CN116143933A - Anti-human CD38 bispecific antibodies and uses thereof - Google Patents

Anti-human CD38 bispecific antibodies and uses thereof Download PDF

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CN116143933A
CN116143933A CN202111400194.0A CN202111400194A CN116143933A CN 116143933 A CN116143933 A CN 116143933A CN 202111400194 A CN202111400194 A CN 202111400194A CN 116143933 A CN116143933 A CN 116143933A
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万姝南
刘志刚
周晓巍
刘玉兰
胡俊杰
郝小勃
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Beijing Wisdomab Biotechnology Co ltd
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Abstract

The present application provides bispecific antibodies against human CD38 comprising two antigen binding fragments that bind different epitopes of human CD38, and uses thereof.

Description

Anti-human CD38 bispecific antibodies and uses thereof
Technical Field
The present invention relates generally to the fields of genetic engineering and antibody pharmaceuticals; in particular, the present application relates to bispecific antibodies against human CD38 and uses thereof.
Background
CD38 is a type II transmembrane protein with a molecular weight of 46KD and comprises a long C-terminal extracellular domain (258 Ammonias)Base acid), one transmembrane region (21 amino acids) and one short N-terminal (21 amino acids). The function of CD38 is manifested in two ways, firstly, the extracellular domain of CD38 is an extracellular enzyme with ADP-ribose cyclase, cyclic ADP-ribose hydrolase, NAD polysaccharide hydrolase activity, involving the formation and hydrolysis of cADPR. When CD38 is present as an ADP ribosyl cyclase, nicotinamide adenine dinucleotide (NAD+) can be converted to nicotinamide, adenosine diphosphate ribose (ADPR), and NAD+ can be cyclized to cyclic adenosine diphosphate ribose (cADPR) [1] . This activity of CD38 is important to maintain homeostasis of NAD, nicotinamide, etc. substances in the body. The substrate of CD38 is mainly beta-NAD and beta-NAD derivatives (such as beta-NMN), but has no catalytic activity on alpha-NAD, NADH and other substances. The products of the catalytic reaction, such as ADPR, cADPR and the like, are taken as important second messengers in cells, and can influence the functions of cell growth, insulin release, T cell activation and the like by regulating and controlling the calcium ions of the cells [2] . ADPR/cADPR produced by CD38 can be continuously produced into adenosine under the action of enzymes such as CD203, CD73 and the like, and the adenosine is a small molecule metabolite with an immunosuppressive function.
In addition, CD38 mediates transmembrane signaling. CD38 acts with ligands to induce transcription of interleukin 1 (IL-1), tumor necrosis factor alpha (TNF alpha) and granulocyte-macrophage colony-stimulating factor (GMCSF) at levels similar to the effect of stimulating the T cell receptor CD 3. CD38 initiates signaling in the Jurkat cell line resulting in the phosphorylation of a series of discrete proteins, the product of protooncogene p120c-cbl being the predominant protein, in which its tyrosine is phosphorylated. CD38 can also signal through T Cell Receptor (TCR) and participate in cellular responses, initiating the switched secretion of immune proteins [3] . Binding of CD38 to the ligand inhibits the growth of immature B lymphocytes cultured in the bone marrow microenvironment.
Expression of CD38 is associated with a variety of diseases, such as HIV infection [4] Systemic lupus erythematosus [5] And tumors. Up-regulation of CD38 expression in various hematological tumors, such as Multiple Myeloma (MM) [6] Acute Lymphoblastic Leukemia (ALL) [7] Chronic Lymphocytic Leukemia (CLL) [8] Etc.Current research on CD38 and tumors basically determines that CD38 has important functions in promoting tumor cell growth and immune escape. The relationship of CD38 to tumor can be summarized essentially in two points:
CD38 is expressed in high quantity in non-solid tumors, especially Multiple Myeloma (MM), and is inversely related to prognosis of the multiple myeloma, so that the CD38 becomes one of good targets for targeting treatment of the non-solid tumors such as multiple myeloma, CLL and the like. Multiple myeloma is a malignant blood tumor caused by abnormal proliferation of plasma cells, and has a proportion of about 10% in blood tumor and about 1% in all tumors [9] . And (3) statistical display: in 2012, there were 114,252 new cases and 80,119 deaths worldwide [10] . The incidence of multiple myeloma has a certain race variability, particularly the incidence in east Asia is lower than that in Europe and America, and the statistics of GLOBOCAN 2012 show that the incidence in America is 3.6/10 ten thousand and the incidence in China is 0.6/10 ten thousand [11] 。
In addition, CD38 is also present in higher expression in certain solid tumors. Because the CD38 and the subsequent enzymes such as CD203, CD73 and the like act together to generate the molecule with the immunosuppressive activity, the CD38 plays an important immunosuppressive function in the tumor microenvironment of the solid tumor, so that the solid tumor treatment targeting the CD38 is expected to relieve the inhibition of the CD38 on immune cells, and the immunotherapy can play a better role.
At the end of 2015, the FDA approved the predatory CD 38-targeting antibody drug Darzalex for multiple myeloma patients who had received at least 3 therapies (four-line therapy). Darzalex is the first CD38 monoclonal antibody worldwide to treat multiple myeloma. Darzalex can kill CD38 by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated phagocytosis (ADCP), and like mechanisms + Myeloma cells [12] . 3 months 2020, U.S. FDA announced approval of the CD38 antibody, sarclisa (isatuximab-irfc), developed by Sanofi, inc., for use in combination with pomalidomide and dexamethasone (dexamethane) in the treatment of adult multiple myelomaAnd (3) a patient. These patients received at least two prior therapies, including lenalidomide and a proteasome inhibitor.
Bispecific antibodies (bispecific antibody, bsAb) are a class of artificial antibodies that contain two different antigen binding sites that can bridge two cell types, or bind two molecules of a cell and two epitopes of a molecule, producing functions that are different from or stronger than monoclonal antibodies, have now become research hotspots in the field of antibody engineering, with broad application prospects in immunotherapy of disease.
The development and use of novel anti-human CD38 bispecific antibodies is desirable in the art. Therefore, in view of the wide applicability of anti-human CD38 bispecific antibodies, the discovery and development of new anti-human CD38 bispecific antibodies based on clinical needs is of great biological and medical significance.
Summary of The Invention
In a first aspect, the present application provides bispecific antibodies comprising a first antigen-binding fragment and a second antigen-binding fragment that bind different epitopes of human CD 38.
In some embodiments of the first aspect, the first antigen binding fragment comprises:
HCDR1 as shown in SEQ ID NO. 23,
HCDR2 as shown in SEQ ID NO. 24,
HCDR3 as shown in SEQ ID NO. 25,
LCDR1 as shown in SEQ ID NO. 26,
LCDR2 as shown in SEQ ID NO 27, and
LCDR3 as shown in SEQ ID NO. 28;
wherein the amino acid sequences of HCDR and LCDR are defined according to Kabat.
