RU2776795C2 - Antibodies to protein cd38, and their use - Google Patents

Abstract

FIELD: biotechnology; biomedicine.

SUBSTANCE: present invention relates to the field of biotechnology and biomedicine, in particular to antibodies and their antigen-binding fragments capable of binding to protein CD38, to an isolated nucleic acid molecule encoding an antibody to CD38, and an expression vector containing the above-mentioned nucleic acid; to a host cell for the production of an antibody to CD38 or its antigen-binding fragments, to a method for the production of an antibody to CD38 or its antigen-binding fragment, and to a pharmaceutical composition for the treatment of tumors. In addition, a method for the prevention or treatment of tumors, and a method for the inhibition of protein CD38 binding to ligand CD38 are proposed.

EFFECT: antibody or its antigen-binding fragment, according to the present invention, can destroy CD38⁺ cells by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and/or apoptosis.

33 cl, 3 tbl, 11 ex, 10 dwg

Description

FIELD OF TECHNOLOGY

The present application relates to the field of biomedicine, in particular to an antibody that is capable of binding to the CD38 protein.

BACKGROUND OF THE INVENTION

The CD38 protein is a bifunctional exonuclease that can catalyze the conversion of NAD ⁺ to cyclic ADP-ribose (cADPR, cyclic ADP-ribose) and hydrolyze cADPR to ADP-ribose. The CD38 protein is one of the antigens expressed on malignant plasma cells and expressed in various malignant hematopoietic cells, including, but not limited to, multiple myeloma cells, chronic B-cell lymphoblastic leukemia cells, and acute B-cell lymphoblastic leukemia cells.

However, anti-CD38 antibodies currently developed have limited recognition activity, limited cytotoxic activity, and limited ability to inhibit tumors, so there is an urgent need to develop a new anti-CD38 antibody for the development of new drugs.

SUMMARY OF THE INVENTION

The present application proposes an anti-CD38 antibody and its use. An anti-CD38 antibody provided herein may have one or more of the following properties: 1) it may bind to the CD38 protein with higher affinity and specificity; 2) it can kill CD38 ⁺ cells by antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and/or apoptosis; 3) it can destroy and damage tumor cells and/or inhibit tumor growth; 4) it can be used to prevent or treat tumors; 5) it can inhibit the binding of the CD38 protein to the CD38 ligand. The present application further provides a method for making and using an anti-CD38 antibody.

On the one hand, the present application provides an antibody, or an antigen-binding fragment thereof, that binds to the CD38 protein at a K _D value of 1×10 ^-9 M or less.

In some embodiments, the antibody or antigen-binding fragment thereof of the present application can kill and damage tumor cells and/or inhibit tumor growth by specifically binding to the CD38 protein.

In some embodiments, the antibody or antigen-binding fragment thereof of the present application can kill CD38 ⁺ cells by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and/or apoptosis.

In some embodiments, the antibody or antigen-binding fragment thereof of the present application does not bind or does not substantially bind to BSA, CD19, TROP2, CD47, AXL or Gas6 and other unrelated antigens.

In some embodiments, the tumors include a CD38 positive tumor. In some embodiments, the CD38 positive tumor is selected from the group consisting of multiple myeloma, lymphoma, and leukemia.

In some embodiments, the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.

In some embodiments, the tumor cells include cells selected from the group consisting of Raja cells, Daudi cells, Ramos cells, and RPMI8226 cells.

In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more residues corresponding to amino acid residues 60-89 of the human CD38 protein when bound to the CD38 protein (wherein the amino acid sequence of amino acids 60-89 of the human CD38 protein is shown in SEQ ID NO: 15) .

In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv, and ScFv.

In some embodiments, the CD38 protein is a human CD38 protein or a monkey CD38 protein. For example, the CD38 protein may not be a murine CD38 protein or may not be a rat CD38 protein.

In some embodiments, an antibody or antigen-binding fragment thereof according to the present application competes with a reference antibody for binding to the CD38 protein, wherein the reference antibody comprises a light chain variable region and a heavy chain variable region, the reference antibody light chain variable region comprises LCDR1, LCDR2, and LCDR3, LCDR1 contains the amino acid sequence shown in SEQ ID NO:1; LCDR2 contains the amino acid sequence shown in SEQ ID NO:2; LCDR3 contains the amino acid sequence shown in SEQ ID NO:3, the heavy chain variable region of the reference antibody contains HCDR1, HCDR2 and HCDR3, HCDR1 contains the amino acid sequence shown in SEQ ID NO:4; HCDR2 contains the amino acid sequence shown in SEQ ID NO:5; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:6.

In some embodiments, the light chain variable region of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:7 and the heavy chain variable region of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:8.

In some embodiments, the implementation of the light chain of the reference antibody contains the amino acid sequence shown in SEQ ID NO:22. For example, the reference antibody light chain, or fragment thereof, contains the amino acid sequence shown in any of SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.

In some embodiments, the implementation of the heavy chain of the reference antibody contains the amino acid sequence shown in SEQ ID NO:23. For example, the reference antibody heavy chain, or fragment thereof, contains the amino acid sequence shown in any of SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21.

In some embodiments, the implementation of the antibody or its antigennegative fragment according to the present application contains a light chain of the antibody or its fragment. In some embodiments, the antibody light chain, or fragment thereof, contains an LCDR1, and the LCDR1 contains the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the antibody light chain, or fragment thereof, contains an LCDR2, and the LCDR2 contains the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the antibody light chain, or fragment thereof, contains an LCDR3, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:3.

In some embodiments, the antibody light chain or fragment thereof comprises a VL light chain variable region, and the VL light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7.

In some embodiments, the antibody light chain or fragment thereof further comprises a human constant region. In some embodiments, the human constant region comprises a human Igκ constant region.

In some embodiments, the antibody light chain, or fragment thereof, contains the amino acid sequence shown in SEQ ID NO:22. For example, an antibody light chain or fragment thereof contains the amino acid sequence shown in any of SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises an antibody heavy chain, or fragment thereof. In some embodiments, the antibody heavy chain or fragment thereof contains HCDR1, and HCDR1 contains the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the antibody heavy chain, or fragment thereof, contains HCDR2, and HCDR2 contains the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the antibody heavy chain or fragment thereof contains HCDR3, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:6.

In some embodiments, the antibody heavy chain or fragment thereof comprises a VH heavy chain variable region, and the VH heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.

In some embodiments, the antibody heavy chain or fragment thereof further comprises a human constant region. In some embodiments, the human constant region comprises a human IgG constant region. In some embodiments, the IgG constant region comprises a human IgG1 constant region.

In some embodiments, the implementation of the heavy chain of the antibody contains the amino acid sequence shown in SEQ ID NO:23. For example, an antibody heavy chain, or fragment thereof, contains the amino acid sequence shown in any one of SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21.

On the other hand, the present application provides an isolated nucleic acid molecule that contains a polynucleotide encoding the antibody or antigen-binding fragment of the present application.

In some embodiments, at least one of the nucleic acid molecules is codon-optimized.

In some embodiments, the nucleic acid molecule contains one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14.

On the other hand, the present application proposes a vector that contains the nucleic acid molecule according to the present application.

On the other hand, the present application proposes a cell that contains the nucleic acid molecule of the present application or the vector of the present application.

On the other hand, the present application provides a method for producing an antibody or antigen-binding fragment thereof. The method includes culturing the cell according to the present application under conditions that allow the expression of the antibody or its antigen-binding fragment.

On the other hand, the present application provides a pharmaceutical composition that contains an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector and/or a cell of the present application, and optionally pharmaceutically acceptable adjuvants.

On the other hand, the present application proposes the use of an antibody or antigen-binding fragment for the production of drugs for the prevention or treatment of tumors.

In some embodiments, the tumors include a CD38 positive tumor. In some embodiments, the CD38 positive tumor is selected from the group consisting of multiple myeloma, lymphoma, and leukemia. In some embodiments, the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.

On the other hand, the antibody or antigen-binding fragment proposed in this application can be used for the prevention or treatment of tumors.

In some embodiments, the tumors include a CD38 positive tumor. In some embodiments, the CD38 positive tumor is selected from the group consisting of multiple myeloma, lymphoma, and leukemia. In some embodiments, the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.

On the other hand, the present application provides a method for preventing or treating tumors. The method includes administering to an individual in need thereof an antibody or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a cell and/or a pharmaceutical composition according to the present application.

In some embodiments, the tumors include a CD38 positive tumor. In some embodiments, the CD38 positive tumor is selected from the group consisting of multiple myeloma, lymphoma, and leukemia. In some embodiments, the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.

On the other hand, the present application provides a method for inhibiting the binding of a CD38 protein to a CD38 ligand, and the method includes administering an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, and/or a cell of the present application.

In some embodiments, the implementation of the CD38 ligand contains CD31.

Additional aspects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present invention are shown and described. Numerous modifications to the embodiments of the invention described herein will now be apparent to those skilled in the art without departing from the disclosure. Accordingly, the drawings and description of the present invention are to be considered as illustrative in nature and not as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The specific features of the invention set forth in this application are presented in the appended claims. The features and advantages of the present invention can be better understood by referring to the illustrative embodiments and the accompanying drawings. A brief description of the drawings is as follows:

In FIG. 1 is a physical map of the assembly of the SG003 antibody of the present application into a vector expressing the full length antibody.

In FIG. 2 shows the binding capacity of the SG003 antibody of the present application to the CD38 protein.

In FIG. 3 shows the results of specific recognition of the CD38 protein by the SG003 antibody of the present application.

Fig. 4A-4D show the binding ability of the SG003 antibody of the present application to the CD38 protein on the surface of the cells shown.

Fig. 5A-5D show the ADCC activity of the SG003 antibody of the present application on the cells shown.

In FIG. 6 shows the inhibitory effect of the SG003 antibody of the present application on tumor growth.

In FIG. 7 shows the effect of administration of the SG003 antibody of the present application on the survival rate of mice.

Fig. 8A-8B show the binding capacity of the administered SG003 antibody of the present application to CD38 molecules from various genera.

In FIG. 9 shows the binding capacity of the SG003 antibody of the present application with random mutations to CD38 molecules.

In FIG. 10 provides details of the binding of epitopes of the SG003 antibody of the present application to the CD38 protein.

DETAILED DESCRIPTION OF EMBODIMENTS

Embodiments of the present invention are described below by way of specific embodiments, and other advantages and effects of the present invention can be readily appreciated by those skilled in the art from the disclosure of the present invention.

As used herein, the term "antibody" generally refers to a peptide capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may include an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains linked to each other via disulfide bonds, and may include any molecule containing an antigen-binding moiety. The term "antibody" includes a monoclonal antibody, an antibody fragment, or an antibody derivative, including, but not limited to, a human antibody (fully human antibody), a humanized antibody, a chimeric antibody, a single chain antibody (e.g., scFv), and an antigen-binding antibody fragment (e.g., Fab, Fab' and (Fab) ₂ fragment). The term "antibody" further includes all recombinant forms of an antibody, such as an antibody expressed in a prokaryotic cell, a non-glycosylated antibody, and any antigen-binding fragment of an antibody of the present application and its derivative. Each heavy chain may consist of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions (known as complementarity determining regions (CDRs)), which are distributed among more conserved regions known as framework regions (FRs). Each VH and VL may be composed of three CDRs and FRs, which may be arranged from amino to carboxy in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The heavy chain and light chain variable regions contain binding domains that interact with antigen. The constant regions of an antibody can mediate the binding of an immunoglobulin to host tissue or a factor found in a variety of cells (eg, effector cells) of the immune system and the first component (Clq) of the classical complement system.

