or antigen-binding portions thereof of the present application have high, medium and low differential affinity binding activity to CD38, while binding to cynomolgus monkey CD38. The present application also provides amino acids of the antibody or antigen-binding fragment thereof, vectors comprising the amino acids, host cells comprising the amino acids or vectors, methods of making and purifying the antibody, and medical and biological applications of the antibody or antigen-binding fragment thereof, such as preventing or treating a CD 38-associated disease or disorder.

To facilitate understanding of the present application, certain terms used herein are first defined.

The term "antibody" as used herein refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by a disulfide bond, as well as multimers thereof (e.g., igM). Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises one domain (CL 1). The VH and VL regions may be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs) into which conserved regions known as Framework Regions (FRs) are interspersed.

As used herein, the term "antigen-binding portion" of an antibody refers to a portion or segment of an intact antibody molecule that is responsible for binding to an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others. Non-limiting examples of antigen-binding moieties include: a Fab fragment; a F (ab') 2 fragment; (ii) a fragment of Fd; (iv) an Fv fragment; single chain Fv (scFv) molecules; a single domain antibody; a dAb fragment and the smallest recognition unit (e.g., an isolated CDR) that consists of amino acid residues that mimic a hypervariable region of an antibody. The term "antigen-binding portion" also includes other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies, and the like.

As used herein, the terms "heavy chain variable region (VH)" and "light chain variable region (VL)" refer to a single antibody variable heavy and light chain region, respectively, that comprises FR1, 2, 3, and 4 and CDR1, 2, and 3.

It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR 3) are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of CDR Sequences for VH or VL, namely the Kabat definition and Chothia definition, see, for example, kabat et al, "Sequences of Proteins of Immunological Interest", national Institutes of Health, bethesda, md. (1991); A1-Lazikani et al, J.mol.biol.273:927-948 (1997); and Martin et al, proc.Natl.Acad.Sci.USA86:9268-9272 (1989). For a given antibody variable region sequence, can according to Kabat definition or Chothia definition to determine VH and VL sequence in CDR region sequence. In embodiments of the present application, the CDR sequences are defined using Kabat. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

The CDR region sequences in the variable region sequences can be analyzed in a variety of ways for the variable region sequences of a given antibody, as can be determined using the online software Abysis (http:// www.abysis.org /).

The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, e.g. binding of an antibody to an epitope of an antigen, e.g. the ability of an antibody to bind to a specific antigen with an affinity that is at least two times greater than its affinity for a non-specific antigen. It will be appreciated, however, that an antibody is capable of specifically binding to two or more antigens whose sequences are related. For example, an antibody of the invention can specifically bind to CD38 in humans and non-humans (e.g., mice or non-human primates).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity (see, U.S. Pat. No. 4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA 81.

The term "homology", as used herein, is defined as the percentage of residues in an amino acid or nucleotide sequence variant that are identical, if necessary to the maximum percentage, after alignment and the introduction of gaps in the sequence. Methods and computer programs for alignment are well known in the art. As used herein, "at least 80% homology" means homology of any value from 80% to 100%, e.g., 85%, 90%, 95%, 99%, etc.

As used herein, the term "CD 38-associated disease" includes multiple myeloma, non-hodgkin's lymphoma, AL-type amyloidosis, T-cell lymphoma, and diffuse large B-cell lymphoma, among others.

In one aspect, the present application provides an antibody, or antigen-binding portion thereof, that specifically binds to CD38, comprising a heavy chain variable region and/or a light chain variable region. Exemplary table 1-3 lists CDR, heavy chain and light chain variable region amino acid sequences suitable for use in the antibodies disclosed herein.

In a specific embodiment, the HCDR3 is selected from the amino acid sequences set forth in SEQ ID NOs:3,6,9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 and 48. In another specific embodiment, the HCDR3 is selected from the amino acid sequences set forth in SEQ ID NOs:3,9, 18, 21, 27, 33, 36, and 45. In a preferred embodiment, the HCDR3 is selected from the group consisting of the amino acid sequences shown as 9, 18, 21, 27, 33, 36 and 45.

In a specific embodiment, the HCDR2 is selected from the amino acid sequences set forth in SEQ ID NOs:2,5,8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, and 47. In another specific embodiment, the HCDR2 is selected from the amino acid sequences set forth in SEQ ID NOs:2,8, 17, 20, 26, 32, 35, and 44. In a preferred embodiment, the HCDR2 is selected from the group consisting of the amino acid sequences shown in 8, 17, 20, 26, 32, 35 and 44.

In a specific embodiment, the HCDR1 is selected from the amino acid sequences set forth in SEQ ID NOs:1,4,7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43 and 46. In another specific embodiment, the HCDR1 is selected from the amino acid sequences set forth in SEQ ID NOs:1,7, 16, 19, 25, 31, 34, and 43. In a preferred embodiment, the HCDR1 is selected from the group consisting of amino acid sequences set forth in 7, 16, 19, 25, 31, 34 and 43.

In some embodiments, the antibody heavy chain variable region disclosed herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:97, 99, 101, 103, 105, 107, 109, and 111. In a specific embodiment, the heavy chain variable region consists of an amino acid sequence selected from the group consisting of SEQ ID NOs:97, 99, 101, 103, 105, 107, 109 and 111.

The antibodies, or antigen-binding portions thereof, disclosed herein may further comprise a light chain variable region in addition to the heavy chain variable region.

In some embodiments, CDR3 (LCDR 3) of the light chain variable region is selected from the amino acid sequences set forth in SEQ ID NOs:51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, and 117. In another specific embodiment the LCDR3 is selected from the amino acid sequences shown in SEQ ID NOs:51, 57, 66, 69, 75, 81, 84 and 117. In a preferred embodiment, the LCDR3 is selected from the amino acid sequences shown in SEQ ID NOs:57, 66, 69, 75, 81, 84 and 117.

In some embodiments, the LCDR2 is selected from the amino acid sequences set forth in SEQ ID NOs:50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, and 116, or from the amino acid sequences set forth in SEQ ID NOs:50, 65, 68, 74, 80, 83, 92, and 116. In a preferred embodiment, LCDR2 is selected from the amino acid sequences set forth in SEQ ID NOs:65, 68, 74, 80, 83, 92, and 116.

In some embodiments, LCDR1 is selected from the amino acid sequences of SEQ ID NOs:49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, and 115, or from the amino acid sequences of SEQ ID NOs:55, 64, 67, 73, 79, 82, 91, and 115. In a preferred embodiment, LCDR1 is selected from the amino acid sequences set forth in SEQ ID NOs:55, 64, 67, 73, 79, 82, and 91.

In some embodiments, the antibody light chain variable region disclosed herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:98, 100, 102, 104, 106, 108, 110, and 112. In a specific embodiment, the light chain variable region consists of an amino acid sequence selected from the group consisting of SEQ ID NOs:98, 100, 102, 104, 106, 108, 110 and 112.