In some embodiments of the first aspect, the second antigen binding fragment comprises:
HCDR1 as shown in SEQ ID NO. 29,
HCDR2 as shown in SEQ ID NO. 30,
HCDR3 as shown in SEQ ID NO. 31,
LCDR1 as shown in SEQ ID NO. 32,
LCDR2 as shown in SEQ ID NO 33, and
LCDR3 as shown in SEQ ID NO. 34;
Wherein the amino acid sequences of HCDR and LCDR are defined according to Kabat.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen binding fragment is set forth in SEQ ID NO. 10 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 11.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen binding fragment is set forth in SEQ ID NO. 12 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 13.
In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are independently selected from a single chain antibody (scFv) or Fab fragment.
In some embodiments of the first aspect, the first antigen-binding fragment is a single chain antibody (ScFv) and the second antigen-binding fragment is a Fab fragment.
In some embodiments of the first aspect, the first antigen-binding fragment is a Fab fragment and the second antigen-binding fragment is a CH1CL substituted Fab fragment.
In a second aspect, the present application provides a nucleic acid molecule encoding the bispecific antibody of the first aspect.
In a third aspect, the present application provides a pharmaceutical composition comprising a bispecific antibody according to the first aspect and a pharmaceutically acceptable excipient, diluent or carrier.
In a fourth aspect, the present application provides the use of a bispecific antibody according to the first aspect, a nucleic acid molecule according to the second aspect or a pharmaceutical composition according to the third aspect for the manufacture of a medicament for the prevention or treatment of a CD38 mediated disease.
Brief Description of Drawings
Figure 1 shows the binding capacity of ELISA assays for recombinant anti-hCD 38 antibodies to different species of CD 38.
FIG. 2 shows the evaluation of ADCC activity of recombinant anti-hCD 38 antibodies in the jurkat-dual-CD16a reporter system.
Figure 3 shows CDC activity of recombinant anti-hCD 38 antibodies.
FIG. 4 shows the inhibition of tumors by anti-hCD 38 antibodies in a model of NOD/SCID mice transplanted with human multiple myeloma cells RPMI 8226.
DESCRIPTION OF THE SEQUENCES
SEQ ID NO. 1 shows the amino acid sequence of the extracellular region (hCD 38) of human (homo sapiens) CD 38.
SEQ ID NO. 2 shows the amino acid sequence of the extracellular domain of cynomolgus monkey (Macaca fascicularis) CD38 (mfCD 38).
SEQ ID NO. 3 shows the amino acid sequence of the His tag (His).
SEQ ID NO. 4 shows the amino acid sequence of the Fc segment (Fc) of human antibody IgG 1.
SEQ ID NO. 5 shows the amino acid sequence of the human IgG1 subtype heavy chain constant region.
SEQ ID NO. 6 shows the amino acid sequence of the human IgG2 subtype heavy chain constant region.
SEQ ID NO. 7 shows the amino acid sequence of the human IgG4 subtype heavy chain constant region.
SEQ ID NO. 8 shows the amino acid sequence of the human kappa subtype light chain constant region.
SEQ ID NO. 9 shows the amino acid sequence of the human lambda subtype light chain constant region.
SEQ ID NO. 10 shows the amino acid sequence of the S5B8 heavy chain humanized S5B8VH-h2 variable region, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO. 23-25, respectively.
SEQ ID NO. 11 shows the amino acid sequence of the S5B8 light chain humanized S5B8VK-h2 variable region, with the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO. 26-28, respectively.
SEQ ID NO. 12 shows the amino acid sequence of the S5A10 heavy chain humanized S5A10VH-h1 variable region, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO. 29-31, respectively.
SEQ ID NO. 13 shows the amino acid sequence of the S5A10 light chain humanized S5A10VK-h1 variable region, with the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO. 32-34, respectively.
SEQ ID NO. 14 shows the amino acid sequence of the heavy chain variable region of the anti-CD 38 monoclonal antibody darimumab.
SEQ ID NO. 15 shows the amino acid sequence of the light chain variable region of the anti-CD 38 monoclonal antibody darimumab.
SEQ ID NO. 16 shows the amino acid sequence of S5B8VH-h 2-scFv-FcH.
SEQ ID NO. 17 shows the amino acid sequence of S5A10VH-h1-CH-IgG 1K.
SEQ ID NO. 18 shows the amino acid sequence of S5A10VK-h 1-CK.
SEQ ID NO. 19 shows the amino acid sequence of S5B8VH-H2-IgG1H 1.
SEQ ID NO. 20 shows the amino acid sequence of S5B8VK-h 2-CK.
SEQ ID NO. 21 shows the amino acid sequence of S5A10VH-h 1-CK-cross-FcK.
SEQ ID NO. 22 shows the amino acid sequence of S5A10VK-h1-CH 1.
Detailed Description
The inventors of the present application have intensively developed antibody drugs binding to human CD38 and have obtained novel bispecific antibodies binding to human CD38 by antibody engineering techniques. In various aspects of the present application, novel bispecific antibodies that bind to human CD38, nucleic acid molecules encoding the bispecific antibodies, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, methods of making and purifying the bispecific antibodies, and medical and biological uses of the bispecific antibodies are provided. According to the sequences of the variable regions of the bispecific antibodies provided herein, full-length bispecific antibody molecules can be constructed as medicaments for clinical use in the prevention or treatment of CD38 mediated diseases.
The practice of the present application employs, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry and immunology techniques which are conventional in the art.
Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.
Definition of the definition
The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. "antibody" as used herein includes not only whole (i.e., full length) antibodies, but also antigen binding fragments thereof (e.g., fab ', F (ab') 2 Fv), variants thereof, fusion proteins comprising an antibody moiety, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, VHH antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of immunoglobulin molecules comprising an antigen recognition site of a desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
Typically, an intact or full length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (VH) and first, second and third constant regions (CH 1, CH2 and CH 3). Each light chain contains a light chain variable region (VL) and a constant region (CL). The full length antibody may be any kind of antibody, such as IgD, igE, igG, igA or IgM (or subclasses thereof described above), but the antibody need not be of any particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant region of the heavy chain. In general, immunoglobulins have five main classes IgA, igD, igE, igG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
"bispecific antibody" refers to an antibody that has both epitope binding capabilities. The two epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have a variety of structural configurations. For example, a bispecific antibody may consist of two Fc fragments and two binding moieties fused to each (similar to a natural antibody, except that the two arms bind different antigen targets or epitopes), the antigen binding moiety may be in the form of a single chain antibody (scfv) or Fab fragment. Two different binding sites of bispecific antibodies each bind to the N-terminus of one Fc fragment, and the two arms antigen binding site configuration may have four combinations of scfv + Fab fragment, fab fragment + scfv, scfv + scfv, fab fragment + Fab fragment. The Fc fragment may contain mutations that ensure heavy chain heteromerisation, a strategy to address heavy chain heteromerisation, is KIH technology (knob-in-hole, KIH). In general, KIH refers to a technology in which the amino acid sequence of the CH3 region is modified to form a structure that facilitates pairing of heterogeneous half antibodies, and the structure of a normal antibody can be maintained as much as possible while a bispecific antibody is formed. For guidance on KIH techniques, see, for example, "An efficient route to human bispecific IgG", a. Margaret Merchant et al, nature Biotechnology, volume 16,1998, which is incorporated herein by reference in its entirety.