As used herein, the term "antibody-binding fragment" generally refers to one or more antibody fragments that serve to specifically bind to an antigen. The antigen-binding function of an antibody can be realized using a full length antibody fragment. The antigen-binding function of an antibody can also be implemented as follows: using a heavy chain containing a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab') ₂ or a light chain containing a fragment of Fv, ScFv, dsFv, Fab, Fab ' or F(ab') ₂ ; (1) a Fab fragment, ie a monovalent fragment containing the VL, VH, CL and CH domains; (2) an F(ab') ₂ fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (3) an Fd fragment containing VH and CH domains; (4) an Fv fragment containing the VL and VH domains in one antibody arm; (5) a dAb fragment containing a VH domain (Ward et al., (1989) Nature 341: 544-546); (6) an isolated complementarity determining region (CDR); and (7) combinations of two or more isolated CDRs that are optionally linked by a linker. In addition, a monovalent single chain Fv molecule (scFv) formed by the pairing of VL and VH can be further included (see Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988)) Proc. Natl.Acad.Sci. 85: 5879-5883). The "antigen binding group" may further comprise an immunoglobulin fusion protein that contains a binding domain selected from the group consisting of (1) a binding domain peptide fused to an immunoglobulin hinge peptide; (2) an immunoglobulin CH2 heavy chain constant region fused to a hinge region; and (3) a CH3 immunoglobulin heavy chain constant region fused to a CH2 constant region.

As used herein, the terms "CD38 protein" and "CD38 antigen" are used interchangeably herein and include any CD38 variant, isotype, and species homologue naturally expressed in cells or expressed in cells transfected with the CD38 gene. According to the present application, CD38 may be human CD38 with GenBank accession number BAA18966.1. In the present application, CD38 may be a simian CD38, such as macaque CD38 with GenBank accession number AAT36330.1. The CD38 protein of the present application may also be known as ADP-ribosylcyclase 1, cADPr hydrolase 1, Cd38-rs1, cyclic ADP-ribose hydrolase 1, I-19 antigen, or NIM-R5. "CD38 ⁺ cell" generally refers to a cell that expresses the CD38 protein. It may also be known as a CD38 positive cell. ^" CD38 cell" refers generally to a cell that does not substantially express the CD38 protein.

As used herein, the term "CD31 protein" refers generally to a 130-140 kDa transmembrane glycoprotein found on the surface of platelets, in cultured endothelial cell junctions, and in myeloid cell lines, and is also known as platelet and endothelial cell adhesion molecule-1. (PECAM-1/CD31). According to the present application, CD31 may be human CD31 with GenBank accession number NP_000433.4. CD31 can be used as a ligand for CD38 to play a role in thrombosis and angiogenesis.

As used herein, the term "ADCC" is antibody-dependent cell-mediated cytotoxicity (ADCC), and generally means that a cell with cell-killing activity recognizes an Fc segment covering a target antigen using an Fc receptor (FcR), expressed on its surface. With ADCC, effector cells of the immune system actively dissolve target cells whose membrane surface antigens have been bound to specific antibodies.

As used herein, the term "CDC" is complement dependent cytotoxicity (CDC) and generally refers to a cytotoxic effect in which complement is involved. That is, the specific antibody binds to the corresponding antigen on the surface of the cell membrane to form a complex and activate the classical complement pathway. The resulting membrane attack complex has a lytic effect on the target cell.

As used herein, the term "cancer" generally refers to or describes a physiological condition in mammals, which may typically be characterized by dysregulation of cell proliferation or survival. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia, as well as lymphoid cancer. For example, the cancer may be a lymphoma.

As used herein, the term "lymphoma" generally refers to a malignant tumor of the lymphatic system. Lymphoma occurs due to the uncontrolled proliferation of lymph node cells or lymph cells and the formation of malignant cells with abnormal abilities that can invade other tissues throughout the body. Lymphoma has many subtypes, where the two main types are Hodgkin's lymphoma and non-Hodgkin's lymphoma. As used herein, the term "Hodgkin's lymphoma" (HL) generally refers to a class of lymphomas produced by lymphocytes from leukocytes. As used herein, the term "non-Hodgkin's lymphoma" (NHL) generally refers to other classes of lymphomas with the exception of Hodgkin's lymphoma.

As used herein, the term "leukemia" generally refers to a malignant proliferative disease of the hematopoietic system, which generally refers to a class of diseases caused by massive proliferation and accumulation of leukemic cells. Clonal leukemic cells proliferate and accumulate to a large extent in the bone marrow and other hematopoietic tissues due to uncontrolled proliferation, impaired differentiation, inhibition of apoptosis or similar mechanisms, infiltrate other non-hematopoietic organs, and thereby suppress normal hematopoietic function. Clinically, anemia of varying degrees, bleeding, fever with infection, as well as enlargement of the liver, spleen and lymph glands, and bone pain can be detected.

As used herein, the term "multiple myeloma" (MM) generally refers to a malignant tumor that affects the bone marrow due to abnormal proliferation of plasma cells. Multiple myeloma causes malignant cells to build up in the bone marrow, crowding out healthy blood cells. Malignant cells instead of normal antibodies produce abnormal proteins that can cause complications.

As used herein, the term "Raja cell" generally refers to a continuous human cell line capable of producing strains of the Epstein-Barr virus. The virus transforms umbilical cord lymphocytes and induces an early antigen in Raja's cell. The Raja cell is widely used as a host for transfection and is also used to understand malignant tumors of hematopoietic cells and other cells. In addition, since the Raja cell expresses several receptors that express certain complement components as well as IgG protein Fc receptors, it is also used to detect immune complexes.

As used herein, the term "Daudi cell" generally refers to a cell line derived from Burkitt's lymphoma. Although the Daudi cell has an intracellular class I heavy chain, class I human leukocyte antigen (HLA) molecules are not expressed on its surface. This is because its gene encoding the β2-microsphere protein (β2m) is defective, so the protein no translated mRNA.

As used herein, the term "Ramos cell" generally refers to the Burkitt's lymphoma cell line. The cell does not carry the Epstein-Barr virus strain and secretes IgM.

As used herein, the term "RPMI8226 cell" generally refers to a multiple myeloma cell line.

As used herein, the term " K _D " is used interchangeably with "KD" and refers generally to the equilibrium dissociation constant for a specific antibody-antigen interaction in M (mol/l). KD can be calculated based on the concentration of substance AB and substances A and B dissociated from it: KD= c(A)×c(B)/c(AB). From this formula, one can learn that the larger the KD value, the greater the dissociation and the weaker the affinity between substances A and B; on the contrary, the smaller the KD value, the smaller the dissociation and the stronger the affinity between substances A and B.

As used herein, the term "monoclonal antibody" generally refers to a population of essentially homologous antibodies, i.e. the different antibodies in that population are the same except for naturally occurring mutations that may exist in minor amounts. The monoclonal antibody is highly specific and directly targets a single antigenic site. For example, a monoclonal antibody can be obtained using hybridoma technology, or a monoclonal antibody can be obtained using a recombinant DNA method in bacteria, eukaryotes, or plant cells. A monoclonal antibody can also be obtained from a phage library using the technology described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., Mol. Biol., 222: 581-597 (1991).

As used herein, the term "single chain antibody" (scFv) generally refers to a molecule formed by linking an antibody heavy chain variable region to a light chain variable region via an oligopeptide linker.

As used herein, the term “chimeric antibody” generally refers to an antibody in which a portion of the amino acid sequence of each heavy chain or light chain is homologous to, or belongs to, the corresponding amino acid sequence of an antibody of a particular species, and the remainder of the chain is homologous to the corresponding sequence in another species. . For example, the light chain variable region and the heavy chain are from the variable region of an animal species (eg, mouse, rat, etc.), while the constant portion is homologous to an antibody sequence of another species (eg, human). For example, to obtain chimeric antibodies, the variable regions can be obtained by using non-human B cells or hybridoma cells, and the constant region associated with them can be obtained from a human. The variable region has the advantage of being easy to manufacture, and its specificity is not affected by the source from which the constant region linked to it is derived. At the same time, since the constant region of a chimeric antibody can be derived from a human, the ability of the antibody to elicit an immune response when injected is lower than that of an antibody in which the constant region is derived from a non-human source.

As used herein, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences derived from non-human immunoglobulin proteins to reduce immunogenicity when the heterologous antibody is administered to humans and maintain full antigen binding affinity and antibody specificity. . For example, CDR grafting and variants thereof (Jones et al., Nature 321: 522 (1986)), including "shaping" (Verhoeyen, et al., 1988 Science 239: 1534-1536; Riechmann, et al., 1988 Nature 332: 323-337; Tempest, et al., Bio/Technol 1991 9: 266-271), "hyperchimerization" (Queen et al., 1989 Proc Natl Acad Sci USA 86: 10029-10033; Co, et al ., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154) and "surface residue masking" (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies". In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312), surface reconstruction (US patent US5639641) and other technical methods for humanization of binding domains from non-human sources. If other regions such as hinge regions and constant region domains are also derived from non-human sources, these regions can also be humanized.

As used herein, the term "epitope" generally refers to an antigenic determinant, that is, a group of molecules recognized by the immune system (eg, by an antibody). For example, an epitope may be discontinuous three-dimensional regions in an antigen that is recognized by the immune system. Epitopes are typically composed of reactive surface groups of molecules (such as amino acid or sugar side chains) and typically have certain three-dimensional structural characteristics as well as certain charge characteristics. Epitopes can be divided into conformational epitopes and non-conformational epitopes (linear epitopes) according to their structure. Epitopes may consist of continuous residues or may be formed by discrete residues that become contiguous due to folding of the antigenic polymer.

As used herein, the term "IgG" generally refers to immunoglobulins G. IgG is one of the human immunoglobulins, which further includes IgA, IgM, IgD, and IgE. In accordance with the antigenic difference of the γ-chain in the IgG molecule, human IgG has four subtypes: IgG1, IgG2, IgG3, IgG4. IgG plays an important role in immunity. As used herein, the term "IgG1" generally refers to the subtype with the highest proportion of IgG that has high affinity for the Fc receptor.

As used herein, the term "nucleic acid molecule" refers generally to an isolated nucleotide, deoxyribonucleotide, ribonucleotide, or analogue thereof, of any length, which may be isolated from the natural environment or artificially synthesized.

As used herein, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host. The vector can carry the inserted nucleic acid molecule into and/or between cells. The vector may include vectors that are primarily used to insert DNA or RNA into a cell, are primarily used to copy DNA or RNA, and are primarily used to transcribe DNA or RNA and/or translate. The vector may be a polynucleotide that can be transcribed and translated into a peptide when introduced into a suitable cell. Generally, the vector can produce the desired expression product by culturing suitable cells that contain the vector.

As used herein, the term “cell” generally refers to a single cell, cell line, or cell culture that may or may contain a plasmid or vector comprising the nucleic acid molecule of the present application, or may express an antibody or antigen-binding fragment thereof of the present invention. application. A "cell" may include the progeny of a single cell. Due to natural, accidental or intentional mutation, the progeny of a cell may not be completely identical to the original parent cell in morphology or genome, provided that the progeny of the cell can express the antibody or antigen-binding fragment of the present application. The cell can be obtained by in vitro transfection of cells with the vector of the present application. The cell may be a prokaryotic cell (eg, E. coli ) or a eukaryotic cell (eg, a yeast cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell ). In some embodiments, the cell is a mammalian cell. For example, a mammalian cell may be a CHO-K1 cell. As used herein, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. A recombinant cell not only includes a certain specific cell, but also includes the progeny of those cells.