In some embodiments, the heavy or heavy chain variable region, the light chain, or the light chain variable region of the antibodies disclosed herein can be substituted, deleted, or added with at least one amino acid based on the respective corresponding specific amino acid sequences listed above, and the resulting variants still retain the activity of binding to human CD38.

In certain embodiments, the number of such amino acid substitutions, deletions or additions is from 1 to 30, preferably from 1 to 20, and more preferably from 1 to 10. In preferred embodiments, the sequence variant differs from the original amino acid sequence by substitutions, deletions and/or additions of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In more preferred embodiments, the sequence variant differs from the original amino acid sequence by a substitution, deletion or addition of about 1, 2, 3, 4 or 5 amino acids. In particular embodiments, the amino acid substitution is a conservative substitution.

In a preferred embodiment, the antibody disclosed herein is

antibody

57H, 105H, 116H, or 145H, wherein the heavy chain variable region sequence of antibody 57H is as set forth in SEQ ID NO:99, the light chain variable region sequence is shown as SEQ ID NO:100, wherein LCDR2 is preferably SEQ ID NO:116, the remaining CDR sequences are identical to those of murine antibody 57; the heavy chain variable region sequence of antibody 105H is set forth in SEQ ID NO:107, and the light chain variable region sequence is shown as SEQ ID NO:108, wherein the CDR sequences are identical to murine antibody 105; the heavy chain variable region sequence of antibody 116H is set forth in SEQ ID NO:109, and the light chain variable region sequence is shown in SEQ ID NO:110, wherein the CDR sequences are identical to murine antibody 116; the heavy chain variable region sequence of antibody 145H is set forth in SEQ ID NO:111, and the light chain variable region sequence is shown as SEQ ID NO:112, wherein LCDR3 is preferably SEQ ID NO:117, the remaining CDR sequences are identical to those of murine antibody 145.

In some embodiments, the antibodies disclosed herein are monoclonal antibodies. In particular embodiments, the antibodies disclosed herein are humanized antibodies.

The antibodies, or antigen-binding portions thereof, disclosed herein are capable of specifically binding to human CD38. In specific embodiments, the antibody, or antigen-binding portion thereof, specifically binds human CD38 or cynomolgus monkey CD38. In a preferred embodiment, the antibody, or antigen-binding portion thereof, specifically binds human CD38.

In some embodiments, any suitable expression vector may be used in the present application. For example, the expression vector may be one of pTT5, pUC57, pDR1, pcDNA3.1 (+), pDFHF and pCHO 1.0. Expression vectors may include fusion DNA sequences with appropriate transcriptional and translational regulatory sequences attached.

In some embodiments, useful host cells are cells containing the above-described expression vectors, which may be eukaryotic cells, such as mammalian or insect host cell culture systems, may be used for expression of the antibodies or antigen-binding portions thereof of the present application. For example, HEK293 cells, COS, CHO, NS0, sf9, sf21, etc. can be used in the present invention. The host cell may be a prokaryotic cell containing the above expression vector, and may be, for example, DH 5. Alpha., BL21 (DE 3), TG1 or the like.

In some embodiments, the methods of preparing an anti-human CD38 monoclonal antibody disclosed herein comprise: culturing the host cell under suitable expression conditions to express the anti-human CD38 monoclonal antibody; the expressed anti-human CD38 monoclonal antibody was isolated and purified. Using the above method, the recombinant protein can be purified as a substantially homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis.

In some embodiments, the anti-human CD38 antibodies disclosed herein can be isolated and purified by affinity chromatography, and the anti-human CD38 antibodies bound to the affinity column can be eluted using conventional methods, such as high salt buffer, PH change, etc., depending on the characteristics of the affinity column used.

In some embodiments, the humanized anti-human CD38 monoclonal antibodies disclosed herein are obtained by the following method: a Balb/c mouse is immunized by using a human CD38 antigen prepared in a laboratory, and after the titer of the immunized mouse is high for multiple times, spleen cells of the mouse are taken to be fused with hybridoma cells, and hybridoma cell strains with different affinities are screened out. More specifically, the inventors of the present application, through a number of experiments, first expressed the human CD38 antigen individually, and then used different adjuvants to mix with the human CD38 antigen to immunize mice, and then further fused the spleen cells of the mice with the hybridoma cell line sp2/0, and the fused hybridomas screened positive cell lines using the human CD38 antigen, and after verifying the binding to human CD38 and Daudi cells, obtained the target cell lines. After the target molecule is humanized and transformed, the light chain and heavy chain genes are cloned into a eukaryotic expression vector pCHO1.0 simultaneously. The expression vector is transfected into CHO cells by a liposome method, positive cell clones are screened by puromycin and methotrexate, the screened high-expression clones are subjected to expansion culture by a serum-free culture medium, and a humanized anti-human CD38 antigen monoclonal antibody is separated or purified by a Protein A affinity column.

In other embodiments, the parent antibody of murine origin may be further altered using techniques conventional in the art, such as PCR mutagenesis, to produce chimeric or humanized or other variant forms of the antibody. Parent antibodies of the present application can be mutated, for example, within the Complementarity Determining Regions (CDR) domains of an antigen to produce variant antibodies that can be screened for the presence of properties of interest, such as binding affinity (lower KD), IC50, specificity, preferential binding, and the like. Preferably, the property of interest in the variant antibody is an improvement over the property in the parent antibody. Amino acid substitution variant antibodies are preferred, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues of the parent antibody molecule are removed and a different residue is inserted in its position. The most interesting sites for substitutional mutagenesis are one or more CDR regions, but Framework Region (FR) alterations are also contemplated. Conservative amino acid substitutions are preferred, and non-conservative amino acid changes can also be introduced and the resulting variant antibodies screened for properties of interest.

The present application provides pharmaceutical compositions comprising an antibody, or antigen-binding portion thereof, disclosed herein and a pharmaceutically acceptable carrier. The above-described anti-human CD38 monoclonal antibody disclosed herein can be formulated into a pharmaceutical preparation together with a pharmaceutically acceptable carrier, thereby exerting a therapeutic effect more stably. In some embodiments, these formulations can ensure the integrity of the conformation of the amino acid core sequence of the anti-human CD38 monoclonal antibodies disclosed herein, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamidation, or oxidation). In some embodiments, for liquid formulations, it may be generally stable for at least one year at 2 ℃ to 8 ℃. In some embodiments, for a lyophilized formulation, it remains stable at 30 ℃ for at least six months.