The term "binding moiety" or "antigen binding fragment" as used herein is used interchangeably to refer to a portion or region of an intact antibody molecule responsible for binding an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3.
It is well known to those skilled in the art that complementarity determining regions (CDRs, typically CDR1, CDR2 and CDR 3) are regions of the variable region that have the greatest influence 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 the Chothia definition (see, e.g., kabat, "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, the CDR region sequences in VH and VL sequences may be determined according to the Kabat definition or Chothia definition. In embodiments of the present application, CDR sequences are defined using Kabat.
For a given antibody variable region sequence, the CDR region sequences in the variable region sequence can be analyzed in a number of ways, for example, as determined using on-line software Abysis (http:// www.abysis.org /).
For a typical antibody, examples of antigen-antigen binding fragments include, but are not limited to, (1) Fab fragments, which can be monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) F (ab') 2 A fragment, which may be a bivalent fragment having two Fab' fragments linked by a disulfide bridge of the hinge region (i.e., a dimer of Fab); (3) Fv fragments having VL and VH domains of a single arm of an antibody; (4) A single chain Fv (scFv), which may be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; (5) (scFv) 2 It may comprise two VH domains connected by a peptide linker and two VL domains combined with the two VH domains via a disulfide bridge.
In bispecific antibody construction, "binding moiety" includes, but is not limited to, the form of a Fab fragment or the form of a single chain antibody (scFv).
The term "single chain antibody (scFv, single chain fragment variable)" as used herein refers to an antibody of single chain structure, typically constructed using genetic engineering techniques, comprising one polypeptide chain of a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker peptide (linker) is typically designed between the heavy and light chain variable regions so that the heavy and light chain variable regions can fold into the correct conformation to be able to bind antigen.
The terms "Fab (fragment antigen binding) fragment", "Fab portion" or similar terms as used herein refer to an antibody fragment capable of binding to an antigen produced by treatment of an intact antibody with papain, including the intact light chain (VL-CL), heavy chain variable region, and CH1 fragment (VH-CH 1).
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein include, inter alia, "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 (U.S. Pat. No. 4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
The term "tumor" as used herein refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors may be benign, premalignant or malignant.
The term "malignancy" as used herein refers to or describes a physiological condition of a mammal that is typically characterized by unregulated cell growth. Exemplary malignancies include: carcinomas, solid tumors, melanoma sarcomas, hematological tumors, germ cell tumors, and blastomas. More specific examples of malignancy include: multiple myeloma, kidney cancer, lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, liver cancer (hepatic carcinoma), stomach cancer including gastrointestinal cancer, prostate cancer, pancreatic cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, ovarian cancer, liver cancer (liver cancer), urinary tract cancer, hepatoma, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous cell carcinoma), vulval cancer, thyroid cancer, anal cancer, penile cancer, melanoma and B-cell lymphoma, brain cancer and head and neck cancer, and related metastases.
The term "hematological neoplasm" as used herein refers to the fact that the site of origin of abnormal cells is in most cases bone marrow, which is also where blood cells are produced, due to uncontrolled growth and proliferation of such abnormal cells. Exemplary hematological tumors include various types of leukemia, multiple myeloma, and malignant lymphoma. More specific examples of hematological neoplasms include: acute onset of diseaseLymphocytic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), hairy Cell Leukemia (HCL), T cell prolymphocytic leukemia, large granule lymphocytic leukemia, juvenile granulo-monocytic leukemia, B cell prolymphocytic leukemia, burkitt's leukemia and adult T cell leukemia, non-Hodgkin's lymphoma, B cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytoid lymphoma, primary macroglobulinemia
Figure BDA0003364543120000101
macrolobulinema), splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, intranodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, burkitt lymphoma, B-cell chronic lymphocytic lymphoma, classical hodgkin's lymphoma, nodular lymphomas are primary hodgkin's lymphoma, adult T-cell lymphoma, extranodal nasal NK/T-cell lymphoma, enteropathic T-cell lymphoma, hepatic splenic T-cell lymphoma, primary skin CD30 positive T-cell lymphoproliferative disease, primary skin anaplastic large cell lymphoma, lymphomatoid eruption, angioimmunoblastic T-cell lymphoma, non-papular peripheral T-cell lymphoma, and anaplastic T-cell lymphoma.
The term "solid tumor" as used herein refers to a tangible tumor that can be reached through clinical examinations such as radiographs, CT scans, B-mode or palpation. Clinically diagnosed solid tumors are classified into malignant and benign. Malignant solid tumors include: hodgkin lymphoma in children: lymphocyte is main type, nodulizing type, mixed cell type and lymphocyte reducing type; non-hodgkin lymphoma in children: pre-lymphoblastic lymphoma, small, non-split cell lymphoma (burkitt/non-burkitt lymphoma), diffuse large B cell lymphoma, anaplastic large cell lymphoma, and the like; renal tumor in children: wilms' tumor, renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primary neuroectoblastoma, etc.; childhood neuroblastoma: neuroblastoma, gangliocytoma, gangliocytoneuroma; children extracranial germ cytoma: mature teratomas, immature teratomas, endoplasmic sinus tumors (yolk sac tumors), seminomas, asexual cytomas, chorionic epithelial cancers, embryonal cancers, etc.; osteosarcoma and chondrosarcoma; rhabdomyosarcoma in children: embryo type, acinus type, polymorphic type, etc.; soft tissue sarcoma in children: fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwannoma, acinar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, synovial sarcoma, and fibroproliferative microcytoma; you Wenshi family sarcoma: ewing's sarcoma, primary neuroectoblastoma; liver tumor in children: hepatoblastomas (embryonic, fetal, undifferentiated), hepatocellular carcinoma; retinoblastoma; other tumors: posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecal carcinoma, mesothelioma, and the like. Benign solid tumors include: lymphangioma, hemangioma, ungulate lingual cyst, etc.
In a first aspect, the present application provides bispecific antibodies comprising a first antigen-binding fragment and a second antigen-binding fragment that bind different epitopes of human CD 38.
In some embodiments of the first aspect, the first antigen binding fragment comprises:
HCDR1 as shown in SEQ ID NO. 23,
HCDR2 as shown in SEQ ID NO. 24,
HCDR3 as shown in SEQ ID NO. 25,
LCDR1 as shown in SEQ ID NO. 26,
LCDR2 as shown in SEQ ID NO 27, and
LCDR3 as shown in SEQ ID NO. 28;
wherein the amino acid sequences of HCDR and LCDR are defined according to Kabat.