As used herein, the terms "Protein A" and "ProA" are used interchangeably herein, and include Protein A derived from its natural source, Protein A obtained by synthesis (e.g., by peptide synthesis or by recombinant technology), and variants that retain protein binding capacity. Protein A is available from Repligen, Pharmacia and Fermatech. Protein A is usually immobilized on a solid phase support. The term "ProA" also refers to an affinity chromatography resin column or solid support chromatographic matrix to which Protein A is covalently bound.

As used herein, the term "approximately" refers, as a rule, to a deviation within 0.5-10% of the specified value, such as a deviation within 0.5%, 1%, 1.5%, 2%, 2.5 %, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% of the indicated amount.

As used herein, the term "comprise/comprising" generally means "including", "comprising", or "covering". In some cases, the term also means "to be" or "to consist of".

Antibody, antigen-binding fragment or variant thereof

In an aspect, the present application provides an antibody, or an antigen-binding fragment or variant thereof, that binds to the CD38 protein with a K _D value of 1×10 ^-9 M or less (for example, a K _D value of not greater than about 1×10 ^-9 M, not more than about 9×10 ^-10 M, not more than about 8×10 ^-10 M, not more than about 7×10 ^-10 M, not more than about 6×10 ^-10 M, not more than about 5× 10 ^-10 M, no more than about ^{4x10 -10} M, no more than about ^{3x10 -10} M, no more than about ^{2x10 -10} M, no more than ^{1x10 -10} M or no more than about 1×10 ^-11 M or less).

The antibody or antigen-binding fragment of the present application can destroy CD38 ⁺ cells by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and/or apoptosis.

An antibody or antigen-binding fragment or variant thereof can kill and damage tumor cells and/or inhibit tumor growth by specifically binding to the CD38 protein. For example, the tumor may include a CD38 positive tumor. For example, a CD38 positive tumor may be selected from the group consisting of multiple myeloma, lymphoma, and leukemia. Alternatively, for example, the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma. The tumor cell can be selected from the group consisting of a Raja cell, a Dowdy cell, a Ramos cell, and an RPMI8226 cell. According to the present application, the antibody or antigen-binding fragment thereof can kill and damage cells of multiple myeloma, lymphoma, leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma or inhibit the growth of multiple myeloma, lymphoma, leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma.

An antibody or antigen-binding fragment thereof herein, when bound to a C38 protein, may bind to one or more residues corresponding to amino acid residues 60-89 of the human CD38 protein. For example, amino acid residues 60-89 of the human CD38 protein are shown in SEQ ID NO:15.

As used herein, an antibody or antigen-binding fragment thereof may also bind to amino acid residues corresponding to amino acid residues 90-119 of the human CD38 protein, corresponding to amino acid residues 150-159 of the human CD38 protein, or corresponding to amino acid residues 180-189 of the human CD38 protein.

As used herein, an antibody or antigen-binding fragment thereof may not substantially bind to amino acid residues corresponding to amino acid residues 170-179 of the human CD38 protein, or not bind to amino acid residues corresponding to amino acid residues 291-300 of the human CD38 protein.

An antibody of the present application may be a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and/or a fully human antibody. The antigen-binding fragment of an antibody of the present application may be a Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv and/or ScFv fragment.

The CD38 protein of the present application may be a human CD38 protein or a simian CD38 protein. For example, a CD38 protein may not be a murine CD38 protein, or may not be a rat CD38 protein. In some embodiments, the antibody or antigen-binding fragment thereof does not substantially bind to mouse CD38 protein or rat CD38 protein.

An antibody or antigen-binding fragment thereof in the present application may compete with a reference antibody for binding to the CD38 protein. The reference antibody may comprise a light chain variable region and a heavy chain variable region. For example, the variable region of the light chain of the reference antibody may contain LCDR1, LCDR2 and LCDR3, LCDR1 may contain the amino acid sequence shown in SEQ ID NO:1; LCDR2 may contain the amino acid sequence shown in SEQ ID NO:2; LCDR3 may contain the amino acid sequence shown in SEQ ID NO:3, the heavy chain variable region of the reference antibody may contain HCDR1, HCDR2 and HCDR3, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:4; HCDR2 may contain the amino acid sequence shown in SEQ ID NO:5; and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:6.

As used herein, the light chain variable region amino acid sequence of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:7, and the heavy chain variable region amino acid sequence of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:8.

According to the present application, the light chain of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:22. For example, the reference antibody light chain may comprise the amino acid sequence shown in any of SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20. The reference antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO:23; For example, the reference antibody heavy chain may comprise the amino acid sequence shown in any of SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21.

For example, a reference antibody light chain may contain the amino acid sequence shown in SEQ ID NO:11 and a reference antibody heavy chain may contain the amino acid sequence shown in SEQ ID NO:13. For example, a light chain of a reference antibody may contain the amino acid sequence shown in SEQ ID NO:16, and a heavy chain of a reference antibody may contain the amino acid sequence shown in SEQ ID NO:17. For example, a light chain of a reference antibody may contain the amino acid sequence shown in SEQ ID NO:18, and a heavy chain of a reference antibody may contain the amino acid sequence shown in SEQ ID NO:19. For example, a reference antibody light chain may contain the amino acid sequence shown in SEQ ID NO:20 and a reference antibody heavy chain may contain the amino acid sequence shown in SEQ ID NO:21.

An antibody or antigen-binding fragment thereof according to the present application may comprise an antibody light chain or a fragment thereof.

For example, an antibody light chain or fragment thereof may contain LCDR1, and LCDR1 may contain the amino acid sequence shown in SEQ ID NO:1. An antibody light chain, or fragment thereof, may contain LCDR2, and LCDR2 may contain the amino acid sequence shown in SEQ ID NO:2. An antibody light chain, or fragment thereof, may contain LCDR3, and LCDR3 may contain the amino acid sequence shown in SEQ ID NO:3.

The light chain of an antibody or fragment thereof of the present application may comprise a VL light chain variable region, and the VL light chain variable region may comprise the amino acid sequence shown in SEQ ID NO:7.

According to the present application, the antibody light chain or fragment thereof may contain an Igκ constant region, for example, may contain a human Igκ constant region.

According to the present application, the light chain of an antibody or its fragment may contain the amino acid sequence shown in SEQ ID NO:22:

EIVMTQSPASLSASLGQRAX ²⁰ ISCRASX ²⁷ SVSX ³¹ SAX ³⁴ SYVHWYQQKSGQPPKLLIYLASX ⁵⁷ X ⁵⁸ X ⁵⁹ SGVPARFSGSGSGTDFTLTIHPVESEDVATYYCHHSRX ⁹⁷ X ⁹⁸ PX ¹⁰⁰ X ¹⁰¹ FGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:22), где X ²⁰ =T или S; X ²⁷ =S or N; X ³¹ =S or T; X ³⁴ =F or Y; X ⁵⁷ =N and D; X ⁵⁸ =L or I; X ⁵⁹ =E or Q; X ⁹⁷ =E or Q; X ⁹⁸ =L or V; X ¹⁰⁰ =F or S; X ¹⁰¹ =T or S.

In some embodiments, compared to the antibody light chain shown in SEQ ID NO:11, the antibody light chain, or fragment thereof, at least contains an amino acid/amino acid substitution selected from the group consisting of:

(a) replacing the amino acid/amino acids at X ²⁰ , X ²⁷ , X ³⁴ , X ⁵⁸ , X ⁵⁹ , X ⁹⁷ and/or X ¹⁰⁰ ;

(b) replacing the amino acid/amino acids at X ³⁴ , X ⁵⁷ , X ⁵⁹ , X ¹⁰⁰ and/or X ¹⁰¹ ; and

(c) replacing the amino acid/amino acids at X ²⁷ , X ³¹ and/or X ⁹⁸ .

In some embodiments, compared to the antibody light chain shown in SEQ ID NO:11, the antibody light chain, or fragment thereof, at least contains an amino acid/amino acid substitution at X ²⁰ , X ²⁷ , X ³⁴ , X ⁵⁸ , X ⁵⁹ , X ⁹⁷ and/or X ¹⁰⁰ , where the amino acid at X ²⁰ may be substituted with T or S; the amino acid at X ²⁷ may be substituted with S or N; the amino acid at X ³⁴ may be substituted with F or Y; the amino acid at X ⁵⁸ may be substituted with L or I; the amino acid at X ⁵⁹ may be substituted with E or Q; the amino acid at X ⁹⁷ may be substituted with E or Q; and the amino acid at X ¹⁰⁰ may be substituted with F or S.

In some embodiments, compared to the antibody light chain shown in SEQ ID NO:11, the antibody light chain, or fragment thereof, at least contains an amino acid/amino acid substitution at X ³⁴ , X ⁵⁷ , X ⁵⁹ , X ¹⁰⁰ and/or X ¹⁰¹ where the amino acid at X ³⁴ may be substituted with F or Y; the amino acid at X ⁵⁷ may be substituted with N or D; the amino acid at X ⁵⁹ may be substituted with E or Q; the amino acid at X ¹⁰⁰ may be substituted with F or S; and the amino acid at X ¹⁰¹ may be substituted with T or S.

In some embodiments, compared to the antibody light chain shown in SEQ ID NO:11, the antibody light chain, or fragment thereof, at least contains an amino acid/amino acid substitution at X ²⁷ , X ³¹ and/or X ⁹⁸ , where the amino acid at X ²⁷ may be substituted with S or N; the amino acid at X ³¹ may be substituted with S or T; and the amino acid at X ⁹⁸ may be substituted with L or V.

For example, an antibody light chain or fragment thereof may comprise the amino acid sequence shown in any of SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.

An antibody or antigen-binding fragment thereof according to the present application may contain an antibody heavy chain or a fragment thereof.

According to the present application, the heavy chain of the antibody or its fragment may contain HCDR1, and the HCDR1 may contain the amino acid sequence shown in SEQ ID NO:4. An antibody heavy chain, or fragment thereof, may contain HCDR2, and HCDR2 may contain the amino acid sequence shown in SEQ ID NO:5. Alternatively, for example, an antibody heavy chain or fragment thereof may contain HCDR3, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:6.

An antibody heavy chain or fragment thereof may comprise a VH heavy chain variable region, and the VH heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:8.

According to the present application, the antibody heavy chain or fragment thereof further comprises a human constant region. For example, a human constant region may comprise a human IgG constant region. For example, an IgG constant region may comprise a human IgG1 constant region.

According to the present application, the heavy chain of the antibody or its fragment may contain the amino acid sequence shown in SEQ ID NO:23:

QVQLLESGGGLVQPGGSLKLSCVASGX ²⁷ X ²⁸ FSLYX ³³ MNWVRQAPGKGLEWIGKIX ⁵² PX ⁵⁴ SSX ⁵⁷ X ⁵⁸ X ⁵⁹ YX ⁶¹ PSX ⁶⁴ KDKFFISRDNAKNTLYLQMTKVRSEDTALYYCARLX ¹⁰⁰ IX ¹⁰² X ¹⁰³ G GX ¹⁰⁶ X ¹⁰⁷ YWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:23), где X ²⁷ =F or Y; X ²⁸ =D or N; X ³³ =W or Y; X ⁵² =N, Q or S; X ⁵⁴ =D, E or N; X ⁵⁷ =T or S; X ⁵⁸ =I or L; X ⁵⁹ =N or Q; X ⁶¹ =T or S; X ⁶⁴ =L or V; X ¹⁰⁰ =W or Y; X ¹⁰² =A or G; X ¹⁰³ =T or S; X ¹⁰⁶ =F or Y; X ¹⁰⁷ =D or N.