In some embodiments, the anti-human CD38 antibody monoclonal antibody preparation may be a suspension, a hydro-acupuncture, a freeze-drying preparation, etc. commonly used in the pharmaceutical field, preferably a hydro-acupuncture or a freeze-drying preparation, and for the anti-human CD38 monoclonal antibody hydro-acupuncture or freeze-drying preparation disclosed herein, pharmaceutically acceptable excipients include but are not limited to: surfactants, solution stabilizers, isotonicity adjusting agents and buffers or combinations thereof. In some embodiments, surfactants include, but are not limited to: nonionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80), poloxamers (such as poloxamer 188), triton, sodium Dodecyl Sulfate (SDS), sodium lauryl sulfate, myristyl, linoleyl or octadecylsarcosine, pluronics, MONAQUATTM, etc., are added in an amount to minimize the tendency of the anti-human CD38 monoclonal antibody to granulate. In some embodiments, the solution stabilizer includes, but is not limited to, one or a combination of the following list: saccharides such as reducing saccharides and non-reducing saccharides; amino acids such as monosodium glutamate or histidine; alcohols such as trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol, and the like. The solution stabilizer should be added in an amount such that the final formulation remains stable for a period of time deemed stable by one skilled in the art. Isotonicity adjusting agents include, but are not limited to, sodium chloride, mannitol, or combinations thereof. Buffers include, but are not limited to: tris, histidine buffer, phosphate buffer, or a combination thereof.

The present application also provides a method for preventing or treating a CD 38-associated disease comprising administering to a subject an anti-human CD38 antibody, or a composition comprising an anti-human CD38 antibody. In particular, the anti-human CD38 antibodies disclosed herein are effective in preventing and/or treating CD 38-associated diseases.

The anti-human CD38 monoclonal antibody and the composition thereof disclosed herein are administered to animals including human, and the administration dose varies depending on the age and body weight of the patient, the nature and severity of the disease and the administration route, and the total administration dose cannot exceed a certain range with reference to the results and general conditions of animal experiments.

The dosage and frequency of administration of the antibody or composition thereof may vary depending on the disease to be prevented or treated. In prophylactic applications, a composition comprising an antibody of the present application or a mixture thereof is administered to a patient who is not already in a disease state to enhance the patient's resistance, this amount being defined as a "prophylactically effective dose". In this use, the specific dosage will depend on the health of the patient and the systemic immunity. Relatively low doses are typically administered at relatively infrequent intervals for extended periods of time. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until disease progression is slowed or terminated, and preferably until the patient shows partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen may be administered to the patient. The specific dosage and frequency can be readily determined by one of ordinary skill in the art based on the actual need.

In this specification and claims, the words "comprise", "comprising" and "contain" mean "including but not limited to", and are not intended to exclude other moieties, additives, components, or steps.

It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application may be applied to any other aspect, embodiment or example described herein unless incompatible therewith.

The foregoing disclosure generally describes the present invention. The following specific examples are intended to further illustrate the invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional methods, which are well known to those of ordinary skill in the art and are described in numerous publications, such as molecular cloning handbooks, antibody technical laboratory manuals from cold spring harbor. The source of the reagent is not indicated as a conventional reagent.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1Fc tag and Flag tag antigen of human CD38 extracellular domain, and cynomolgus monkey CD38 extracellular domain Flag tag egg Preparation of white and reference antibody Daratumumab

The human CD38 antigen sequence was purchased from Yiqiao Shenzhou (cat # HG 10818-M). The 43 th-300 th amino acid fragment of the N end of the human CD38 is spliced with an hFc fragment and a Flag tag (DYKDDDDK) respectively by a PCR method, the hFc fragment and the Flag tag are positioned at the C end and are constructed on a pTT5 expression vector (stored in a laboratory) to obtain pTT5 (hCD 38-ECD-hFc) and pTT5 (hCD 38-ECD-Flag), and a clone with a completely correct sequencing result is selected for plasmid extraction and then transfection.

The cynomolgus monkey CD38 sequence was purchased from Yiqiao Shenzhou (cat # CG 90050-G). Splicing the 44 th-301 th amino acid fragment of the N end with a Flag tag (DYKDDDDK) by a PCR method, wherein the Flag tag is positioned at the C end and is constructed on a pTT5 expression vector (stored in a laboratory) to obtain pTT5 (cyno-CD 38-ECD-Flag), and selecting a clone with a completely correct sequencing result for plasmid extraction and transfection.

The amino acid sequence of Daratumumab comes from IMGT, and the nucleotide sequences of the variable regions of heavy chain and light chain are synthesized by the whole gene after codon optimization. Linking the variable region of the Daratumumab heavy chain to the constant region of IgG1 by PCR; the Daratumumab light chain variable region is linked to the constant region of the Kappa chain. These fragments were then cloned into the pTT5 expression vector and plasmid extraction was performed for transfection after confirmation of sequencing verification.

Plasmids were transfected into HEK293E cell line (laboratory preservation) by the PEI method. After 5 days of culture in Freestyle293 medium (from Gibco) containing 3mM valproic acid, the Protein of interest was purified from the cell culture supernatant by Protein A affinity chromatography (from Pharmacia) or Flag affinity chromatography (from Genscript). The protein was quantified by the Bicinchoninic acid (BCA) method, and the resulting protein was purified for further analysis and study as follows. The purified protein was used for the following immunization of mice and further analysis and study.

Example 2Immunization of hCD38-ECD-hFc

100 mu g/mouse of hCD38-ECD-hFc antigen was diluted to 75. Mu.l with physiological saline, mixed with equal volume of Freund's complete adjuvant, and subjected to ultrasonic emulsification to complete subcutaneous multi-site injection of 4-5 week-old Balb/c mice (purchased from Shanghai Ling Biotech Co., ltd., animal production license number: SCXK 2013-0018). Three weeks later, 50. Mu.g/mouse of protein was similarly diluted to 75. Mu.l and mixed with an equal volume of Freund's incomplete adjuvant, and after completion of the phacoemulsification, the mice were injected subcutaneously in multiple spots, and the immunization was repeated two weeks later. All mice were bled at their tail one week after the third immunization and serum titers were determined by ELISA coated with hCD38-ECD-hFc antigen. For mice with serum antibody titers >10000, shock immunization was performed one week after blood draw: tail vein injection of 10 μ g antigen/100 μ l saline/mouse.

The detection of the titres was carried out by ELISA: the ELISA plates were coated with hCD38-ECD-hFc antigen at a concentration of 1. Mu.g/ml, 100. Mu.l per well, overnight at 4 ℃. PBST (PBS containing 0.5% Tween-20) was washed 2 times and then patted dry. Each well was blocked with 200. Mu.l of a coating solution containing 1% BSA at room temperature for 4 hours, then patted dry, and stored in a freezer at-20 ℃ until use. In the detection process, 100 mu l of mouse serum with different concentrations is added into each hole of an ELISA plate, 2 multiple holes are arranged, and the mixture is incubated for 1.5 hours at room temperature. PBST was washed 3 times and then patted dry. 100. Mu.l of HRP-labeled rabbit anti-mouse Ig antibody (purchased from Sigma) diluted 10000-fold in PBST1 was added and incubated at room temperature for 1 hour. PBST was washed 3 times and then patted dry. Adding 100 ul of color developing solution into each well (mixing ELISA color developing solution A and color developing solution B according to the volume ratio of 1:1 for color development immediately before use), and adding 100 ul of 2M H2SO4 stop solution into each well to stop reaction. OD values of each well were immediately measured at a wavelength of 450nm with a microplate reader (Molecular Device).