In some embodiments of the first aspect, the second antigen binding fragment comprises:
HCDR1 as shown in SEQ ID NO. 29,
HCDR2 as shown in SEQ ID NO. 30,
HCDR3 as shown in SEQ ID NO. 31,
LCDR1 as shown in SEQ ID NO. 32,
LCDR2 as shown in SEQ ID NO 33, and
LCDR3 as shown in SEQ ID NO. 34;
wherein the amino acid sequences of HCDR and LCDR are defined according to Kabat.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen binding fragment is set forth in SEQ ID NO. 10 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 11.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen binding fragment differs from the amino acid sequence set forth in SEQ ID NO. 10 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.
In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the first antigen binding fragment differs from the amino acid sequence set forth in SEQ ID NO. 11 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID No. 10.
In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the first antigen binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID No. 11.
In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO. 10 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functions of the heavy chain variable region of the first antigen binding fragment.
In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added at the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO. 10, the resulting amino acid sequence still retaining similar functions of the heavy chain variable region of the first antigen binding fragment.
In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-or N-terminus of the amino acid sequence shown in SEQ ID NO. 10, provided that the altered amino acid sequence substantially retains similar function of the heavy chain variable region of the first antigen binding fragment.
In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO. 11 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functions of the light chain variable region of the first antigen binding fragment.
In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added at the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO. 11, the resulting amino acid sequence still retaining similar functions of the light chain variable region of the first antigen binding fragment.
In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-or N-terminus of the amino acid sequence shown in SEQ ID NO. 11, provided that the altered amino acid sequence substantially retains similar function of the light chain variable region of the first antigen binding fragment.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen binding fragment is set forth in SEQ ID NO. 12 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 13.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen binding fragment differs from the amino acid sequence set forth in SEQ ID NO. 12 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.
In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen binding fragment differs from the amino acid sequence set forth in SEQ ID NO. 13 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID No. 12.
In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID No. 13.
In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO. 12 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functions of the heavy chain variable region of the second antigen binding fragment.
In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added at the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO. 12, the resulting amino acid sequence still retaining similar functions of the heavy chain variable region of the second antigen binding fragment.
In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-or N-terminus of the amino acid sequence shown in SEQ ID NO. 12, provided that the altered amino acid sequence substantially retains similar function of the heavy chain variable region of the second antigen binding fragment.
In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO. 13 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functions of the light chain variable region of the second antigen binding fragment.
In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added at the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO. 13, the resulting amino acid sequence still retaining similar functions of the light chain variable region of the second antigen binding fragment.
In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-or N-terminus of the amino acid sequence shown in SEQ ID NO. 13, provided that the altered amino acid sequence substantially retains similar function of the light chain variable region of the second antigen binding fragment.
In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are independently selected from a single chain antibody (scFv) or Fab fragment.
In some specific embodiments of the first aspect, the first antigen-binding fragment is a single chain antibody (ScFv) and the second antigen-binding fragment is a Fab fragment.
In some embodiments of the first aspect, the bispecific antibody comprises the amino acid sequence of SEQ ID No. 16, the amino acid sequence of SEQ ID No. 17 and the amino acid sequence of SEQ ID No. 18.
In some specific embodiments of the first aspect, the first antigen-binding fragment is a Fab fragment and the second antigen-binding fragment is a CH1CL substituted Fab fragment.
In some embodiments of the first aspect, the bispecific antibody comprises the amino acid sequence of SEQ ID NO. 19, the amino acid sequence of SEQ ID NO. 20, the amino acid sequence of SEQ ID NO. 21 and the amino acid sequence of SEQ ID NO. 22.
In some embodiments of the first aspect, the bispecific antibody has Complement Dependent Cytotoxicity (CDC).
In a second aspect, the present application provides a nucleic acid molecule encoding the bispecific antibody of the first aspect.
In some embodiments of the second aspect, the nucleic acid molecule is operably linked to regulatory sequences that are recognized by a host cell transformed with the vector.
In a third aspect, the present application provides a pharmaceutical composition comprising a bispecific antibody according to the first aspect and a pharmaceutically acceptable excipient, diluent or carrier.
In some embodiments of the third aspect, the pharmaceutical composition is for use in preventing or treating a CD38 mediated disease.
In some embodiments of the third aspect, the CD38 mediated disease is a tumor.
In some embodiments of the third aspect, the CD38 mediated disease is a malignancy, such as multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia.
In some embodiments of the third aspect, the pharmaceutical composition may further comprise one or more of a lubricant such as talc, magnesium stearate and mineral oil; a wetting agent; an emulsifying agent; a suspending agent; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, etc.
In some embodiments of the third aspect, the pharmaceutical compositions herein may be formulated in the form of tablets, pills, powders, troches, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules and the like.
In some embodiments of the third aspect, the pharmaceutical compositions of the present application may be delivered using any physiologically acceptable mode of administration, including, but not limited to, oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
In some embodiments of the third aspect, the pharmaceutical composition for therapeutic use may be formulated for storage in the form of a lyophilized formulation or an aqueous solution by mixing an agent of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or the like.
In a fourth aspect, the present application provides the use of a bispecific antibody according to the first aspect, a nucleic acid molecule according to the second aspect, a pharmaceutical composition according to the third aspect for the manufacture of a medicament for the prevention or treatment of a CD38 mediated disease.
In some embodiments of the fourth aspect, the CD38 mediated disease is a tumor.
In some embodiments of the fourth aspect, the CD38 mediated disease is a malignancy, such as multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia.
In a fifth aspect, the present application provides a method of preventing or treating a CD38 mediated disease comprising administering to a subject in need thereof a bispecific antibody according to the first aspect or a pharmaceutical composition according to the third aspect.
In some embodiments of the fifth aspect, the CD38 mediated disease is a tumor.
In some embodiments of the fifth aspect, the CD38 mediated disease is a malignancy, such as multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia.
In other aspects, the application also provides a vector comprising an isolated nucleic acid molecule encoding the bispecific antibody of the first aspect and a host cell comprising the nucleic acid molecule or vector. In other aspects, the present application also provides a method of producing a bispecific antibody of the first aspect. In some embodiments, the method of producing a bispecific antibody of the first aspect comprises culturing a host cell so that the nucleic acid molecule is expressed. In some embodiments, the method of producing a bispecific antibody of the first aspect further comprises recovering the bispecific antibody from the host cell culture medium.
It should be understood that the foregoing detailed description is only for the purpose of making the contents of the present application more clearly apparent to those skilled in the art, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will occur to those skilled in the art.
The following examples are for the purpose of illustration only and are not intended to limit the scope of the present application.