In some embodiments, compared to the antibody light chain shown in SEQ ID NO: 13, the antibody light chain, or fragment thereof, at least contains an amino acid/amino acid substitution selected from the group consisting of:

(a) replacing the amino acid/amino acids at X ²⁷ , X ²⁸ , X ³³ , X ⁵² , X ⁵⁴ , X ⁵⁹ , X ¹⁰² and/or X ¹⁰³ ;

(b) replacing the amino acid/amino acids at X ⁵² , X ⁵⁴ , X ⁶¹ , X ¹⁰⁰ and/or X ¹⁰³ ; and

(c) replacing the amino acid/amino acids at X ⁵² , X ⁵⁷ , X ⁵⁸ , X ⁶⁴ , X ¹⁰⁶ and/or X ¹⁰⁷ .

In some embodiments, compared to the antibody light chain shown in SEQ ID NO: 13, the antibody light chain, or fragment thereof, at least contains an amino acid/amino acid substitution at X ²⁷ , X ²⁸ , X ³³ , X ⁵² , X ⁵⁴ , X ⁵⁹ , X ¹⁰² and/or X ¹⁰³ where the amino acid at X ²⁷ may be substituted with F or Y; the amino acid at X ²⁸ may be substituted with D or N; the amino acid at X ³³ may be substituted with W or Y; the amino acid at X ⁵² may be substituted with N or Q; the amino acid at X ⁵⁴ may be substituted with D or E; the amino acid at X ⁵⁹ may be substituted with N or Q; the amino acid at X ¹⁰² may be substituted with A or G; and the amino acid at X ¹⁰³ may be substituted with T or S.

In some embodiments, compared to the antibody light chain shown in SEQ ID NO: 13, the antibody light chain, or fragment thereof, at least contains an amino acid/amino acid substitution at X ⁵² , X ⁵⁴ , X ⁶¹ , X ¹⁰⁰ and/or X ¹⁰³ where the amino acid at X ⁵² may be substituted with N or S; the amino acid at X ⁵⁴ may be substituted with D or N; the amino acid at X ⁶¹ may be substituted with T or S; the amino acid at X ¹⁰⁰ may be substituted with W or Y; and the amino acid at X ¹⁰³ may be substituted with T or S.

In some embodiments, compared to the antibody light chain shown in SEQ ID NO: 13, the antibody light chain, or fragment thereof, at least contains an amino acid/amino acid substitution at X ⁵² , X ⁵⁷ , X ⁵⁸ , X ⁶⁴ , X ¹⁰⁶ and/or X ¹⁰⁷ where, the amino acid at X ⁵² may be substituted with N or S; the amino acid at X ⁵⁷ may be substituted with T or S; the amino acid at X ⁵⁸ may be substituted with I or L; the amino acid at X ⁶⁴ may be substituted with L or V; the amino acid at X ¹⁰⁶ may be substituted with F or Y; and the amino acid at X ¹⁰⁷ may be substituted with D or N.

In some embodiments, the antibody heavy chain may comprise the amino acid sequence shown in any of SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21.

In some embodiments, in an antibody or antigen-binding fragment thereof of the present application, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1 or a variant thereof; LCDR2 may contain the amino acid sequence shown in SEQ ID NO:2 or a variant thereof; the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:3 or a variant thereof; and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:4 or a variant thereof; HCDR2 may contain the amino acid sequence shown in SEQ ID NO:5 or a variant thereof; HCDR3 may contain the amino acid sequence shown in SEQ ID NO:6 or a variant thereof. For example, an antibody or antigen-binding fragment thereof may comprise an SG003 antibody or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the implementation of the light chain of the antibody or its antigennegative fragment according to the present application may contain the variable region of the light chain, the variable region of the light chain may contain the amino acid sequence shown in SEQ ID NO:7 or a variant thereof; and the heavy chain may comprise a heavy chain variable region, the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:8 or a variant thereof. For example, an antibody or antigen-binding fragment thereof may comprise an SG003 antibody or an antibody with the same light chain variable region and heavy chain variable region. In some embodiments, an antibody or antigen-binding fragment thereof of the present application may comprise a light chain and a heavy chain, the light chain may comprise the amino acid sequence shown in SEQ ID NO:11, and the heavy chain may contain the amino acid sequence shown in SEQ ID NO:13 . For example, an antibody or antigen-binding fragment thereof may comprise an SG003 antibody or an amino acid sequence of the same light chain and heavy chain.

In some embodiments, the implementation of the antibody according to the present application may be SG003. LCDR1, LCDR2 and LCDR3 from the SG003 antibody may contain the amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively; VL may contain the amino acid sequence as shown in SEQ ID NO.7; HCDR1, LCDR2 and LCDR3 may contain amino acid sequences as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively; VH may contain the amino acid sequence as shown in SEQ ID NO.8; the light chain may contain an amino acid sequence as shown in SEQ ID NO.11; and the heavy chain may contain the amino acid sequence as shown in SEQ ID NO.13.

In some embodiments, an antibody or antigen-binding fragment thereof of the present application competes with a reference antibody for binding to a CD38 protein (eg, human CD38 protein or simian CD38 protein). The reference antibody may contain LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3, and LCDR1 may contain the amino acid sequence as shown in SEQ ID NO:1; LCDR2 may contain the amino acid sequence as shown in SEQ ID NO:2; LCDR3 may contain the amino acid sequence as shown in SEQ ID NO:3; HCDR1 may contain the amino acid sequence as shown in SEQ ID NO:4; HCDR2 may contain the amino acid sequence as shown in SEQ ID NO:5; and HCDR3 may contain the amino acid sequence as shown in SEQ ID NO:6. In some embodiments, the reference antibody may comprise an SG003 antibody or an antibody with the same LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3. In some embodiments, the reference antibody may comprise a light chain variable region and a heavy chain variable region, the light chain variable region may comprise an amino acid sequence as shown in SEQ ID NO:7; and the heavy chain variable region may comprise an amino acid sequence as shown in SEQ ID NO:8. For example, an antibody or antigen-binding fragment thereof may comprise an SG003 antibody or an antibody with the same light chain variable region and heavy chain variable region. In some embodiments, the reference antibody may comprise a light chain and a heavy chain, the light chain may comprise the amino acid sequence shown in SEQ ID NO:11, and the heavy chain may contain the amino acid sequence as shown in SEQ ID NO:13. For example, a reference antibody or antigen-binding fragment thereof may contain an SG003 antibody or an antibody with the same light chain and heavy chain.

An antibody or antigen-binding fragment thereof herein may further comprise one or more random mutations (eg, one or more, one or more amino acid substitutions) in the light chain and/or heavy chain amino acid sequences. For example, an antibody or antigen-binding fragment thereof may contain one or more random mutations (eg, one or more, one or more amino acid substitutions), and/ or contain one or more random mutations (eg, one or more, one or more amino acid substitutions) at one or more sites in the H-FR1~H-FR4 framework region of the heavy chain variable region. For example, after random mutation, the light chain of an antibody or antigen-binding fragment thereof may contain the amino acid sequence shown in any of SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20; and/or the heavy chain of an antibody or antigen-binding fragment thereof may comprise the amino acid sequence shown in any of SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21. The randomly mutated anti-CD38 antibody or antigen-binding fragment thereof still has the ability to specifically bind to human CD38 protein and simian CD38 protein.

In some embodiments, the implementation of the light chain of the antibody or antigennegative fragment according to the present application may contain an amino acid sequence, as shown in SEQ ID NO:16; and the heavy chain may contain the amino acid sequence as shown in SEQ ID NO:17; alternatively, the light chain of the antibody or antigennegative fragment of the present application may contain the amino acid sequence as shown in SEQ ID NO:18; and the heavy chain may contain the amino acid sequence as shown in SEQ ID NO:19; alternatively, the light chain of the antibody or antigennegative fragment of the present application may contain the amino acid sequence as shown in SEQ ID NO:20; and the heavy chain may contain the amino acid sequence as shown in SEQ ID NO:21.

Proteins, peptides and/or amino acid sequences provided herein should also be understood to encompass the following scope: a variant or homologue having the same or similar function as a protein or peptide.

According to the present application, a variant may be a protein or peptide obtained by substitution, deletion or addition of one or more amino acids in the amino acid sequence of a protein and/or peptide (for example, an antibody or fragment thereof that specifically binds to the CD38 protein). For example, a functional variant may contain a protein or peptide with modification of amino acids by substitution, deletion and/or insertion of at least one, for example, 1-30, 1-20, or 1-10, or for example, 1, 2, 3 , 4 or 5 amino acids. A functional variant may substantially retain the biological activity of the protein or peptide prior to modification (eg, substitution, deletion, or addition). For example, a functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen-binding capacity) of the original protein or peptide. For example, the substitution may be a conservative substitution.

As used herein, a homologue may be a protein or peptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94 %, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of a protein and/or peptide (for example, an antibody or fragment thereof that specifically binds to the CD38 protein).

As used herein, homology generally refers to similarity, analogy, or association between two or more sequences. "Percent sequence homology" can be calculated as follows: two sequences for alignment are compared in a comparison window to determine the number of positions with the same nucleic acid base (e.g. A, T, C, G, I) or with the same amino acid residues (e.g. Ala , Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) in two sequences to get the number of matching positions. The number of matching positions is divided by the total number of positions in the comparison window (ie, window size) and the result is multiplied by 100 to obtain the percent sequence homology. Alignment to determine percent sequence homology can be performed according to various methods known in the art using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve maximum alignment within the full length sequence being compared or within the target sequence region. Homology can also be determined in the following ways: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Pearson and D. J. Lipman's "Improved Tools for Biological Sequence Comparison", Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and D. J. Lipman and W. R. Pearson's "Fast and Sensitive Protein Mass Similarity Search", Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers and D. Lipman , "A Basic Local Alignment Search Tool", Journal of Molecular Biology, 215: 403-410, 1990.

Nucleic acid, vector, cell, and production method

In another aspect of the present application, one or more isolated nucleic acid molecules are further provided. One or more nucleic acid molecules may contain a polynucleotide encoding the antibody or antigen-binding fragment of the present application. For example, a polynucleotide in one or more nucleic acid molecules may encode the entire antibody, or an antigen-binding fragment thereof, and may also encode a portion of it (e.g., one or more of HCDR1-3, LCDR1-3, VL, VH, light chain, or heavy chain) .

At least one of the nucleic acid molecules of the nucleic acid molecules of the present application may be codon-optimized. For example, the codon optimization method includes, but is not limited to, rare codon removal, GC content correction, mRNA stability enhancement, mRNA secondary structure correction, rational linker design, and start codon environment correction.

In some embodiments, the implementation of the nucleic acid molecule may contain one or more polynucleotide sequences selected from the group including SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14., where SEQ ID NO: 9 may encode the light chain variable region of the SG003 antibody of the present application. SEQ ID NO:10 may encode the heavy chain variable region of the SG003 antibody of the present application. SEQ ID NO:12 may encode the light chain of the SG003 antibody of the present application. SEQ ID NO:14 may encode the heavy chain of the SG003 antibody of the present application.

The nucleic acid molecule of the present application may be isolated. For example, a nucleic acid molecule can be obtained or synthesized in the following ways: (i) a nucleic acid molecule can be amplified in vitro , for example, obtained by polymerase chain reaction (PCR) amplification, (ii) a nucleic acid molecule can be obtained by cloning and recombination, ( iii) the nucleic acid molecule can be purified, eg separated by cleavage and gel electrophoretic fractionation, or (iv) the nucleic acid molecule can be synthesized, eg by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule produced using recombinant DNA technology.

According to the present application, a nucleic acid encoding an antibody or antigen-binding fragment can be obtained using various methods known in this field. These methods include, but are not limited to, fragment restriction or overlapping PCR using synthetic oligonucleotides. For details, see Sambrook et al., Molecular cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.