Example 3Hybridoma fusion and screening

Hybridoma sp2/0 cells (from the cell bank of the culture Collection of type culture of Chinese academy of sciences, accession number TCM-18) at 37 ℃ in 5% CO ₂ Culturing in an incubator, and changing the culture solution the day before fusion. Three days after the mice are subjected to impact immunization, and the splenocytes of the mice are taken for fusion. The fusion and screening method is as follows: spleen cells were counted after taking the mouse spleen, grinding and washing. Spleen cells and sp2/0 cells were mixed at a ratio of 10. The supernatant was washed off. 1ml of PEG (1450) was added within 1 minute, the mixture was gently shaken for 90 seconds, 5ml of serum-free DMEM medium (purchased from Gibco) was added within 2.5 minutes, and 5ml of serum-free DMEM was added once again to terminate the reaction, and the mixture was left to stand for 5 minutes and centrifuged at 1280rpm for 8 minutes. Cells were seeded evenly into 96-well plates at 200. Mu.l per well, in the number of two million sp2/0 cells per 96-well plate. The culture medium is first screened with HAT culture medium containing hypoxanthine (H), methotrexate (A) and thymidine (T), and the culture medium is changed to HT culture medium every 3-4 days. After 10 days, when the hybridoma cells are more than 10% of the bottom of the 96-well plate, taking the supernatant and carrying out ELISA detection by using an ELISA plate coated by hCD38-ECD-hFc antigen. The ELISA detection method was the same as described in example 2. Selecting positive hybridoma to clone in 24-well plateAnd (3) performing amplification culture and subcloning by a limiting dilution method to obtain a hybridoma strain for stably expressing the target antibody, and then performing seed preservation and library construction.

Example 4Determination of sequence of mouse-derived anti-human CD38 monoclonal antibody

The positive hole with higher affinity is subcloned for 2-3 rounds to obtain 150 hybridoma strains. Antibody sequences were prepared from these hybridoma cell lines. Total RNA of each hybridoma cell line was extracted using Trizol, and mRNA was reverse-transcribed into cDNA using a reverse transcription kit (purchased from ABI). The light chain variable region and heavy chain variable region genes of the mouse anti-human CD38 monoclonal antibody are amplified by PCR with primers reported in literature, then the PCR products are cloned into pGEM-T vector, and the variable region gene sequences are sequenced and analyzed. The obtained sequences were analyzed by alignment in GenBank, and all the sequences were consistent with the characteristics of mouse IgG variable region genes. Table 1 and table 2 list the amino acid sequences of the CDR regions of preferred antibodies.

Table 1: heavy chain CDR amino acid sequences of exemplary murine anti-human CD38 antibodies

Example 5Humanization of anti-human CD38 monoclonal antibodies

Based on the results of the sequence analysis, antibodies nos. 1, 57, 71, 73, 77, 78, 105, 116 and 145 were picked and subjected to the construction of chimeric antibodies and humanized antibodies. The chimeric antibody is constructed by intercepting the heavy chain variable region and the light chain variable region of a murine antibody and connecting the heavy chain variable region and the light chain variable region with the light chain constant region of human IgG1 respectively by overlapping PCR.

Table 2: light chain CDR amino acid sequences of exemplary murine anti-human CD38 antibodies

The amino acid sequences of the light chain variable region and the heavy chain variable region of the mouse-derived anti-human CD38 monoclonal antibody were analyzed according to the Kabat's rule and 3 CDRs and 4 FRs were determined. Taking antibody 105 as an example, heavy chain CDR-grafted antibodies were constructed by homology comparison between NCBI IgBlast and human IgG Germline sequences (Germinne), selecting IGHV 1-46X 01 as a heavy chain CDR-grafted template, and grafting the heavy chain CDR region of murine anti-human CD38 monoclonal antibody 105 into the IGHV 1-46X 01 framework region. Similarly, by comparing the sequence homology with human IgG germline, IGKV1-16 × 01 was selected as a light chain CDR-grafted template, and the light chain CDR region of mouse-derived anti-human CD38 monoclonal antibody No. 105 was grafted into the framework region of IGKV1-16 × 01 to construct a light chain CDR-grafted antibody. Meanwhile, on the basis, the amino acid sites of some framework regions are subjected to back mutation. In the case of back-mutation, the amino acid sequence is Kabat-encoded and the position of the site is indicated by the Kabat code. Preferably, for the light chain variable region sequence, kabat encodes reversion of F at position 36 to murine L, a mutation of a at position 43 to T, P at position 44 to murine I, S at position 46 to murine R, G at position 66 to murine R, and T at position 69 to murine S. For the heavy chain variable region sequence, kabat encoded M at position 48 reverted to murine I, V at position 67 reverted to murine A, M at position 69 reverted to murine L, R at position 71 reverted to murine A, T at position 73 reverted to murine K, and V at position 78 reverted to murine A. The above-mentioned variable region gene sequence was codon-optimized and synthesized by an organism according to the codon usage preference of Cricetulus griseus. The synthetic humanized variable region sequences were linked to human IgG1 constant regions, defined as the humanized antibody 105-Humanization, 105H) of antibody number 105.

The remaining antibodies were also humanized using the same principles described above. The amino acid sequence numbering of the variable region of the humanized antibody is shown in table 3, and the amino acid sequence of the heavy chain constant region is SEQ ID NO:113, the amino acid sequence of the light chain constant region is SEQ ID NO:114, respectively. Transient expression vectors of humanized heavy chains and light chains are respectively constructed by utilizing pTT5 vectors, and the light chains and the heavy chains are combined to carry out transient transfection by utilizing a HEK293E system and express antibodies. HEK293E cells in Free Style293 Expression Medium (purchased from Gibco company) Medium culture, using PEI transfection method to plasmid transfer into cells for 5 days after cell supernatant, using Protein A purification to obtain each humanized monoclonal antibody.

Finally, the amino acid sequence of the humanized heavy chain variable region of the No. 1 antibody is shown as SEQ ID NO:97 is shown; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO:98, respectively. After ligation to the human IgG1 constant region, the antibody sequence of 1H was finally obtained.

The amino acid sequence of the humanized heavy chain variable region of the No. 57 antibody is shown as SEQ ID NO: 99; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO: shown at 100. After ligation to the human IgG1 constant region, the antibody sequence of 57H was finally obtained.

The amino acid sequence of the humanized heavy chain variable region of the antibody No. 71 is shown as SEQ ID NO:101 is shown; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO:102, respectively. After ligation to the human IgG1 constant region, the antibody sequence of 71H was finally obtained.