Examples
EXAMPLE 1 preparation of recombinant proteins
A number of different recombinant proteins are required for the preparation of anti-CD 38 antibodies, including the human CD38 extracellular domain (hCD 38, SEQ ID NO: 1), and the cynomolgus CD38 extracellular domain (mfCD 38, SEQ ID NO: 2). These proteins all have a number of post-translational modifications (e.g., glycosylation or disulfide bonds, etc.), and thus the use of mammalian cell expression systems would be more advantageous in maintaining the structure and function of the recombinant protein. In addition, the addition of His tag (His, SEQ ID: 3) or Fc segment (Fc, SEQ ID NO: 4) of human antibody IgG1 at the C-terminal of these recombinant proteins would be more advantageous for purification of recombinant proteins and identification of monoclonal antibody functions. The antibody heavy chain constant region may be of the human IgG1 subtype (SEQ ID NO: 5), the human IgG2 subtype (SEQ ID NO: 6), the human IgG4 subtype (SEQ ID NO: 7), and the light chain constant region may be of the human kappa subtype (SEQ ID NO: 8), the human lambda subtype (SEQ ID NO: 9).
Genes (coding genes containing His tag or Fc) of the above recombinant proteins were designed and synthesized based on the amino acid sequences of the recombinant proteins of various purposes of Uniprot database. The synthesized recombinant protein genes are cloned into proper eukaryotic expression vectors (such as pcDNA3.1 of the company of invitrogen) by using conventional molecular biology technology, and then the prepared recombinant protein expression plasmids are transfected into HEK293 cells (such as HEK293F of the company of invitrogen) by using liposomes (such as 293fectin of the company of invitrogen) or other cationic transfection reagents (such as PEI and the like), and are cultured for 3-4 days under serum-free suspension culture conditions. The culture supernatant is then harvested by centrifugation or the like.
The recombinant protein expressed by His tag fusion is purified in one step by using metal chelating affinity chromatography column (such as HisTrap FF of GE company). The Fc fusion expressed recombinant protein was further purified using a ProteinA/G affinity column (e.g., mabSelect SURE, GE, etc.). The recombinant protein preservation buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (e.g., GE company Hitrap desaulting, etc.). If necessary, the recombinant protein samples may be subjected to filtration sterilization and then sub-packaged for storage at-20 ℃.
EXAMPLE 2 preparation of recombinant anti-human CD38 bispecific and monoclonal antibodies
Referring to example 1, igG1 subtype whole antibodies of monoclonal antibody S5B8h2 (the amino acid sequence of heavy chain variable region S5B8VH-h2 is shown as SEQ ID NO:10, the amino acid sequence of light chain variable region S5B8VK-h2 is shown as SEQ ID NO: 11) and S5A10h1 (the amino acid sequence of heavy chain variable region S5A10VH-h1 is shown as SEQ ID NO:12, the amino acid sequence of light chain variable region S5A10VK-h1 is shown as SEQ ID NO: 13) and control antibody darimumab (the amino acid sequence of heavy chain variable region is shown as SEQ ID NO:14, the amino acid sequence of light chain variable region is shown as SEQ ID NO: 15) in Chinese patent application No. 201810615165.8 were prepared. Bispecific antibodies consisting of monoclonal antibodies S5B8h2 and S5A10h1 based on different CD38 binding epitopes were prepared simultaneously.
To investigate the differences between bispecific antibody activities comparing different structures, bispecific antibodies based on the Fab-FcK/scFv-FcH structure were constructed on the basis of the "Knob-into-hole" technique. Specifically, a bispecific antibody S5A10h1Fab+S5B8h2ScFv was formed by fusing S5A10h1-Fab to the N-terminus of Fc containing Knob mutation and S5B8h2-scFv to the N-terminus of Fc containing Hole mutation, and the three chains were S5B8VH-h2-scFv-FcH1 (SEQ ID NO: 16), S5A10VH-h1-CH-IgG1K (SEQ ID NO: 17) and S5A10VK-h1-CK (SEQ ID NO: 18), respectively. Meanwhile, on the basis of the Knob-into-hole and the cross mab technology, a bispecific antibody S5A10H1cross+S5B8h2 based on the structure of Fab-FcK/Fab-cross mab-FcH is constructed, specifically, S5B8H2-Fab is fused at the N end of Fc containing hole mutation, CH1 and CK sequences of S5A10H1-Fab are replaced, and then fused at the N end of Fc containing Knob mutation, and four chains forming the bispecific antibody are respectively S5B8VH-H2-IgG1H1 (SEQ ID NO: 19), S5B8VK-H2-CK (SEQ ID NO: 20), S5A10VH-H1-CK-cross-FcK (SEQ ID NO: 21) and S5A10VK-H1-CH1 (SEQ ID NO: 22).
EXAMPLE 3 protein level identification of recombinant anti-human CD38 bispecific and monoclonal antibodies
1. Affinity analysis of recombinant anti-human CD38 bispecific and monoclonal antibodies
Affinity was determined by surface plasmon resonance technique using Biacore X100. Amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), CM5 chip ((BR 100530) and pH7.4 10 XHBS-EP (BR 100669) related reagents and consumables were purchased from GE healthcare, according to the instructions in the kit, an amino coupling method was used to couple an anti-human Fc antibody (capture antibody) to the CM5 chip surface, then the recombinant anti-human CD38 antibody was diluted to a suitable concentration, ensuring that about 50-100RU of the recombinant anti-human CD38 antibody was captured by the anti-human Fc antibody, the human CD38-His was set to a series of concentration gradients (100 nM, 33nM, 11nM, 3.7nM and 1.23 nM) across the stationary phase surface, and MgCl 3M was used 2 The chip surface was regenerated and the affinity of each recombinant anti-human CD38 antibody was determined at 25 ℃. Biacore data was analyzed using Biacore X100 evaluation software version 2.0.1 and the fit results are shown in Table 1.
TABLE 1 affinity constants of different recombinant anti-human CD38 bispecific antibodies and monoclonal antibodies for binding to human CD38
Ka Kd KD
S5A10h1Fab+S5B8h2ScFv 1.496E+6 2.266E-4 1.514E-10
S5A10h1 1.291E+6 1.724E-3 1.335E-9
S5B8h2 1.111E+6 2.367E-5 2.131E-11
Darifenacin 8.665E+5 2.829E-3 3.265E-9
S5A10h1cross+S5B8h2 1.434E+6 3.102E-5 2.164E-11
2. Binding of recombinant anti-human CD38 bispecific antibodies and monoclonal antibodies to different species of CD38
The prepared hCD38-His and mfCD38-His were coated on 96-well ELISA plates (1.5. Mu.g/ml, 100. Mu.l/well) respectively, and at 4℃overnight. Benefit (benefit)The coated 96-well ELISA plates were blocked with blocking solution (2% milk powder-PBST) at 37℃for 1 hour, then each recombinant anti-human CD38 antibody and the unrelated control DP47 were added separately and bound at 37℃for 1 hour. The ELISA plates were then washed with PBST, HRP mouse anti-human IgG was added and allowed to bind for an additional 1 hour at 37 ℃. The ELISA plate was then washed with PBST and OPD substrate chromogenic solution was added, 5-10 minutes later with 1M H 2 SO 4 The solution was stopped to develop color, and the optical density value was measured at 492nm/630nm dual wavelength using an enzyme-labeled instrument. As a result, as shown in FIG. 1, each of the recombinant anti-human CD38 bispecific antibody and the monoclonal antibody was able to recognize the human CD38 extracellular region, wherein monoclonal antibody S5B8h2, bispecific antibody S5A10h1Fab+S5B8h2scFv and S5A10h1cross+S5B8h2 were able to cross-recognize the cynomolgus CD38 extracellular region.