In another aspect of the present application, one or more vectors are provided that contain one or more nucleic acid molecules of the present application. Each vector may contain one or more nucleic acid molecules. In addition, the vector may contain other genes, such as a marker gene, which allows selection of the vector in appropriate cells and under appropriate conditions. In addition, the vector may further comprise an expression control element that allows said coding region to be properly expressed in the appropriate host. Such a control element is well known to those skilled in the art and may include a promoter, a ribosome binding site, an enhancer, and other control elements that regulate gene transcription or mRNA translation, etc. In some embodiments, the expression control sequence is a regulated element. The specific structure of the expression control sequence may vary depending on the functions of species or cell types, but generally contains a 5' non-transcribed sequence and a 5' and 3' non-translated sequence involved in transcription and translation initiation, such as TATA- box, capping sequence, CAAT sequence, etc. For example, a 5' non-transcribed sequence to control expression may include a promoter region, and the promoter region may contain a promoter sequence to control transcription of a functionally linked nucleic acid. The expression control sequence may also include an enhancer sequence or a precursor activator sequence. One or more nucleic acid molecules of the present application may be operably linked to an expression control element.

The vector may contain, for example, plasmids, cosmids, viruses, bacteriophages, or other vectors widely used, for example, in genetic engineering. For example, the vector is an expression vector. For example, the expression vector may be a T-easy vector.

In another aspect of the present application, a cell is provided that may contain one or more nucleic acid molecules of the present application and/or one or more vectors of the present application. In some embodiments, each cell or cell type may contain one nucleic acid molecule or one vector or one type of nucleic acid molecule or vector of the present application. In some embodiments, each cell or cell type may contain more than one (eg, two or more) nucleic acid molecules or vectors, or more than one type (eg, two or more types) of nucleic acid molecules or vectors of the present application. For example, the vector of the present application can be introduced into a cell such as eukaryotic cells such as plant cells, fungal or yeast cells, etc. The vector of the present application can be introduced into a cell by a method known in the art, such as electroporation, transfection with lipofectin, transfection with lipofectamine, etc. For example, the cell may be a CHO-S cell.

In another aspect of the present application, a method for producing an antibody or antigen-binding fragment thereof is provided. The method may include culturing the cell according to the present application under conditions that allow the expression of the antibody or its antigen-binding fragment. For example, it is appropriate to use appropriate culture media, appropriate incubation temperature and time, and so on. These methods are known to those skilled in the art.

In some cases, the method may further include the step of isolating and/or purifying the antibody or antigen-binding fragment thereof. For example, protein G or protein A agarose and antibodies in gel and/or high performance liquid chromatography and the like can be used for affinity chromatography. can also be used to purify and isolate antibodies or antigen-binding fragments of the present application. For example, protein A affinity purification can also be used.

Pharmaceutical composition and application

In another aspect of the present application, a pharmaceutical composition is provided that may comprise an antibody, an antigen-binding fragment thereof, or a variant thereof, a nucleic acid molecule, a vector, or a cell, and optionally a pharmaceutically acceptable adjuvant.

The pharmaceutically acceptable adjuvant may contain buffers, antioxidants, preservatives, low molecular weight peptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or nonionic surfactants, etc.

According to the present application, the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the site of a tumor, inhalation, rectal administration, vaginal administration, transdermal administration, or administration through a subcutaneous depot.

The pharmaceutical composition can be used to inhibit tumor growth. For example, the pharmaceutical composition of the present application may inhibit or alleviate the development or progression of diseases, reduce the size of a tumor (or even substantially eliminate a tumor), and/or reduce and/or stabilize the manifestation of a disease.

The pharmaceutical composition according to the present application may contain a therapeutically effective amount of an antibody or antigen-binding fragment. A therapeutically effective amount may be the dose required to prevent and/or treat (at least partially treat) a disorder or condition (e.g., cancer) and/or any complications in an individual with the disorder or condition or at risk of developing the disorder. or states.

On the other hand, the present application proposes the use of an antibody or antigen-binding fragment for the production of drugs for the prevention or treatment of tumors.

On the other hand, the present application provides an antibody or antigen-binding fragment for the prevention or treatment of tumors.

On the other hand, the present application provides a method for preventing or treating tumors. The method includes administering to an individual in need thereof an antibody or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a cell and/or a pharmaceutical composition of the present application.

According to the present application, the tumor may include a CD38-positive tumor. For example, a CD38 positive tumor may be selected from the group consisting of multiple myeloma, lymphoma, and leukemia.

In some embodiments, the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.

On the other hand, the present application provides a method for inhibiting the binding of a CD38 protein to a CD38 ligand, and the method includes administering an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, and/or a cell of the present application.

According to the present application, the CD38 ligand may contain the CD31 protein.

In some embodiments, the method for inhibiting the binding of a CD38 protein to a CD38 ligand is an in vitro or ex vivo method.

The present application may include the following embodiment:

1. An antibody or antigen-binding fragment thereof that binds to the CD38 protein with a K _D value of 1×10 ^-9 M or less.

2. An antibody or antigen-binding fragment thereof according to embodiment 1, which can kill and damage tumor cells and/or inhibit tumor growth by specifically binding to the CD38 protein.

3. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-2, which can kill CD38 ⁺ cells by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and/or apoptosis.

4. An antibody or antigen-binding fragment thereof according to embodiment 3, wherein the tumors comprise a CD38 positive tumor.

5. The antibody or antigen-binding fragment thereof according to embodiment 4, wherein the CD38 positive tumor is selected from the group consisting of multiple myeloma, lymphoma, and leukemia.

6. An antibody or antigen-binding fragment thereof according to any one of embodiments 3-5, wherein the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.

7. An antibody or antigen-binding fragment thereof according to any one of embodiments 3-6, wherein the tumor cells include cells selected from the group consisting of a Raji cell, a Dowdy cell, a Ramos cell, and an RPMI8226 cell.

8. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-7, which, when bound to the CD38 protein, binds to one or more residues corresponding to amino acid residues 60-89 of the human CD38 protein.

9. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-8, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

10. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-9, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv and ScFv fragments.

11. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-10, wherein the CD38 protein is a human CD38 protein or a simian CD38 protein.

12. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-11 that competes with a reference antibody for binding to the CD38 protein, wherein the reference antibody comprises a light chain variable region and a heavy chain variable region, the reference antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3, LCDR1 contains the amino acid sequence shown in SEQ ID NO:1; LCDR2 contains the amino acid sequence shown in SEQ ID NO:2; LCDR3 contains the amino acid sequence shown in SEQ ID NO:3, the heavy chain variable region of the reference antibody contains HCDR1, HCDR2 and HCDR3, HCDR1 contains the amino acid sequence shown in SEQ ID NO:4; HCDR2 contains the amino acid sequence shown in SEQ ID NO:5; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:6.

13. An antibody or antigen-binding fragment thereof according to embodiment 12, wherein the light chain variable region of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:7 and the heavy chain variable region of the reference antibody comprises the amino acid sequence shown in SEQ ID NO: eight.

14. An antibody or antigen-binding fragment thereof according to any one of embodiments 12-13, wherein the light chain of the reference antibody contains the amino acid sequence shown in SEQ ID NO:22; and the heavy chain of the reference antibody contains the amino acid sequence shown in SEQ ID NO:23.

15. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-14 that competes with a reference antibody for binding to the CD38 protein, wherein the reference antibody comprises a light chain and a heavy chain, wherein the reference antibody light chain comprises the amino acid sequence shown in any of SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20; and the heavy chain of the reference antibody contains the amino acid sequence shown in any of SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:21.

16. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-15, wherein the antibody comprises an antibody light chain or fragment thereof.

17. An antibody or antigen-binding fragment thereof according to embodiment 16, wherein the antibody light chain or fragment thereof comprises an LCDR1 and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.

18. An antibody or antigen-binding fragment thereof according to any one of embodiments 16-17, wherein the antibody light chain or fragment thereof contains LCDR2 and LCDR2 contains the amino acid sequence shown in SEQ ID NO:2.

19. An antibody or antigen-binding fragment thereof according to any one of embodiments 16-18, wherein the antibody light chain or fragment thereof contains an LCDR3 and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:3.

20. An antibody or antigen-binding fragment thereof according to any one of embodiments 16-19, wherein the antibody light chain or fragment thereof comprises a VL light chain variable region and the VL light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7.

21. An antibody or antigen-binding fragment thereof according to any one of embodiments 16-20, wherein the antibody light chain or fragment thereof further comprises a human constant region.

22. An antibody or antigen-binding fragment thereof according to embodiment 21, wherein the human human constant region comprises a human Igκ constant region.

23. An antibody or antigen-binding fragment thereof according to any one of embodiments 16-22, wherein the antibody light chain or fragment thereof comprises the amino acid sequence shown in SEQ ID NO:22.

24. An antibody or antigen-binding fragment thereof according to embodiment 27, wherein the antibody light chain or fragment thereof comprises the amino acid sequence shown in any of SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO :twenty.

25. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-24, wherein the antibody comprises an antibody heavy chain or fragment thereof.

26. An antibody or antigen-binding fragment thereof according to embodiment 25, wherein the antibody heavy chain or fragment thereof contains HCDR1 and HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

27. An antibody or antigen-binding fragment thereof according to any one of embodiments 25-26, wherein the antibody heavy chain or fragment thereof contains HCDR2 and HCDR2 contains the amino acid sequence shown in SEQ ID NO:5.

28. An antibody or antigen-binding fragment thereof according to any one of embodiments 25-27, wherein the antibody heavy chain or fragment thereof contains HCDR3 and HCDR3 contains the amino acid sequence shown in SEQ ID NO:6.

29. An antibody or antigen-binding fragment thereof according to any one of embodiments 25-28, wherein the antibody heavy chain or fragment thereof comprises a VH heavy chain variable region and the VH heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.

30. An antibody or antigen-binding fragment thereof according to any one of embodiments 25-29, wherein the antibody heavy chain or fragment thereof further comprises a human constant region.

31. An antibody or antigen-binding fragment thereof according to embodiment 30, wherein the human constant region comprises a human IgG constant region.

32. An antibody or antigen-binding fragment thereof according to embodiment 31, wherein the IgG constant region comprises a human IgG1 constant region.

33. An antibody or antigen-binding fragment thereof according to any one of embodiments 25-32, wherein the heavy chain of the antibody contains the amino acid sequence shown in SEQ ID NO:23.

34. An antibody or antigen-binding fragment thereof according to embodiment 33, wherein the heavy chain of the antibody comprises the amino acid sequence shown in any of SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21.

35. One or more isolated nucleic acid molecules comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of embodiments 1-34.

36. A nucleic acid molecule according to embodiment 35, wherein at least one of the nucleic acid molecules is codon-optimized.

37. A nucleic acid molecule according to any one of embodiments 35-36, wherein the nucleic acid molecule comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14.

38. A vector containing a nucleic acid molecule according to any of embodiments 35-37.

39. A cell containing a nucleic acid molecule according to any of embodiments 35-37 or a vector according to embodiment 38.

40. A method for producing an antibody or antigen-binding fragment thereof according to any one of Embodiments 1-34, comprising culturing the cell according to Embodiment 39 under conditions that allow expression of the antibody or antigen-binding fragment thereof.

41. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any of embodiments 1-34, a nucleic acid molecule according to any of embodiments 35-37, a vector according to any of embodiments 38, or a cell according to any of embodiments 38, with Embodiment 39, and optionally a pharmaceutically acceptable adjuvant.

42. The use of an antibody or antigen-binding fragment thereof according to any of embodiments 1-34 for the preparation of medicaments for the prevention or treatment of tumors.