The amino acid sequence of the humanized heavy chain variable region of the antibody No. 73 is shown as SEQ ID NO:103 is shown; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO:104, respectively. After ligation to the human IgG1 constant region, the antibody sequence of 73H was finally obtained.

The amino acid sequence of the heavy chain variable region of the humanized antibody No. 78 is shown as SEQ ID NO:105, respectively; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO:106, respectively. After ligation to the human IgG1 constant region, the antibody sequence of 78H was finally obtained.

The amino acid sequence of the humanized heavy chain variable region of the antibody No. 105 is shown as SEQ ID NO:107 is shown in the figure; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO: shown at 108. After ligation to the human IgG1 constant region, the 105H antibody sequence was finally obtained.

The amino acid sequence of the humanized heavy chain variable region of the antibody No. 116 is shown as SEQ ID NO:109 is shown in the specification; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO: 110. After ligation to the human IgG1 constant region, the antibody sequence of 116H was finally obtained.

The amino acid sequence of the humanized heavy chain variable region of the No. 145 antibody is shown as SEQ ID NO:111 is shown; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO:112, respectively. After ligation to the human IgG1 constant region, the 145H antibody sequence was finally obtained.

Table 3: amino acid sequences of exemplary humanized anti-human CD38 antibody heavy and light chain variable regions

Example 6Binding of humanized CD38 monoclonal antibody to Daudi cells

Sampling and counting Daudi cells (stored in laboratory) in good growth state, centrifuging, resuspending with PBS containing 1% FBS, and adjusting density to 2X 10 ⁷ . Antibody samples were loaded at a maximum concentration of 10. Mu.g/ml after dilution in three-fold gradients (8 gradients total). In 96-well culture plates, 20 ul/well (4X 10) was added ⁵ Per well) Daudi cells and 30 μ l of the antibody drug diluted in a gradient was added and incubated at 4 ℃ for 30 minutes. Add 180 u l 1% FBS PBS washing, 1200rpm centrifugation for 6min. After the supernatant was aspirated by a vacuum pump, 50. Mu.l of goat anti-human IgG-FITC (diluted with 1% FBS PBS 1, 1000) was added and incubated at 4 ℃ for 30 minutes. After adding 180. Mu.l of 1-cell FBS PBS, the mixture was centrifuged at 1200rpm for 6 minutes and then resuspended in 160. Mu.l of PBS. And finally, detecting the fluorescence intensity of the cell surface by using a flow cytometer. Data analysis was performed using GraphPad Prism6 software. The results are shown in FIG. 1 and Table 4.

Table 4: binding of humanized CD38 antibody to Daudi cells

Example 7Humanized CD38 antibody induces apoptosis in Daudi lymphoma cells

In an experimental system of 96-well culture plate 200. Mu.l/well, 1X 10 was added ⁵ Daudi cells/well, and 1.85 and 1.03. Mu.g/well of antibody drug, after the incubation for 24 hours, after incubationDirectly add 1:100 of PI (Propidium Iodid) solution (2. Mu.l), further incubation for 30 minutes, centrifugation to remove supernatant, washing with PBS, centrifugation to remove supernatant, then adding 160. Mu.l PBS to resuspend, using flow cytometry to complete the assay. Unstained wells were set as blank control and the reference antibody was Daratumumab (abbreviated Dara in fig. 2). The results in fig. 2 indicate that 73H showed stronger apoptosis-inducing activity than the reference antibody, and the remaining antibodies were identical to the reference antibody, and no significant apoptosis-inducing activity was seen.

Example 8anti-Fc crosslinking of humanized CD38 antibody induces apoptosis in Daudi lymphoma cells

In a 96-well culture plate 200. Mu.l/well experiment system, 1X 105Daudi cells/well and 1.85 and 1.03. Mu.g/well antibody drug were added and incubated in a cell incubator for 30 minutes. Then 1. Mu.g/Kong Kangren Fc antibody was added and incubation continued for 24 hours. The culture was carried out for 24 hours. Directly adding 1:100 μ l of PI solution (2 μ l), incubation for 30 min, centrifugation to remove supernatant, washing with PBS, centrifugation to remove supernatant, addition of 160 μ l PBS for resuspension, and flow cytometry to complete the assay. Unstained wells were set as blank controls and the reference antibody was Daratumumab (abbreviated Dara in fig. 3). The results in fig. 3 show that the 5 preferred antibodies have consistently stronger anti-Fc cross-linking induced apoptotic activity compared to the reference antibody.

Example 9Affinity assay for humanized CD38 monoclonal antibody

The affinity of the expressed purified humanized antibody was measured by Biacore T200 (GE healthcare). The specific experimental method comprises the following steps: a Protein-A CM5 sensor chip (GE healthcare) was used, FC1 (Flow cell 1) was used as a reference channel, and FC2 (Flow cell 2) was used as a sample channel. Human or control antibody was captured in the FC2 channel, respectively, followed by injection of different concentrations of hCD38-Flag or cyno-CD38-Flag. The circulation conditions are as follows: surface regeneration was performed by injecting 50. Mu.l/min of 4min analyte in all channels of FCs with a dissociation time of 20min, and 6M guanidine hydrochloride (national drug group chemical Co., ltd.) at a rate of 10. Mu.l/min for 30s, and then calculating the difference between the signal of captured antibody and the signal of no captured antibody and the affinity of the interaction using Biacore T200 Evaluation Software Ver 1.0. As shown in tables 5 and 6. The experimental results show that the preferred antibodies are both capable of binding to human and cynomolgus monkey CD38 with different affinities of high, medium and low.

Table 5: affinity of humanized CD38 antibodies for binding to human CD38

Antibody numbering	Ka(1/Ms)	Kd(1/s)	KD(nM)
				57H	2.641E+05	1.009E-04	0.382
105H	2.976E+04	9.748E-04	32.755
				116H	1.472E+06	9.530E-05	0.065
145H	6.125E+05	1.975E-03	3.224

Table 6: affinity of humanized CD38 antibody for binding to monkey CD38

Antibody numbering	Ka(1/Ms)	Kd(1/s)	KD(nM)
				57H	5.492E+04	9.201E-05	1.675
105H	6.113E+04	2.080E-04	3.402
				116H	2.513E+05	1.019E-04	0.405
145H	9.782E+04	3.930E-03	40.176

And (4) conclusion: the inventors of the present invention have conducted a number of experiments to obtain a group of monoclonal antibodies that specifically bind to cell surface CD38, and these antibodies can bind to cynomolgus monkey CD38 at the same time. A series of anti-human CD38 antibodies with different affinities of high, medium and low were obtained, which were able to kill tumor cells by Fc-dependent immune effector mechanisms.

It is to be understood that while the application is illustrated in certain forms, it is not limited to what has been shown and described herein. It will be apparent to those skilled in the art that various changes can be made in the embodiments and/or in a feature or parameter without departing from the scope of the application. Such variations are within the scope of the claims of this application.