3. Epitope identification of recombinant anti-human CD38 monoclonal antibodies
Antibody binding was determined by surface plasmon resonance techniques using Biacore T200. Amino coupling kit (BR-1000-50), his capturing kit (28-9950-56), CM5 chip ((BR 100530) and 10 XHBS-EP (BR 100669) pH 7.4) related reagents and consumables are purchased from GE healthcare, according to the instructions in the kit, an anti-His antibody (capture antibody) is coupled to the CM5 chip surface by amino coupling method, the saturated about 50RU human CD38-His is captured by the capture antibody and then the first antibody is diluted to a proper concentration, the saturated first antibody is guaranteed to be fully bound to human CD38-His, the first antibody is fully bound and then the second antibody is passed through the stationary phase surface, and it is determined whether the first antibody and the second antibody bind to the same epitope according to the binding amount (RU) of the second antibody, when the second antibody binds to the first antibody, the second antibody cannot bind to the chip, the binding amount is about 0, finally MgCl 3M is used 2 The chip surface was regenerated and Biacore data was analyzed using Biacore T200 evaluation software version 3.0, and the fitting results are shown in table 2. The results show that S5A10h1 and S5B8h2, and darifenacin and S5B8h2 are different binding epitopes. The darifenacin and S5A10h1 are the same binding epitope.
TABLE 2 epitope identification of different recombinant anti-human CD38 monoclonal antibodies
Figure BDA0003364543120000201
Example 4 in vitro Activity of recombinant anti-human CD38 antibodies
1. ADCC Activity of recombinant anti-human CD38 antibodies
The inventors constructed a jurkat-dual-CD16a reporter cell line by transferring the CD16a plasmid into a jurkat-dual cell of Invivogen. When ADCC effects occur, the activation signal passes through the CD16a molecule to the downstream nfkb pathway, and ADCC activity is detected by the luciferase.
1640+5% inactivated serum was used as test medium. jurkat-dual-CD16a cells were resuspended to 2 x 10 after counting with test medium 6 Target cells (e.g., daudi cells) that highly express human CD38 were counted and resuspended to 4 x 10 cells per ml 5 Individual cells/ml. Mu.l of jurkat-dual-CD16a cells and 50. Mu.l of daudi cells were mixed in an effective target ratio of 10:1 and incubated with various recombinant anti-human CD38 monoclonal antibodies and bispecific antibodies in gradient dilutions (starting at a final concentration of 10nM, 3-fold gradient dilutions, 10 concentration points) for 20h in an incubator. 20. Mu.l of the supernatant was centrifuged, an automatic loading program was set in the microplate reader, 50. Mu.l of luciferase assay solution quat-luc (invivogen, cat. Rep-qlc 2) was added to each well and the fluorescence intensity was measured by full-wavelength detection.
As shown in FIG. 2, the ADCC activities of the bispecific antibodies S5A10h1Fab+S5B8h2ScFv and S5A10h1cross+S5B8h2 were significantly stronger than that of the monoclonal antibodies S5A10h1 and S5B8h2 alone, and were comparable to that of darimumab.
2. CDC Activity of recombinant anti-human CD38 antibodies
Blood (10 mL) was collected from healthy volunteers, where the collected blood was provided by the inventors and their colleagues as volunteers, all of whom had signed a known consent. Inclusion criteria for volunteers were:
1. age above 18 years old;
2. no infection of HIV and HBV;
3. normal blood routine detection;
4. non-pregnant or lactating women.
Standing blood at room temperature for 2 hr until agglutination is completed, centrifuging at 3000rpm for 10min, carefully sucking out upper serum, and packaging.
1640+5% inactivated serum was used as test medium. The recombinant anti-human CD38 bispecific antibody and monoclonal antibody were diluted with test medium to a final concentration of 100nM and then diluted with a 3-fold concentration gradient to give 10 dilutions in total. Target cells (e.g., daudi cells) that highly express human CD38 are counted and resuspended to 5 x 10 5 Individual cells/ml. Mu.l of various concentration dilutions of recombinant anti-human CD38 antibodies and 90. Mu.l of target cells highly expressing human CD38 were pre-incubated for 15min, then 10. Mu.l of fresh human serum (volunteer donation) was added and mixed well and incubated in an incubator for 20h. A series of control wells were set up for spontaneous release of target cells (only target cells), maximum release of target cells (complete lysis of target cells), spontaneous release of target cells + effector cells (only target cells + effector cells), background of medium (only medium), and volume correction (medium + lysate), and the control wells were kept consistent with each experimental group volume with medium.
Equal volumes of 10 Xlysate (Cytotox) were added to both the maximum release and volume correction wells of the target cells 50min prior to detection
Figure BDA0003364543120000211
Nonradioactive cytotoxicity detection kit, promega, cat No. G1780), centrifuging after complete lysis of target cells, taking 50 μl of supernatant and adding 50 μl of LDH detection solution (CytoTox>
Figure BDA0003364543120000212
Nonradioactive cytotoxicity detection kit, promega, cat No. G1780), incubation in the dark for 30min, and addition of 50 μl of stop solution (CytoTox +.>
Figure BDA0003364543120000213
Nonradioactive cytotoxicity assay kit, promega, cat No. G1780), absorbance was measured with a microplate reader at 490/492nm wavelength and the killing rate was calculated. Calculation formula of killing rate (%)The method comprises the following steps:
killing (%) = { absorbance of experimental group- (absorbance of target cell + effector cell spontaneous release) }/{ (absorbance of target cell maximum release-volume correction) - (absorbance of target cell spontaneous dilution-absorbance of medium background) } 100.
As shown in FIG. 3, the single monoclonal antibodies S5A10h1 and S5B8h2 have no CDC activity, but the two bispecific antibodies S5A10h1Fab+S5B8h2ScFv and S5A10h1cross+S5B8h2 constructed by S5A10h1 and S5B8h2 have obvious CDC effect and are strong in activity Yu Dalei.
EXAMPLE 5 tumor-inhibiting effect of recombinant anti-human CD38 antibodies
Myeloma cell line RPMI8226 cells in exponential growth phase were collected, resuspended to appropriate concentration with PBS and mixed with matrigel at a ratio of 1:1, and inoculated subcutaneously into 6-8 week female NOD/SCID mice. The day before cell inoculation, mice were subjected to a 60Co irradiation treatment (150 rad). The average volume of the tumor is 100-120mm 3 At the time, the selected animals are randomly grouped according to the weight and the tumor volume of the animals, so that the tumor volumes among different groups are ensured to be similar. The first administration was performed on the day of the group, which was defined as day 0. The dosing regimen is shown in table 3.