43. Use according to Embodiment 42, wherein the tumors comprise a CD38 positive tumor.

44. Use according to Embodiment 43, wherein the CD38 positive tumor is selected from the group consisting of multiple myeloma, lymphoma, and leukemia.

45. Use according to embodiments 42-44 wherein the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.

46. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-34 for the prevention or treatment of tumors.

47. An antibody or antigen-binding fragment thereof according to embodiment 46, wherein the tumors comprise a CD38 positive tumor.

48. The antibody or antigen-binding fragment thereof according to embodiment 47, wherein the CD38 positive tumor is selected from the group consisting of multiple myeloma, lymphoma, and leukemia.

49. Use according to any one of embodiments 46-48 wherein the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.

50. A method for preventing or treating tumors in an individual in need thereof, comprising administering to the individual an antibody or an antigen-binding fragment thereof according to any one of embodiments 1-34, a nucleic acid molecule according to any one of embodiments 35-37, a vector according to embodiment 38, a cell according to embodiment 39, and/or a pharmaceutical composition according to embodiment 40.

51. The method of embodiment 50 wherein the tumors comprise a CD38 positive tumor.

52. The method of embodiment 51 wherein the CD38 positive tumor is selected from the group consisting of multiple myeloma, lymphoma, and leukemia.

53. The method according to any one of embodiments 50-52, wherein the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.

54. A method for inhibiting the binding of a CD38 protein to a CD38 ligand, comprising administering an antibody or antigen-binding fragment thereof according to any one of embodiments 1-34, a nucleic acid molecule according to any one of embodiments 35-37, a vector according to embodiment 38, cells according to embodiment 39.

55. The method according to option 54, wherein the CD38 ligand comprises CD31.

Without being limited by any theory, the following examples are only intended to explain the mode of operation of the products, methods or systems of the present application, but are not intended to limit the scope of the invention of the present application.

EXAMPLES

Example 1: Preparation of hybridoma antibodies and gene cloning

Immunization of mice: balb/c mice (purchased from Beijing Vital River Labs Animal Technology Co., Ltd.) subcutaneously (s.c.) were immunized three times with Freund's adjuvant or incomplete Freund's adjuvant mixed with 100 µg of soluble CD38 antigen (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.). On day 0 complete Freund's adjuvant was used and on days 14 and day 28 incomplete Freund's adjuvant was used. Spleen cells from immunized mice were fused with SP2/0 myeloma cells (ATCC) according to the standard method.

Hybridoma Fusion: Mouse spleens were fused according to the currently conventional hybridoma fusion method, and fused hybridoma cells (10 ⁵ cells per well) were screened according to the HAT screening method. After 12 days, the supernatant was subjected to ELISA analysis using a microplate coated with CD38 antigen. Preferred clones were subjected to a second round of subcloning by the serial dilution method. The resulting hybridoma strain, which stably expressed the antibody of interest, was stored and used to construct libraries.

RNA Preparation: RNA was prepared using the TRIzol Reagent RNA Isolation Kit (purchased from Life technologies). A reverse transcription kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) was used to obtain cDNA encoding an antibody gene, and cDNA was used as a template for PCR amplification of the antibody variable region gene. The T-easy vector was cloned, and after cloning, the resulting heavy chain and light chain variable region sequences were sequenced to then obtain the corresponding amino acid sequences.

Example 2 Humanization and Antibody Production

The mouse antibody heavy and light chain variable region sequences obtained in Example 1 were subjected to sequence alignment with human antibody sequences from the NCBI database using the NCBI Online Sequence Alignment (IgBlast) method to identify potential sites for humanization. Next, a three-dimensional structure of the mouse antibody variable region was constructed using the SwissModel to determine sites for humanization. Humanization was carried out at the appropriate humanization sites to obtain the humanized antibody sequence.

The humanized antibody was named SG003, and the sequencing results showed that the LCDR1-3 amino acid sequences of the SG003 antibody are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the amino acid sequence of VL is shown in SEQ ID NO.7; the nucleotide sequence encoding VL obtained by codon optimization and reverse translation is shown in SEQ ID NO.9; the amino acid sequences of HCDR1-3 of antibody SG003 are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively; the amino acid sequence of VH is shown in SEQ ID NO.8; and the nucleotide sequence encoding the VH obtained by codon optimization and reverse translation is shown in SEQ ID NO.10.

The light chain amino acid sequence of antibody SG003 is shown in SEQ ID NO.11; and the nucleotide sequence encoding it is shown in SEQ ID NO.12. The amino acid sequence of the heavy chain of antibody SG003 is shown in SEQ ID NO.13; and the nucleotide sequence encoding it is shown in SEQ ID NO.14.

The resulting humanized antibody variable region genes were cloned into the pCMV-163 eukaryotic expression vector containing the human IgG constant region gene to construct a whole antibody expression vector with the physical map shown in FIG. 1. In FIG. 1 shows the various components of the pCMV-163 eukaryotic expression vector known in the art and recombined in the order shown.

The ExpiCHOTM Expression System Kit (purchased from Thermo Fisher Scientific) was used to transfect a eukaryotic expression vector encoding the SG003 antibody into CHO-S cells for expression, and cell culture supernatant containing the protein of interest was harvested. The target antibody was purified using conventional protein A affinity purification.

Example 3 Binding of Humanized Antibody to Target Antigen

The ELISA plate was covered with a PBS solution containing 1 μg/ml CD38-His fusion protein at 4°C overnight. The plate was then washed with a PBS solution containing 0.01% Tween-20 (PBST). 10% fetal calf serum was added to PBST and blocked at 37°C for one hour. Then, various concentrations of SG003 antibody and control antibody daratumumab (DARZALEX) were added and reacted at 37° C. for one hour. After washing with PBST, horseradish peroxidase (HRP) labeled secondary goat anti-human IgG antibodies (purchased from Thermo Fisher Scientific) were added and reacted at 37° C. for 30 minutes. The plate was then washed with PBST (5 times) and the remaining drops of liquid were removed as far as possible with blotting paper. 100 μl TMB (purchased from eBioscience) was added to each well and placed at room temperature (20±5°C) in the dark for 1-5 minutes. 100 μl of 2N H ₂ SO ₄ stopping solution was added to each well to quench the reaction with the substrate. The OD value was read at 450 nm with a microplate reader and the binding capacity of the antibody to the target CD38-His antigen was analyzed. The results show that the SG003 antibody showed stronger binding activity than the control antibody daratumumab, and the results are shown in FIG. 2. In FIG. 2, the EC50 value is 35.1±10.5 ng/mL, while the EC50 value of the daratumumab antibody is 150.9±105.8 ng/mL.

Example 4 Affinity Determination of a Humanized Antibody

Antibody affinity was analyzed using the BIACORE Biomacromolecular Interaction Instrument (GE). An anti-human IgG-Fab (purchased from Abcam) was linked to the chip and the anti-human IgG was used to capture the SG003 antibody. The antibody concentration was set to 1 μg/ml, the injection time was 60-150 seconds; CD38 antigen was used as mobile phase; six concentration gradients were used (3.125, 6.25, 12.5, 25, 50, 100 nM), association time was 120 seconds; the dissociation time was 1200 seconds; used 10 mM glycine-hydrochloric acid buffer (pH 2.1) to regenerate within 60 seconds. The results showed that the SG003 antibody has the affinity shown in Table 1.

Table 1: Affinity of SG003 antibody Antibody Association constant
(10 ⁶ ⋅1/Ms) Dissociation constant
(10 ^-4 ⋅1/s) Relative affinity
( ^10-10M ) SG003 2.86±0.00 9.18±0.01 3.21±0.01

Example 5 Specific Recognition of a Target Antigen by a Humanized Antibody

Various proteins such as milk protein (Beijing Bomed Biotechnology Co., Ltd.), BSA (BOVOGEN), CD19 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), TROP2 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), CD47 (Beijing Magpel Biotech Technology Co., Ltd.), CD38 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), Gas6 (R&D), and AXL (ACRO Biosystems) were respectively used to coat ELISA strips at 1 mg/mL at 4°C during the night. After washing with PBST, 10% fetal calf serum was added and blocked at 37° C. for one hour. SG003 antibody was added and reacted at 37°C for one hour. After washing with PBST, horseradish peroxidase (HRP) labeled secondary goat anti-human IgG antibodies (purchased from Thermo Fisher Scientific) were added and reacted at 37° C. for 30 minutes. The plate was then washed with PBST (5 times) and the remaining drops of liquid were removed as far as possible with blotting paper. 100 μl TMB (eBioscience) was added to each well and placed at room temperature (20±5°C) in the dark for 1-5 minutes. 100 μl of 2N H ₂ SO ₄ stopping solution was added to each well to quench the reaction with the substrate, and the OD value at 450 nm was read using a microspectrophotometer to read antibody-protein binding assay plates.

The results are shown in FIG. 3. Results in FIG. 3 shows that the SG003 antibody can specifically recognize the target CD38 antigen, but does not show a specific binding reaction with milk protein, BSA, CD19, TROP2, CD47, AXL, Gas6, and other proteins.

Example 6 Binding of Humanized Antibody to Cell Surface Antigen

Flow analysis technology was used to detect the binding of CD38 proteins on the surfaces of Raji cells, Daudi cells, Ramos cells and RPMI8226 cells to the SG003 antibody and the control antibody daratumumab. Collected cells in the phase of logarithmic growth, adjusted the cell density to 5×10 ⁶ cells/ml, and pre-cooled on ice. The SG003 antibody and the control antibody daratumumab were diluted to various concentrations with pre-chilled saline containing 2% FBS. Took 100 μl of cells, and added an equal volume of diluted antibody to carry out the reaction at 4°C for 30 minutes in the dark. After completion of the reaction, the cells were washed twice. Cells were resuspended in 100 μl of diluted phycoerythrin-labeled anti-IgG secondary mouse antibodies (purchased from BD Pharmingen) and reacted at 4° C. in the dark for 30 minutes. After completion of the reaction, the cells were washed twice with pre-chilled saline containing 2% FBS. Cells were resuspended in 400 μl of 1% paraformaldehyde. Flow cytometry (BD Calibur) was used to analyze the binding capacity of antibodies to antigens on the cell surface.

The results show that the SG003 antibody can specifically recognize CD38 on the surface of Raji cells, Daudi cells, Ramos cells, and RPMI8226 cells. This recognition activity is significantly higher than the control antibody Daratumumab and is dose dependent, and the EC50 value for SG003 and Raji cells (Fig. 4A) was 34.4 ng/mL and for Daratumumab was 49.3 ng/mL. The EC50 value for SG003 and Daudi cells (Fig. 4B) was 36.7 ng/mL and for Daratumumab it was 50.9 ng/mL. The EC50 value for SG003 and Ramos cells (Fig. 4C) was 81.5 ng/mL and for Daratumumab it was 95.1 ng/mL. The EC50 value for SG003 and RPMI8226 cells (Fig. 4D) was 140.3 ng/mL and for Daratumumab it was 176.5 ng/mL.