SEQUENCE LISTING

<110> Shanghai economic Biotechnology Limited

<120> anti-human CD38 antibody, preparation method and application thereof

<130> anti-CD38

<160> 114

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Mus musculus

<400> 1

Ser Ser Trp Met His

1 5

<210> 2

<211> 14

<212> PRT

<213> Mus musculus

<400> 2

Glu Ile His Pro Asp Ser Gly Lys Thr Asn Tyr Asn Glu Lys

1 5 10

<210> 3

<211> 11

<212> PRT

<213> Mus musculus

<400> 3

Leu Ser Ser Pro Tyr Tyr Tyr Val Met Asp Tyr

1 5 10

<210> 4

<211> 5

<212> PRT

<213> Mus musculus

<400> 4

Asn Tyr Trp Ile Gln

1 5

<210> 5

<211> 17

<212> PRT

<213> Mus musculus

<400> 5

Glu Met Phe Pro Gly Thr Gly Lys Thr Tyr Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 6

<211> 10

<212> PRT

<213> Mus musculus

<400> 6

Ser Gly Tyr Asp Tyr Asp Gly Phe Thr Tyr

1 5 10

<210> 7

<211> 5

<212> PRT

<213> Mus musculus

<400> 7

Ser Phe Gly Met His

1 5

<210> 8

<211> 17

<212> PRT

<213> Mus musculus

<400> 8

Tyr Ile Gly Gly Asp Ser Asn Ile Ile Tyr Tyr Ala Asp Thr Val Lys

1 5 10 15

Gly

<210> 9

<211> 8

<212> PRT

<213> Mus musculus

<400> 9

Ser His Asp Tyr Thr Met Asp Tyr

1 5

<210> 10

<211> 5

<212> PRT

<213> Mus musculus

<400> 10

Ser Tyr Trp Ile Gln

1 5

<210> 11

<211> 17

<212> PRT

<213> Mus musculus

<400> 11

Glu Ile Phe Pro Gly Thr Gly Lys Thr Tyr Tyr Asn Gly Lys Phe Lys

1 5 10 15

Gly

<210> 12

<211> 10

<212> PRT

<213> Mus musculus

<400> 12

Ser Pro Tyr Asp Ile Val Gly Phe Asp Tyr

1 5 10

<210> 13

<211> 5

<212> PRT

<213> Mus musculus

<400> 13

Gly Tyr Asn Met Asn

1 5

<210> 14

<211> 17

<212> PRT

<213> Mus musculus

<400> 14

Asn Ile Asp Pro His Phe Gly Gly Thr Arg Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 15