Body weight and tumor size were measured twice weekly. Tumor size calculation formula:
tumor volume (mm) 3 ) =0.5× (tumor long diameter×tumor short diameter 2 )。
The last tumor dose was performed on day 32 after the initial dose and all experimental animals were euthanized and weighed. Tumor tissue was dissected and tumor weights were measured, and all experimental results were expressed as mean tumor volume ± SEM (mean standard error). Statistical analysis was performed on each experimental group, and the relative tumor volume and tumor weight of the treatment group (groups 2 and 3) and the relative tumor volume and tumor weight of the control group (group 1) were compared by an independent sample T-test method to see whether the differences were significant.
TABLE 3 Experimental dosing regimen for evaluating anti-human CD38 antibody tumor inhibiting Activity
Numbering device Drug administration quality Dosage (mg/kg) Administration mode Time of administration
1 Isotype control IgG1 10 i.p. BIW.times.4.5 weeks
2 S5A10h1Fab+S5B8h2ScFv 8.3 i.p. BIW.times.4.5 weeks
3 S5A10h1cross+S5B8h2 10 i.p. BIW.times.4.5 weeks
Tumor volume results show (FIG. 4), group 1 control mice had a mean tumor volume of 556mm at the 32 nd balance of group administration 3 . Statistically significant differences (p)<0.01). Tumor volume was averaged at dosing balance 32 for group 2S 5A10h1Fab+S5B8h2ScFv treatment group216mm 3 The relative tumor inhibition TGI (%) was 61.1%. The average tumor volume of the 3 rd group S5A10h1cross+S5B8h2 treatment group at the dosing 32 nd balance was 221mm 3 The relative tumor inhibition TGI (%) was 60.3%. The tumor re-analysis results are consistent with the tumor volume analysis results.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Reference to the literature
1.Hoshino S,et al.Mapping of the catalytic and epitopic sites of human CD38/NAD glycohydrolase to a functional domain in the carboxyl terminus.J Immunol 158:741–747,1997.
2.Hamblin TJ,et al.CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia,but CD38 expression may vary during the course of the disease.Blood.2002；99:1023-1029.
3.Hon Cheung Lee,et al.Structure and Enzymatic Functions of Human CD38.J Mol Med.2006,12(11-12):317–323.
4.Ho HN,et al.Circulating HIV-specific CD8+cytotoxic T cells express CD38 and HLA-DR antigens.J Immunol 1993；150:3070–3079.
5.Henriques A,et al.CD38,CD81 and BAFFR combined expression by transitional B cells distinguishes active from inactive systemic lupus erythematosus.Clin Exp Med(2016)16:227.
6.Niels W.C.J.van de Donk,et al.CD38 antibodies in multiple myeloma:back to the future.J Blood 2018 131:13-29.
7.Zhiwu Jiang,et al.CD34 and CD38 are prognostic biomarkers for acute B lymphoblastic leukemia.J Biomarker Research 2016 4:23.
8.Tiziana Vaisitti,et al.Multiple Metamorphoses of CD38 from Prognostic Marker to Disease Modifier to Therapeutic Target in Chronic Lymphocytic Leukemia.J Curr Top Med Chem.2013；13(23):2955-64.
9.Rebecca L.Siegel,et al.Cancer statistics,2017.J CA Cancer J Clin 2017；67:7–30.
10.Lindsey A.Torre,et al.Global cancer statistics,2012.J CA Cancer J Clin 2015；65:87-108.
11.Wanqing Chen,et al.Cancer statistics in China,2015.J CA Cancer J Clin 2016；66:115-132.
12.Colin Phipps,et al.Daratumumab and its potential in the treatment of multiple myeloma:overview of the preclinical and clinical development.J Ther Adv Hematol 2015,Vol.6(3)120–127.
Sequence listing
<110> Beijing fructus Alpinae Oxyphyllae Meibo Biotechnology Co., ltd
<120> anti-human CD38 bispecific antibodies and uses thereof
<160> 34
<170> CNIPASequenceListing 1.0
<210> 1
<211> 258
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 1
Val Pro Arg Trp Arg Gln Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg
1 5 10 15
Phe Pro Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Ile His
20 25 30
Pro Glu Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys
35 40 45
Gly Ala Phe Ile Ser Lys His Pro Cys Asn Ile Thr Glu Glu Asp Tyr
50 55 60
Gln Pro Leu Met Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Ile
65 70 75 80
Leu Leu Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val
85 90 95
Gln Arg Asp Met Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala
100 105 110
Asp Asp Leu Thr Trp Cys Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr
115 120 125
Gln Ser Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser
130 135 140
Val Phe Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp
145 150 155 160
Val Val His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys
165 170 175
Asn Ser Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys
180 185 190
Val Gln Thr Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser
195 200 205
Arg Asp Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile
210 215 220
Ser Lys Arg Asn Ile Gln Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp
225 230 235 240
Lys Phe Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser
245 250 255
Glu Ile
<210> 2
<211> 258
<212> PRT
<213> cynomolgus monkey (Macaca fascicularis)
<400> 2
Leu Pro Arg Trp Arg Gln Gln Trp Ser Gly Ser Gly Thr Thr Ser Arg
1 5 10 15
Phe Pro Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Val His
20 25 30
Pro Glu Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys
35 40 45
Gly Ala Phe Ile Ser Lys Tyr Pro Cys Asn Ile Thr Glu Glu Asp Tyr
50 55 60
Gln Pro Leu Val Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Thr
65 70 75 80
Leu Leu Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val
85 90 95
Gln Arg Asp Met Phe Thr Leu Glu Asp Met Leu Leu Gly Tyr Leu Ala
100 105 110
Asp Asp Leu Thr Trp Cys Gly Glu Phe Asn Thr Phe Glu Ile Asn Tyr
115 120 125
Gln Ser Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser
130 135 140
Val Phe Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Thr Ala Cys Gly
145 150 155 160
Val Val His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys
165 170 175
Asn Ser Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys
180 185 190
Val Gln Ala Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser
195 200 205
Arg Asp Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile
210 215 220
Ser Lys Arg Asn Ile Arg Phe Phe Cys Lys Asn Ile Tyr Arg Pro Asp
225 230 235 240
Lys Phe Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Leu Ser
245 250 255
Gly Ile
<210> 3
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
His His His His His His
1 5
<210> 4
<211> 232
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 4
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 5
<211> 330
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 5
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
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 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 Glu Glu
225 230 235 240
Met 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> 6
<211> 326
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 6
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro Gly Lys
325
<210> 7
<211> 327
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 7
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 8
<211> 107
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 8
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> 9
<211> 106
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 9
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
20 25 30
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
35 40 45
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65 70 75 80
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 10
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
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 Ala Phe Thr Asn Ser
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Glu Ile His Pro Gln Ser Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly 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 Asp Leu Leu Gln Phe Gly Asn Gln Tyr Tyr Tyr Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 11
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Asp Ile Val Met Thr Gln Thr 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
Asn 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 Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 12
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Asp Tyr Pro Phe Ile Ser Tyr
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Asn Pro Ser Asn Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile 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 Arg Lys Tyr Gly Asn Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 13
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
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 Glu Asn Ile Tyr Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asn Ala Lys Thr Leu Ala Glu 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 His His Tyr Gly Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 14
<211> 122
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
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> 15
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
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> 16
<211> 483
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Asp Ile Val Met Thr Gln Thr 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
Asn 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 Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Ser Trp