Example 7: Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) of a Humanized Antibody

First, the density of target cells (Raji cells, Daudi cells, Ramos cells and RPMI8226 cells) required for the experiment was adjusted to 2×10 ⁵ cells/ml, resuspended in ADCC buffer (MEM without phenol red + 1% FBS) , and added to a 96-well plate (50 μl/well). Then, 100 μl of SG003 antibody at various concentrations was added to each well, mixed until homogeneous and incubated at 37°C in an incubator containing 5% CO ₂ for 30 minutes. Then, the density of the desired NK92MI-CD16a effector cells (purchased from Huabo Bio) was adjusted to 1.2×10 ⁶ cells/ml, and added to the target cell wells at a ratio of target cells to effector cells of 1:6. After mixing, the mixture was incubated at 37°C for 4-6 hours in an incubator containing 5% CO ₂ and then part of the stock solution (100 μl/well) was removed in a 96-well plate and the LDH reaction mixture was added to each well. from the LDH Detection Kit (Cytotoxicity Detection Kit, purchased from Roche) (100 µl/well). The mixture was reacted at 37° C. for 10 minutes. Then, stop solution (50 µl/well) was added and mixed gently. The OD value was read at 492 nm using a microplate reader, and the OD value at 650 nm was used as the background value. The following control groups were also used in the experiment: control 1 was ADCC buffer, control 2 was target cells+ADCC buffer, control 3 was target cells+lysate+ADCC buffer, and control 4 was target cells+effector cells+ buffer for ADCC. Specific lysis rate (%) = ((experimental group-control 4)/(control 3-control 2))×100%. The dose-response curve was analyzed using GraphPad Prism, version 5.

The results show that when tested with NK92MI-CD16a effector cells and Raji target cells, SG003 showed very efficient ADCC activity (see Figure 5A), with an EC50 value of 6.97 ng/mL for SG003 and 6.97 ng/mL for Daratumumab. 17.0 ng/ml. When Daudi cells were used as the target cell, the EC50 value for SG003 was 0.72 ng/ml and for Daratumumab was 4.6 ng/ml (see Fig. 5B). At the same time, the SG003 antibody also showed effective ADCC activity against Ramos cells (see Fig. 5C). The EC50 value for SG003 was 0.69±0.007 ng/mL and for Daratumumab it was 3.01 ng/mL. The SG003 antibody also showed effective ADCC activity against RPMI8226 cells (see Fig. 5D) with an EC50 value for SG003 of 1.46 ng/mL and for Daratumumab 4.94 ng/mL.

Example 8: In vivo inhibition of tumor activity with a humanized antibody

A tumor model was created by inoculating Raji-Luc cells into CB17 SCID mice to evaluate the effect of the SG003 antibody in inhibiting tumor activity.

CB17 female mice aged 5-8 weeks (purchased from Beijing Biocytogen Co., Ltd.) were selected for the experiment. The Raji-Luc cell is a stable cell line obtained by Beijing Biocytogen Co., Ltd. based on a Raja cell that was transformed with a luciferin reporter gene. After reconstitution and cultivation to the required number, cells in the logarithmic growth phase were harvested and suspended to a concentration of 5×10 ⁶ cells/0.2 ml. CB17 SCID mice were inoculated with cells via the tail vein at 0.2 ml/mouse. After inoculation, tumor growth and body weight were monitored with a small animal imager on Day 0 and Day 7. On Day 7, 18 mice with moderate tumor imaging signals were selected and randomly assigned to three groups, six mice per group. Then the animals were injected. There was a saline control group, a positive control group (Daratumumab, purchased from Johnson & Johnson) and an experimental group (SG003) at 200mcg/kg, once every two weeks, twice in total. The mice were then monitored for body weight, tumor growth, and survival rate.

The results show that both daratumumab and SG003 can be tolerated in experimental animals. Animals in each group showed a significant inhibitory effect on tumor growth compared to the control group (see Fig. 6). On Day 7 after the first injection, the mean fluorescence intensity in the daratumumab and SG003 antibody treatment groups were respectively (mean 3.92×10 ⁷ , SEM 4.04×10 ⁶ ), (mean 3.19×10 ⁷ , SEM 9 .32×10 ⁶ ), which is significantly lower than the average tumor fluorescence intensity in the control group (mean 3.04×10 ⁸ , standard error 4.29×10 ⁷ ).

Then, the survival rate of the animals was additionally monitored. The median survival time in the control group was 12 days after grouping, for Daratumumab it was 21 days, and for the SG003 group it was 28 days. The SG003 group outperformed the Daratumumab group (see FIG. 7).

Example 9 Species Recognition Specificity for a Chimeric Antibody

Extracellular proteins CD38 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) of different species - human (Human), mouse (Mus), rat (Rattus), cynomolgus monkey (Cynomolgus), etc. - used to coat ELISA strips at a concentration of 1 mg/ml at 4°C overnight. After washing with PBST, 10% fetal calf serum was added and blocked at 37° C. for one hour. Test antibodies SG003 and control antibodies Daratumumab were added and reacted at 37° C. for one hour. After washing with PBST, horseradish peroxidase (HRP) labeled secondary goat anti-human IgG (purchased from Thermo Fisher Scientific) was added and reacted at room temperature for 30 minutes. The plate was washed with PBST (5 times) and the remaining drops were removed as far as possible with blotting paper. 100 μl TMB (purchased from eBioscience) was added to each well and placed at room temperature (20±5°C) in the dark for 1-5 minutes. 100 µl of 2N H ₂ SO ₄ stopping solution was added to each well to quench the reaction with the substrate, and the OD value at 450 nm was read using a microspectrophotometer for reading plates to analyze the binding ability of antibodies to CD38 protein of various species.

The results show that the SG003 antibody can recognize human and cynomolgus CD38 protein molecules, but does not bind to mouse and rat CD38 protein molecules; the results are shown in FIG. 8A.

The control antibody Daratumumab only recognizes human CD38 protein molecules and does not bind to CD38 protein molecules from mice, rats, and cynomolgus monkeys. The results are shown in FIG. 8B.

Example 10: Maintaining the biological activity of a humanized antibody after mutation

Using genetic engineering technology, the SG003 antibody variable region sequences can be randomly mutated to generate new antibody variable region sequences, and further generate new antibody light chain and heavy chain sequences. For example, for an antibody resulting from a random mutation based on the SG003 antibody, the light chain amino acid sequence may be SEQ ID NO:16 and the heavy chain amino acid sequence may be SEQ ID NO:17; or, the light chain amino acid sequence may be SEQ ID NO:18 and the heavy chain amino acid sequence may be SEQ ID NO:19; alternatively, the light chain amino acid sequence may be SEQ ID NO:20 and the heavy chain amino acid sequence may be SEQ ID NO:21. Antibodies containing the above-described combination of light chain and heavy chain after mutation are referred to as SG003M1-3 in the sequence (for example, the light chain amino acid sequence of the antibody SG003M1 is SEQ ID NO:16, and the amino acid sequence of the heavy chain is SEQ ID NO:17).

A new antibody was prepared using the method of Example 2, and the activity of the antibody was evaluated using the method of Example 4. The randomly mutated SG003M1-3 antibody can recognize the CD38 antigen (see Fig. 9). The EC50 value of SG003M1 was determined to be 15.72 ng/ml, the EC50 value of SG003M2 was 27.75 ng/ml, and the EC50 value of SG003M3 was 6.10 ng/ml. SG003M1 was randomly selected and affinity results determined as shown in Table 2.

Table 2: Affinity analysis of SG003 mutants Antibody Association constant
(10 ⁶ ⋅1/Ms) Dissociation constant
(10 ^-4 ⋅1/s) affinity
( ^10-10M ) SG003M1 2.66±0.01 6.36±0.05 2.39±0.03

Example 11: Confirmation of epitopes for a humanized antibody

An epitope mapping method was used to analyze the epitopes of the SG003 antibody recognizing the CD38 antigen. Mutants of the CD38 antigen (Table 3, where all mutated amino acids are alanine) were designed using the alanine substitution method, and wild-type CD38 as well as various mutant genes were generated by polymerase chain reaction (PCR) and overlapping primers (overlapping) and other methods. molecular biology. After separation and recovery by agarose gel electrophoresis, the antibody or mutants were digested with restriction enzymes HindIII and NheI and cloned into the pEGFP-N1 vector. After sequencing the correct sequences, the HighGene Transfection Kit (ABclonal) was used to transfect the vector containing the wild-type CD38 or mutant gene into 293T cells according to the method described in the kit instructions. 48 hours after transfection, cells were harvested by trypsin digestion to prepare a single cell suspension. Target cells and SG003 antibody were incubated at a working concentration of 10 μg/ml for 30 minutes at a constant temperature of 4°C. Then, GAH-IgG Fc PE (Invitrogen, Cat No: 12-4998-82) was added and incubated at a constant temperature of 4°C for 30 minutes, and the fluorescence intensity of the channels for FITC and PE was measured by flow cytometry, where the fluorescence intensity for FITC represents the level of expression of the CD38 molecule and its mutants, and the fluorescence intensity for PE represents the binding strength of the SG003 antibody to the antigen.

The flow cytometry results in Fig. 10 show that the SG003 antibody does not bind to mutants 21, 22, and 23, suggesting that the amino acid region 60-89 of the CD38 protein is the main epitope that is recognized by the SG003 antibody.

Table 3: Mutants and Mutation Sites for the CD38 Protein Name Mutation site CD38 wild-type N/A CD38M4 291-300 CD38M5 281-290 CD38M6 256-266 CD38M7 247-255 CD38M8 210-219 CD38M9 200-209 CD38M10 190-199 CD38M11 180-189 CD38M12 170-179 CD38M13 160-169 CD38M14 150-159 CD38M15 140-149 CD38M16 130-139 CD38M17 120-129 CD38M18 110-119 CD38M19 100-109 CD38M20 90-99 CD38M21 80-89 CD38M22 70-79 CD38M23 60-69 CD38M24 50-59 CD38M25 43-49

The foregoing detailed description is provided by way of explanation and example, and is not intended to limit the scope of the appended claims. Many of the embodiments listed herein are obvious to those skilled in the art and are within the scope of the appended claims and their equivalents.

--->

SEQUENCE LIST

<110> HANGZHOU SUMGEN BIOTECH CO., LTD.; SUMGENMAB (BEIJING)

BIOTECH CO., LTD.

<120> ANTIBODIES TO CD38 PROTEIN AND THEIR USE

<130> 0070-PA-008EN

<160> 23

<170>PatentIn version 3.5

<210> 1

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR1

<400> 1

Arg Ala Ser Ser Ser Val Ser Ser Ser Ala Phe Ser Tyr Val His

1 5 10 15

<210> 2

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR2

<400> 2

Leu Ala Ser Asn Leu Glu Ser

fifteen

<210> 3

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR3

<400> 3

His His Ser Arg Glu Leu Pro Phe Thr

fifteen

<210> 4

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR1

<400> 4

Leu Tyr Trp Met Asn

fifteen

<210> 5

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR2

<400> 5

Lys Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu Lys

1 5 10 15

asp

<210> 6

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR3

<400> 6

Leu Trp Ile Ala Thr Gly Gly Phe Asp Tyr

1 5 10

<210> 7

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> VL

<400> 7

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Ala Phe Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

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

100 105 110

<210> 8

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> VH

<400> 8

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Trp Ile Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 9

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized VL nucleotide sequence

<400> 9

gagatcgtga tgacccagag ccctgccagc ctgagcgcca gcctgggcca gagggccacc

60

atcagctgca gggccagcag cagcgtgagc agcagcgcct tcagctacgt gcactggtac

120

cagcagaaga gcggccagcc tcctaagctg ctgatctacc tggccagcaa cctggagagc

180

ggcgtgcctg ccaggttcag cggcagcggc agcggcaccg acttcaccct gaccatccac

240

cctgtggaga gcgaggacgt ggccacctac tactgccacc acagcaggga gctgcctttc

300

accttcggca gcggcaccaa gctggagatc aag

333

<210> 10

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized VH nucleotide sequence

<400> 10

caggtgcagc tgctggagag cggcggcggc ctggtgcagc ctggcggcag cctgaagctg

60

agctgcgtgg ccagcggctt cgacttcagc ctgtactgga tgaactgggt gaggcaggcc

120

cctggcaagg gcctggagtg gatcggcaag atcaaccctg acagcagcac catcaactac

180

acccctagcc tgaaggacaa gttcttcatc agcagggaca acgccaagaa caccctgtac

240

ctgcagatga ccaaggtgag gagcgaggac accgccctgt actactgcgc caggctgtgg

300

atcgccaccg gcggcttcga ctactggggc cagggcacca ccctgaccgt gagcagc

357

<210> 11

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> light chain SG003

<400> 11

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Ala Phe Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Glu Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 12