<211> 10

<212> PRT

<213> Mus musculus

<400> 15

Gln His Gly Asn Trp Val Gly Phe Ala Tyr

1 5 10

<210> 16

<211> 5

<212> PRT

<213> Mus musculus

<400> 16

Lys Tyr Trp Ile Glu

1 5

<210> 17

<211> 14

<212> PRT

<213> Mus musculus

<400> 17

Glu Ile Ser Pro Gly Ile Asp Lys Thr Asn Tyr Asn Glu Lys

1 5 10

<210> 18

<211> 14

<212> PRT

<213> Mus musculus

<400> 18

Gly Glu Glu Tyr Gly Asn Ser Val Arg Tyr Ala Met Asp Tyr

1 5 10

<210> 19

<211> 5

<212> PRT

<213> Mus musculus

<400> 19

Gly Tyr Asn Met Asn

1 5

<210> 20

<211> 17

<212> PRT

<213> Mus musculus

<400> 20

Asn Ile Asp Pro Tyr Phe Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 21

<211> 10

<212> PRT

<213> Mus musculus

<400> 21

Gln Tyr Gly Asn Trp Val Gly Phe Ala Tyr

1 5 10

<210> 22

<211> 5

<212> PRT

<213> Mus musculus

<400> 22

Asp Arg Ala Val His

1 5

<210> 23

<211> 17

<212> PRT

<213> Mus musculus

<400> 23

Val Ile Ser Pro Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 24

<211> 10

<212> PRT

<213> Mus musculus

<400> 24

Val Asp Phe Phe Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 25

<211> 6

<212> PRT

<213> Mus musculus

<400> 25

Ser Gly Tyr Tyr Trp Asn

1 5

<210> 26

<211> 16

<212> PRT

<213> Mus musculus

<400> 26

Tyr Arg Ser Tyr Asp Gly Ser Tyr Asn Tyr Asn Pro Ser Leu Glu Asn

1 5 10 15

<210> 27

<211> 12

<212> PRT

<213> Mus musculus

<400> 27

Glu Glu Arg Tyr Ile Leu Arg Tyr Ala Met Asp Tyr

1 5 10

<210> 28

<211> 5

<212> PRT

<213> Mus musculus

<400> 28

Asp Arg Ala Val His

1 5

<210> 29

<211> 17

<212> PRT

<213> Mus musculus

<400> 29

Val Ile Ser Pro Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 30

<211> 10

<212> PRT

<213> Mus musculus

<400> 30

Val Asp Phe Tyr Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 31

<211> 5

<212> PRT

<213> Mus musculus

<400> 31

Ser Tyr Trp Met His

1 5

<210> 32

<211> 17

<212> PRT

<213> Mus musculus

<400> 32

His Ile Asn Pro Ser Ser Gly Tyr Ser Glu Tyr Ser Gln Lys Phe Lys

1 5 10 15

Asp

<210> 33

<211> 11

<212> PRT

<213> Mus musculus

<400> 33

Tyr Tyr Tyr Val Ser Arg Tyr Val Gly Asp Ser

1 5 10

<210> 34

<211> 7

<212> PRT

<213> Mus musculus

<400> 34

Thr Ser Arg Met Gly Val Gly

1 5

<210> 35

<211> 16

<212> PRT

<213> Mus musculus

<400> 35

His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser

1 5 10 15

<210> 36

<211> 11

<212> PRT

<213> Mus musculus

<400> 36

Ile Glu Val Gly Arg Gly Ala Trp Phe Ala Tyr

1 5 10

<210> 37

<211> 5

<212> PRT

<213> Mus musculus

<400> 37

Ser Tyr Trp Ile Gln

1 5

<210> 38

<211> 17

<212> PRT

<213> Mus musculus

<400> 38

Glu Ile Phe Pro Gly Thr Gly Lys Thr Tyr Tyr Asn Gly Lys Phe Lys

1 5 10 15

Gly

<210> 39

<211> 10

<212> PRT

<213> Mus musculus

<400> 39

Ser Pro Tyr Asp Ile Val Gly Phe Asp Tyr

1 5 10

<210> 40

<211> 5

<212> PRT

<213> Mus musculus

<400> 40

Ser Tyr Trp Ile Gln

1 5

<210> 41

<211> 17

<212> PRT

<213> Mus musculus

<400> 41

Glu Ile Phe Pro Gly Thr Gly Lys Thr Tyr Tyr Asn Gly Lys Phe Lys

1 5 10 15

Gly

<210> 42

<211> 10

<212> PRT

<213> Mus musculus

<400> 42

Ser Pro Tyr Asp Ile Val Gly Phe Asp Tyr

1 5 10

<210> 43

<211> 5

<212> PRT

<213> Mus musculus

<400> 43

Asn Tyr Leu Ile Asp

1 5

<210> 44

<211> 17

<212> PRT

<213> Mus musculus

<400> 44

Leu Ile Asn Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 45

<211> 6

<212> PRT

<213> Mus musculus

<400> 45

Thr Gly Thr Leu Pro Tyr

1 5

<210> 46

<211> 5

<212> PRT

<213> Mus musculus

<400> 46

Ser Tyr Trp Ile His

1 5

<210> 47

<211> 14

<212> PRT

<213> Mus musculus

<400> 47

Glu Ile Ser Pro Ser Asn Gly Arg Arg Asn Asn Asn Glu Lys

1 5 10

<210> 48

<211> 11

<212> PRT

<213> Mus musculus

<400> 48

Glu Gln Gln Gly Tyr Tyr Glu Gly Phe Ala Ser

1 5 10

<210> 49

<211> 16

<212> PRT

<213> Mus musculus

<400> 49

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 50

<211> 7

<212> PRT

<213> Mus musculus

<400> 50

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 51

<211> 9

<212> PRT

<213> Mus musculus

<400> 51

Ser Gln Ser Thr His Val Pro Tyr Thr

1 5

<210> 52

<211> 16

<212> PRT

<213> Mus musculus

<400> 52

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 53

<211> 7

<212> PRT

<213> Mus musculus

<400> 53

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 54

<211> 9

<212> PRT

<213> Mus musculus

<400> 54

Ser Gln Ser Ser His Val Pro Arg Thr

1 5

<210> 55

<211> 17

<212> PRT

<213> Mus musculus

<400> 55

Lys Ser Ser Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 56

<211> 7

<212> PRT

<213> Mus musculus

<400> 56

Trp Ala Ser Thr Arg Asp Ser

1 5

<210> 57

<211> 9

<212> PRT

<213> Mus musculus

<400> 57

Gln Arg Tyr Tyr Asn Tyr Pro Tyr Thr

1 5

<210> 58

<211> 16

<212> PRT

<213> Mus musculus

<400> 58

Arg Ser Ser Gln Ser Ile Val His Ile Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 59