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Glu Ile His Pro Gln Ser Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys
180 185 190
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Asp Leu Leu Gln Phe Gly Asn Gln Tyr Tyr Tyr Ala Met Asp Tyr
225 230 235 240
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Ser
245 250 255
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
260 265 270
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
275 280 285
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
290 295 300
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
305 310 315 320
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
325 330 335
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
340 345 350
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
355 360 365
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
370 375 380
Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
385 390 395 400
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
405 410 415
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
420 425 430
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
435 440 445
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
450 455 460
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
465 470 475 480
Pro Gly Lys
<210> 17
<211> 449
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Asp Tyr Pro Phe Ile Ser Tyr
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Asn Pro Ser Asn Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile 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 Arg Lys Tyr Gly Asn Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 18
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
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 Glu Asn Ile Tyr Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asn Ala Lys Thr Leu Ala Glu 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 His His Tyr Gly Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 19
<211> 454
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
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 Ala Phe Thr Asn Ser
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Glu Ile His Pro Gln Ser Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly 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 Asp Leu Leu Gln Phe Gly Asn Gln Tyr Tyr Tyr Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
355 360 365
Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly Lys
450
<210> 20
<211> 219
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Asp Ile Val Met Thr Gln Thr 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
Asn 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 Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 21
<211> 453
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Asp Tyr Pro Phe Ile Ser Tyr
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Asn Pro Ser Asn Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile 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 Arg Lys Tyr Gly Asn Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe
115 120 125
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
130 135 140
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
145 150 155 160
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
165 170 175
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
180 185 190
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
195 200 205
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
210 215 220
Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
225 230 235 240
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln
355 360 365
Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly Lys
450
<210> 22
<211> 212
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 22
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 Glu Asn Ile Tyr Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asn Ala Lys Thr Leu Ala Glu 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 His His Tyr Gly Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr
100 105 110
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
115 120 125
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
130 135 140
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
145 150 155 160
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
180 185 190
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
195 200 205
Pro Lys Ser Cys
210
<210> 23
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Asn Ser Trp Met His
1 5
<210> 24
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Glu Ile His Pro Gln Ser Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 25
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
Asp Leu Leu Gln Phe Gly Asn Gln Tyr Tyr Tyr Ala Met Asp Tyr
1 5 10 15
<210> 26
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<210> 27
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 28
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Ser Gln Ser Thr His Val Pro Leu Thr
1 5
<210> 29
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 29
Ser Tyr Thr Ile His
1 5
<210> 30
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 30
Tyr Ile Asn Pro Ser Asn Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210> 31
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 31
Lys Tyr Gly Asn Phe Tyr Ala Met Asp Tyr
1 5 10
<210> 32
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 32
Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala
1 5 10
<210> 33
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 33
Asn Ala Lys Thr Leu Ala Glu
1 5
<210> 34
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 34
Gln His His Tyr Gly Thr Pro Tyr Thr
1 5

Claims (10)

1. A bispecific antibody comprising a first antigen-binding fragment and a second antigen-binding fragment that bind different epitopes of human CD 38.
2. The bispecific antibody of claim 1, wherein the first antigen-binding fragment comprises:
HCDR1 as shown in SEQ ID NO. 23,
HCDR2 as shown in SEQ ID NO. 24,
HCDR3 as shown in SEQ ID NO. 25,
LCDR1 as shown in SEQ ID NO. 26,
LCDR2 as shown in SEQ ID NO 27, and
LCDR3 as shown in SEQ ID NO. 28;
wherein the amino acid sequences of HCDR and LCDR are defined according to Kabat.
3. The bispecific antibody of claim 1, wherein the second antigen-binding fragment comprises:
HCDR1 as shown in SEQ ID NO. 29,
HCDR2 as shown in SEQ ID NO. 30,
HCDR3 as shown in SEQ ID NO. 31,
LCDR1 as shown in SEQ ID NO. 32,
LCDR2 as shown in SEQ ID NO 33, and
LCDR3 as shown in SEQ ID NO. 34;
wherein the amino acid sequences of HCDR and LCDR are defined according to Kabat.
4. The bispecific antibody of claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the first antigen binding fragment is shown in SEQ ID No. 10 and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 11.
5. The bispecific antibody of claim 1 or 3, wherein the heavy chain variable region of the second antigen binding fragment has the amino acid sequence of SEQ ID No. 12 and the light chain variable region has the amino acid sequence of SEQ ID No. 13.
6. The bispecific antibody of any one of claims 1-5, wherein the form of the first antigen-binding fragment and the second antigen-binding fragment is independently selected from a single chain antibody (scFv) or Fab fragment.
7. The bispecific antibody of claim 6, wherein
The first antigen-binding fragment is a single chain antibody (ScFv) and the second antigen-binding fragment is a Fab fragment; preferably, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO. 16, the amino acid sequence shown in SEQ ID NO. 17 and the amino acid sequence shown in SEQ ID NO. 18; or alternatively
The first antigen binding fragment is a Fab fragment, and the second antigen binding fragment is a CH1CL substituted Fab fragment; preferably, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO. 19, the amino acid sequence shown in SEQ ID NO. 20, the amino acid sequence shown in SEQ ID NO. 21 and the amino acid sequence shown in SEQ ID NO. 22.
8. A nucleic acid molecule encoding the bispecific antibody of any one of claims 1-7.
9. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-7, and a pharmaceutically acceptable excipient, diluent or carrier.
10. Use of the bispecific antibody of any one of claims 1-7, the nucleic acid molecule of claim 8 or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the prevention or treatment of a CD38 mediated disease; preferably, the disease is a tumor; more preferably, the disease is a malignancy, such as multiple myeloma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia.
CN202111400194.0A 2021-11-19 2021-11-19 Anti-human CD38 bispecific antibodies and uses thereof Pending CN116143933A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111400194.0A CN116143933A (en) 2021-11-19 2021-11-19 Anti-human CD38 bispecific antibodies and uses thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111400194.0A CN116143933A (en) 2021-11-19 2021-11-19 Anti-human CD38 bispecific antibodies and uses thereof

Publications (1)

Publication Number Publication Date
CN116143933A true CN116143933A (en) 2023-05-23

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (1)

Country Link
CN (1) CN116143933A (en)

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