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> light chain nucleotide sequence SG003

<400> 12

gagatcgtga tgacccagag ccctgccagc ctgagcgcca gcctgggcca gagggccacc

60

atcagctgca gggccagcag cagcgtgagc agcagcgcct tcagctacgt gcactggtac

120

cagcagaaga gcggccagcc tcctaagctg ctgatctacc tggccagcaa cctggagagc

180

ggcgtgcctg ccaggttcag cggcagcggc agcggcaccg acttcaccct gaccatccac

240

cctgtggaga gcgaggacgt ggccacctac tactgccacc acagcaggga gctgcctttc

300

accttcggca gcggcaccaa gctggagatc aagcgtacgg tggctgcacc atctgtcttc

360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg

420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg

480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc

540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc

600

acccatcagg gcctgagctc gcccgtcaca aaggcttca acaggggaga gtgttag

657

<210> 13

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> heavy chain SG003

<400> 13

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Trp Ile Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

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> 14

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> heavy chain nucleotide sequence SG003

<400> 14

caggtgcagc tgctggagag cggcggcggc ctggtgcagc ctggcggcag cctgaagctg

60

agctgcgtgg ccagcggctt cgacttcagc ctgtactgga tgaactgggt gaggcaggcc

120

cctggcaagg gcctggagtg gatcggcaag atcaaccctg acagcagcac catcaactac

180

acccctagcc tgaaggacaa gttcttcatc agcagggaca acgccaagaa caccctgtac

240

ctgcagatga ccaaggtgag gagcgaggac accgccctgt actactgcgc caggctgtgg

300

atcgccaccg gcggcttcga ctactggggc cagggcacca ccctgaccgt gagcagcgct

360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc

420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg

480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga

540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac

600

atctgcaacg tgaatcacaa gccagcaac accaaggtgg acaagaaagt tgagcccaaa

660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg

720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag

780

gtcacgtgcg tggtggtgga cgtgagccac gaagaccccg aggtcaagtt caactggtac

840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc

900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag

960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa

1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg

1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc

1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg

1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag

1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag

1320

aagagcctct ccctgtctcc gggtaaatga

1350

<210> 15

<211> 30

<212> Protein

<213> Homo sapiens

<400> 15

Pro Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro

1 5 10 15

Glu Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe

20 25 30

<210> 16

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Mutated light chain 1

<400> 16

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

1 5 10 15

Gln Arg Ala Ser Ile Ser Cys Arg Ala Ser Asn Ser Val Ser Ser Ser

20 25 30

Ala Tyr Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Ile Gln Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Gln Leu Pro Ser Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 17

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Mutated heavy chain 1

<400> 17

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Tyr Asn Phe Ser Leu Tyr

20 25 30

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

35 40 45

Gly Lys Ile Gln Pro Glu Ser Ser Thr Ile Gln Tyr Thr Pro Ser Leu

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Trp Ile Gly Ser Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

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> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Mutated light chain 2

<400> 18

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Ala Tyr Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asp Leu Gln Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

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

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 19

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Mutated heavy chain 2

<400> 19

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Tyr Ile Ala Ser Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

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> 20

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Mutated light chain 3

<400> 20

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

1 5 10 15

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

20 25 30

Ala Phe Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Glu Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Mutated heavy chain 3

<400> 21

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

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

35 40 45

Gly Lys Ile Ser Pro Asp Ser Ser Ser Leu Asn Tyr Thr Pro Ser Val

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Trp Ile Ala Thr Gly Gly Tyr Asn Tyr Trp Gly Gln Gly

100 105 110

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

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> 22

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> light chain formula

<220>

<221>X20

<222> (20)..(20)

<223> X20=T or S

<220>

<221>X27

<222> (27)..(27)

<223> X27=S or N

<220>

<221>X31

<222> (31)..(31)

<223> X31=S or T

<220>

<221>X34

<222> (34)..(34)

<223> X34=F or Y

<220>

<221> X57

<222> (57)..(57)

<223> X57=N or D

<220>

<221> X58

<222> (58)..(58)

<223> X58=L or I

<220>

<221>X59

<222> (59)..(59)

<223> X59=E or Q

<220>

<221> X97

<222> (97)..(97)

<223> X97=E or Q

<220>

<221>X98

<222> (98)..(98)

<223> X98=L or V

<220>

<221> X100

<222> (100)..(100)

<223> X100=F or S

<220>

<221> X101

<222> (101)..(101)

<223> X101=T or S

<400> 22

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

1 5 10 15

Gln Arg Ala Xaa Ile Ser Cys Arg Ala Ser Xaa Ser Val Ser Xaa Ser

20 25 30

Ala Xaa Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Xaa Xaa Xaa Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Xaa Xaa Pro Xaa Xaa Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 23

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> heavy chain formula

<220>

<221>X27

<222> (27)..(27)

<223> X27=F or Y

<220>

<221>X28

<222> (28)..(28)

<223> X28=D or N

<220>

<221>X33

<222> (33)..(33)

<223> X33=W or Y

<220>

<221>X52

<222> (52)..(52)

<223> X52=N, Q or S

<220>

<221>X54

<222> (54)..(54)

<223> X54=D, E or N

<220>

<221> X57

<222> (57)..(57)

<223> 57=T or S

<220>

<221>X58

<222> (58)..(58)

<223> X58=I or L

<220>

<221> X59

<222> (59)..(59)

<223> X59=N or Q

<220>

<221> X61

<222> (61)..(61)

<223> X61=T or S

<220>

<221>X64

<222> (64)..(64)

<223> X64=L or V

<220>

<221> X100

<222> (100)..(100)

<223> X100=W or Y

<220>

<221> X102

<222> (102)..(102)

<223> X102=A or G

<220>

<221> X103

<222> (103)..(103)

<223> X103=T or S

<220>

<221> X106

<222> (106)..(106)

<223> X106=F or Y

<220>

<221> X107

<222> (107)..(107)

<223> X107=D or N

<400> 23

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Xaa Xaa Phe Ser Leu Tyr

20 25 30

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

35 40 45

Gly Lys Ile Xaa Pro Xaa Ser Ser Xaa Xaa Xaa Tyr Xaa Pro Ser Xaa

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Xaa Ile Xaa Xaa Gly Gly Xaa Xaa Tyr Trp Gly Gln Gly

100 105 110

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

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

<---

Claims (58)

1. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, containing the light chain of the antibody or fragment thereof and the heavy chain of the antibody or fragment thereof, where the light chain of the antibody or its fragment contains LCDR1, LCDR2 and LCDR3 and the heavy chain of the antibody or its fragment contains HCDR1, HCDR2 and HCDR3, LCDR1 contains the amino acid sequence of SEQ ID NO:1, LCDR2 contains the amino acid sequence of SEQ ID NO:2, LCDR3 contains the amino acid sequence SEQ ID NO:3, HCDR1 contains the amino acid sequence of SEQ ID NO:4, HCDR2 contains the amino acid sequence of SEQ ID NO:5, and HCDR3 contains the amino acid sequence of SEQ ID NO:6. 2. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, containing LCDR1, LCDR2 and LCDR3 of the antibody light chain and HCDR1, HCDR2 and HCDR3 of the antibody heavy chain, the antibody light chain contains the amino acid sequence of SEQ ID NO:16 and the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:17. 3. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, containing LCDR1, LCDR2 and LCDR3 of the antibody light chain and HCDR1, HCDR2 and HCDR3 of the antibody heavy chain, the antibody light chain contains the amino acid sequence of SEQ ID NO:18 and the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:19. 4. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, containing LCDR1, LCDR2 and LCDR3 of the antibody light chain and HCDR1, HCDR2 and HCDR3 of the antibody heavy chain, the antibody light chain contains the amino acid sequence of SEQ ID NO:20 and the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:21. 5. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, containing the light chain variable region VL of the light chain of the antibody and the variable region of the heavy chain VH of the heavy chain of the antibody, the antibody light chain contains the amino acid sequence of SEQ ID NO:16 and the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:17. 6. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, comprising the light chain variable region VL of the light chain of the antibody and the variable region of the heavy chain VH of the heavy chain of the antibody, the antibody light chain contains the amino acid sequence of SEQ ID NO:18 and the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:19. 7. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, containing the light chain variable region VL of the light chain of the antibody and the variable region of the heavy chain VH of the heavy chain of the antibody, the antibody light chain contains the amino acid sequence of SEQ ID NO:20 and the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:21. 8. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, containing an antibody light chain and an antibody heavy chain, the antibody light chain contains the amino acid sequence of SEQ ID NO:16 and the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:17. 9. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, containing an antibody light chain and an antibody heavy chain, the antibody light chain contains the amino acid sequence of SEQ ID NO:18 and the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:19. 10. An antibody or antigen-binding fragment thereof that binds to the CD38 protein, containing an antibody light chain and an antibody heavy chain, the antibody light chain contains the amino acid sequence of SEQ ID NO:20 and the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:21. 11. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, which binds to CD38 with a K _D value of 1×10 ^-9 M or less. 12. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof kills and damages tumor cells and/or inhibits tumor growth through specific binding to the CD38 protein. 13. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. 14. An antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv and ScFv fragments. 15. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the CD38 protein is a human CD38 protein or a simian CD38 protein. 16. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody light chain or fragment thereof comprises a VL light chain variable region and the VL light chain variable region comprises the amino acid sequence of SEQ ID NO:7. 17. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:22. 18. An antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:11. 19. An antibody or antigen-binding fragment thereof according to claim 1 or 16, wherein the heavy chain of the antibody or fragment thereof comprises a VH heavy chain variable region and the VH heavy chain variable region comprises the amino acid sequence of SEQ ID NO:8. 20. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO:23. 21. An antibody or antigen-binding fragment thereof according to claim 1 or 18, wherein the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO:13. 22. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-21. 23. A nucleic acid molecule according to claim 22, wherein the nucleotide is codon-optimized. 24. A nucleic acid molecule according to any one of claims 22-23, wherein the nucleic acid molecule contains one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14. 25. An expression vector containing a nucleic acid molecule according to any one of claims 22-24. 26. A host cell for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, containing a nucleic acid molecule according to any one of claims 22 to 24 or a vector according to claim 25. 27. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, comprising culturing the cell according to claim 26 under conditions that allow expression of the antibody or antigen-binding fragment thereof. 28. A pharmaceutical composition for the treatment of tumors, comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21, a nucleic acid molecule according to any one of claims 22 to 24, a vector according to claim 25 and / or a cell according to claim 26, and optionally a pharmaceutically acceptable adjuvant. 29. A method for preventing or treating tumors, comprising administering to a patient an antibody or an antigen-binding fragment thereof according to any one of claims 1-21. 30. The method of claim 29, wherein the tumors include a CD38 positive tumor. 31. The method of claim 30, wherein the CD38 positive tumor is selected from the group consisting of multiple myeloma, lymphoma, and leukemia. 32. A method for inhibiting the binding of a CD38 protein to a CD38 ligand, comprising administering an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21, a nucleic acid molecule according to any one of claims 22 to 24, a vector according to claim 25 and/or a cell according to item 26. 33. The method of claim 32 wherein the CD38 ligand comprises CD31.

Images