<211> 7

<212> PRT

<213> Mus musculus

<400> 59

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 60

<211> 9

<212> PRT

<213> Mus musculus

<400> 60

Phe Gln Gly Ser His Val Pro Arg Thr

1 5

<210> 61

<211> 11

<212> PRT

<213> Mus musculus

<400> 61

Lys Ala Ser Gln Asp Val Phe Thr Ala Val Ala

1 5 10

<210> 62

<211> 7

<212> PRT

<213> Mus musculus

<400> 62

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210> 63

<211> 9

<212> PRT

<213> Mus musculus

<400> 63

Gln Gln His Tyr Phe Phe Pro Tyr Thr

1 5

<210> 64

<211> 10

<212> PRT

<213> Mus musculus

<400> 64

Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr

1 5 10

<210> 65

<211> 7

<212> PRT

<213> Mus musculus

<400> 65

Arg Thr Ser Asn Leu Ala Ser

1 5

<210> 66

<211> 10

<212> PRT

<213> Mus musculus

<400> 66

Gln Glu Arg Ser Ser Tyr Pro Pro Phe Thr

1 5 10

<210> 67

<211> 11

<212> PRT

<213> Mus musculus

<400> 67

Lys Ala Ser Gln Asp Val Thr Thr Ala Val Ala

1 5 10

<210> 68

<211> 7

<212> PRT

<213> Mus musculus

<400> 68

Ser Ala Ser Ser Arg Tyr Thr

1 5

<210> 69

<211> 9

<212> PRT

<213> Mus musculus

<400> 69

Gln Gln His Phe Phe Phe Pro Tyr Thr

1 5

<210> 70

<211> 16

<212> PRT

<213> Mus musculus

<400> 70

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 71

<211> 7

<212> PRT

<213> Mus musculus

<400> 71

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 72

<211> 9

<212> PRT

<213> Mus musculus

<400> 72

Ser Gln Ser Thr His Val Pro Pro Thr

1 5

<210> 73

<211> 11

<212> PRT

<213> Mus musculus

<400> 73

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 74

<211> 7

<212> PRT

<213> Mus musculus

<400> 74

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 75

<211> 9

<212> PRT

<213> Mus musculus

<400> 75

Gln Gln Gly Asn Thr Leu Pro Phe Thr

1 5

<210> 76

<211> 16

<212> PRT

<213> Mus musculus

<400> 76

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 77

<211> 7

<212> PRT

<213> Mus musculus

<400> 77

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 78

<211> 9

<212> PRT

<213> Mus musculus

<400> 78

Ser Gln Ser Thr His Val Pro Pro Thr

1 5

<210> 79

<211> 11

<212> PRT

<213> Mus musculus

<400> 79

Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser

1 5 10

<210> 80

<211> 7

<212> PRT

<213> Mus musculus

<400> 80

Ala Ala Ser Thr Leu Asp Ser

1 5

<210> 81

<211> 9

<212> PRT

<213> Mus musculus

<400> 81

Leu Gln Tyr Asp Ser Tyr Pro Leu Thr

1 5

<210> 82

<211> 11

<212> PRT

<213> Mus musculus

<400> 82

Lys Ala Ser Gln Asp Val Val Thr Thr Val Ala

1 5 10

<210> 83

<211> 7

<212> PRT

<213> Mus musculus

<400> 83

Ser Ala Ser Tyr Arg Tyr Ser

1 5

<210> 84

<211> 9

<212> PRT

<213> Mus musculus

<400> 84

Gln Gln Tyr Tyr Thr Tyr Pro Phe Thr

1 5

<210> 85

<211> 16

<212> PRT

<213> Mus musculus

<400> 85

Arg Ser Ser Gln Ser Ile Val His Ile Asn Gly Lys Thr Tyr Leu Glu

1 5 10 15

<210> 86

<211> 7

<212> PRT

<213> Mus musculus

<400> 86

Lys Ile Ser Asn Arg Phe Ser

1 5

<210> 87

<211> 9

<212> PRT

<213> Mus musculus

<400> 87

Phe Gln Gly Ser His Val Pro Arg Thr

1 5

<210> 88

<211> 16

<212> PRT

<213> Mus musculus

<400> 88

Arg Ser Ser Gln Ser Ile Val His Ile Asn Gly Lys Thr Tyr Leu Glu

1 5 10 15

<210> 89

<211> 6

<212> PRT

<213> Mus musculus

<400> 89

Lys Ile Ser Asn Arg Phe

1 5

<210> 90

<211> 9

<212> PRT

<213> Mus musculus

<400> 90

Phe Gln Gly Ser His Val Pro Arg Thr

1 5

<210> 91

<211> 11

<212> PRT

<213> Mus musculus

<400> 91

Lys Ala Ser Gln Asn Val Gly Thr Ser Val Ala

1 5 10

<210> 92

<211> 7

<212> PRT

<213> Mus musculus

<400> 92

Ser Thr Ser Tyr Arg Ser Ser

1 5

<210> 93

<211> 9

<212> PRT

<213> Mus musculus

<400> 93

Gln Gln Tyr Asn Ser Tyr Pro Leu Thr

1 5

<210> 94

<211> 11

<212> PRT

<213> Mus musculus

<400> 94

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 95

<211> 7

<212> PRT

<213> Mus musculus

<400> 95

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 96

<211> 9

<212> PRT

<213> Mus musculus

<400> 96

Gln Gln Gly Lys Thr Leu Pro Trp Thr

1 5

<210> 97

<211> 122

<212> PRT

<213> Synthetic

<400> 97

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 Ser Ser

20 25 30

Trp Met His Trp Ala Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile His Pro Asp Ser Gly Lys Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Leu Ser Ser Pro Tyr Tyr Tyr Val Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 98

<211> 112

<212> PRT

<213> Synthetic

<400> 98

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Gly Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 99

<211> 117

<212> PRT

<213> Synthetic

<400> 99

Glu 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 Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Gly Gly Asp Ser Asn Ile Ile Tyr Tyr Ala Asp Thr 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 Lys Ser His Asp Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 100

<211> 113

<212> PRT

<213> Synthetic

<400> 100

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Thr Asn Gln Lys Asn Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Arg

85 90 95

Tyr Tyr Asn Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 101

<211> 123

<212> PRT

<213> Synthetic

<400> 101

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 Lys Phe Thr Lys Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Ser Pro Gly Ile Asp Lys Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Arg Ala Thr Phe Thr Ala Asp Thr Ser Thr Asn 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 Gly Glu Glu Tyr Gly Asn Ser Val Arg Tyr Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 102

<211> 107

<212> PRT

<213> Synthetic

<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 Leu Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Glu Arg Ser Ser Tyr Pro Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 103

<211> 119

<212> PRT

<213> Synthetic

<400> 103

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 Gly Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Tyr Phe Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Gly Asn Trp Val Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 104

<211> 107

<212> PRT

<213> Synthetic

<400> 104

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 Gln Asp Val Thr Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Arg Tyr 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 His Phe Phe Phe Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 105

<211> 121

<212> PRT

<213> Synthetic

<400> 105

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Gly

20 25 30

Tyr Tyr Trp Asn Trp Phe Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp

35 40 45

Ile Gly Tyr Arg Ser Tyr Asp Gly Ser Tyr Asn Tyr Asn Pro Ser Leu

50 55 60

Glu Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Glu Arg Tyr Ile Leu Arg Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 106

<211> 107

<212> PRT

<213> Synthetic

<400> 106

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 107

<211> 120

<212> PRT

<213> Synthetic

<400> 107

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 Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly His Ile Asn Pro Ser Ser Gly Tyr Ser Glu Tyr Ser Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Glu Ile Tyr Tyr Tyr Val Ser Arg Tyr Val Gly Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 108

<211> 107

<212> PRT

<213> Synthetic

<400> 108

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Lys Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 109

<211> 121

<212> PRT

<213> Synthetic

<400> 109

Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Arg Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Lys Glu Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Gly Arg Ile Glu Val Gly Arg Gly Ala Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 110

<211> 107

<212> PRT

<213> Synthetic

<400> 110

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asp Val Val Thr Thr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Thr Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val 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 Tyr Tyr Thr Tyr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 111

<211> 115

<212> PRT

<213> Synthetic

<400> 111

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

Leu Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Thr Gly Thr Leu Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 112

<211> 107

<212> PRT

<213> Synthetic

<400> 112

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 Gln Asn Val Gly Thr Ser

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Thr Ser Tyr Arg Ser Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 113

<211> 330

<212> PRT

<213> Homo sapiens

<400> 113

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 Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser 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

Lys 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 Lys 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 Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 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> 114

<211> 107

<212> PRT

<213> Homo sapiens

<400> 114

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

Claims

1. An antibody, or antigen-binding portion thereof, that specifically binds human CD38,

the amino acid sequence of the HCDR1 of the antibody or antigen-binding portion thereof is set forth in SEQ ID NO:34, the amino acid sequence of HCDR2 is shown as SEQ ID NO:35, the amino acid sequence of HCDR3 is shown as SEQ ID NO:36, the amino acid sequence of LCDR1 is shown as SEQ ID NO:82, the amino acid sequence of LCDR2 is shown as SEQ ID NO:83, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: as shown at 84.

2. The antibody, or antigen-binding portion thereof, of claim 1, wherein:

the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO:109, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 110.

3. The antibody or antigen-binding portion thereof of any one of claims 1-2, wherein the antigen-binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab') 2 fragment, a Fv fragment, and a scFv fragment.

4. The antibody or antigen-binding portion thereof of any one of claims 1-2, wherein the antibody is a murine monoclonal antibody or a humanized antibody.

5. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 1-4 and a pharmaceutically acceptable carrier.

6. An expression vector comprising a nucleotide molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-4.

7. The expression vector of claim 6, wherein the expression vector is pTT5, pUC57, pDR1, pcDNA3.1 (+), pDFHF or pCHO 1.0.

8. A host cell comprising the antibody or antigen-binding portion thereof of any one of claims 1-4, or comprising the expression vector of claim 6 or 7.

9. The host cell of claim 8, wherein the host cell is HEK293, COS, CHO, NS0, sf9, sf21, DH5 α, BL21 (DE 3), or TG1.

10. A method of producing the antibody, or antigen-binding portion thereof, of any one of claims 1-4, comprising:

a) Culturing the host cell of claim 8 or 9 under expression conditions such that the host cell is capable of producing the antibody, or antigen-binding portion thereof, thereby expressing the antibody, or antigen-binding portion thereof; and

b) Isolating and purifying the antibody or antigen-binding portion thereof expressed in step a).

11. Use of the antibody or antigen-binding portion thereof of any one of claims 1-4 or the pharmaceutical composition of claim 5 in the manufacture of a medicament for preventing or treating a CD 38-associated disease.

12. The use according to claim 11, wherein said CD38 related disease is selected from the group consisting of multiple myeloma, non-hodgkin's lymphoma, AL-type amyloidosis, T-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, plasma cell leukemia, acute myelogenous leukemia, non-small cell lung cancer, pancreatic cancer, colorectal cancer and triple negative breast cancer.

13. The use according to claim 11, wherein said CD38 associated disease is selected from the group consisting of multiple myeloma, non-hodgkin's lymphoma, AL-type amyloidosis, T-cell lymphoma, and diffuse large B-cell lymphoma.