Antibodies that bind to human CD38, methods for their preparation and uses

Abstract

An antibody or antigen-binding fragment thereof that binds to human CD38, which can effectively bind to human CD38 and is used in the preparation of drugs for treating diseases with strong CD38 expression (such as multiple myeloma), has good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of tumor treatment, and relates to an antibody that binds to human CD38, a preparation method thereof, and uses thereof. Background Art

[0002] Multiple myeloma (MM) is a malignant tumor of plasma cells, accounting for about 1% of all cancers. It is prone to involve the bones and kidneys, and is liable to cause pathological fractures, bone pain, spinal cord compression, and renal failure. For the treatment of multiple myeloma, traditional treatment methods such as chemotherapy and radiotherapy are difficult to achieve satisfactory curative effects. In the past decade, in-depth studies on myeloma biology have revealed many new potential therapeutic targets.

[0003] CD38 is a 46 kDa type II transmembrane glycoprotein. CD38 has been found to have multiple functions, including extracellular enzyme activity and regulation of receptor-mediated cell adhesion and signal transduction. The enzymatic activity of CD3 involves the conversion of nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) into cyclic adenosine diphosphate ribose (CADPR), ADPR, and nicotinic acid adenine dinucleotide phosphate (NAADP), which are substrates necessary for regulating intracellular calcium signaling. In the preliminary study of the CD38 receptor function, it was found that CD38 mediates the binding of cells to endothelial cells, plays a role in lymphocyte migration, and has a functional connection with surface molecules of T, B, and natural killer (NK) cells. CD38 is highly expressed only in progenitor bone marrow, B lymphocytes in germinal centers, terminally differentiated plasma cells, and activated tonsils, while mature and memory B lymphocytes express low levels of CD38. CD38 is strongly expressed on multiple myeloma cells, making it an ideal target for the treatment of multiple myeloma. Currently, the marketed Daratumumab and Isatuximab have shown excellent performance in the treatment of multiple myeloma. At present, there are no related products in China, and the demand for antibody therapy remains unmet. Summary of the Invention

[0004] In order to solve the above technical problems, the inventors of the present invention conducted a large number of experiments. From antigen immunization, hybridoma preparation and screening, antibody expression and purification to bioactivity identification, a murine antibody that specifically binds to human CD38 was screened. On this basis, its chimeric antibody and humanized antibody were further constructed.

[0005] Accordingly, the object of the present invention is to provide an antibody or an antigen-binding fragment thereof that binds to human CD38; to provide a nucleotide molecule encoding the antibody or an antigen-binding fragment thereof that binds to human CD38; to provide an expression vector containing the nucleotide molecule; to provide a host cell of the expression vector; to provide a method for preparing the antibody or an antigen-binding fragment thereof that binds to human CD38; to provide a pharmaceutical composition containing the antibody or an antigen-binding fragment thereof that binds to human CD38; and to provide the use of the antibody or an antigen-binding fragment thereof that binds to human CD38 in the preparation of a drug.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides an antibody or an antigen-binding fragment thereof that binds to human CD38, including:

[0008] (a) heavy chain complementarity-determining regions H-CDR1, H-CDR2, H-CDR3, wherein the amino acid sequence of H-CDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of H-CDR2 is as shown in SEQ ID NO: 2, and the amino acid sequence of H-CDR3 is as shown in SEQ ID NO: 3, and

[0009] (b) light chain complementarity-determining regions L-CDR1, L-CDR2, L-CDR3, wherein the amino acid sequence of L-CDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of L-CDR2 is as shown in SEQ ID NO: 5, and the amino acid sequence of L-CDR3 is as shown in SEQ ID NO: 6.

[0010] The "antibody (Ab)" of the present invention is a heterotetrameric glycoprotein of approximately 150,000 daltons, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. The antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed by at least two antibodies (such as bispecific antibodies), etc.

[0011] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor variations resulting from naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, in contrast to conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0012] As used herein, the term "antigen-binding fragment" refers to a fragment of an antibody that is capable of specifically binding to human CD38. Examples of antigen-binding fragments of the present invention include Fab fragments, F(ab')2 fragments, Fv fragments, and the like. Fab fragments are fragments produced by digesting an antibody with papain. F(ab')2 fragments are fragments produced by digesting an antibody with pepsin. Fv fragments are dimers composed of the variable regions of the heavy and light chains of an antibody, which are tightly associated non-covalently.

[0013] Preferably, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0014] As used herein, the term "murine antibody" refers to an antibody derived from a rat or a mouse, preferably a mouse. The murine antibodies of the present invention are obtained by immunizing a mouse with human CD38 as an antigen and screening for hybridoma cells.

[0015] As used herein, the term "chimeric antibody" refers to an antibody that comprises variable region sequences of the heavy and light chains derived from one species and constant region sequences derived from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions. Preferably, the chimeric antibodies of the present invention are obtained by splicing the variable region sequences of the heavy and light chains of the murine antibody 50G12 to human constant regions. More preferably, the chimeric antibodies of the present invention are selected from 50G12-Chimeric.

[0016] As used herein, the term "humanized antibody" refers to an antibody in which the CDRs are derived from antibodies of a non-human species (preferably a mouse), and the remaining portions of the antibody molecule (including the framework regions and the constant regions) are derived from human antibodies. In addition, framework region residues may be altered to maintain binding affinity. Preferably, the humanized antibodies of the present invention are obtained by recombining the CDR regions of the murine antibody 50G12 and the non-CDR regions derived from human antibodies, adding a fourth framework region, and mutating some residues that have an important impact. More preferably, the humanized antibodies of the present invention are selected from 50G12-Humanized.

[0017] As a preferred embodiment, the antigen-binding fragment includes a Fab fragment, an F(ab’)2 fragment, and an Fv fragment.

[0018] As a preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment that binds to human CD38 is as shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8.

[0019] As a preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment that binds to human CD38 is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 10.

[0020] As a preferred embodiment, the amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment that binds to human CD38 is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 12.

[0021] On the other hand, the present invention provides a nucleotide molecule that encodes the above-mentioned antibody that binds to human CD38 or its antigen-binding fragment.

[0022] As a preferred embodiment, the nucleotide sequence encoding the heavy chain variable region of the nucleotide molecule is as shown in SEQ ID NO: 13, and the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO: 14.

[0023] As a preferred embodiment, the nucleotide sequence encoding the heavy chain variable region of the nucleotide molecule is as shown in SEQ ID NO: 15, and the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO: 16.

[0024] As a preferred embodiment, the nucleotide sequence encoding the heavy chain of the nucleotide molecule is as shown in SEQ ID NO: 17, and the nucleotide sequence encoding the light chain is as shown in SEQ ID NO: 18.

[0025] The preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the art. Preferably, it includes the following preparation methods: obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody through gene cloning techniques such as the PCR method, or obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody through the method of artificial total sequence synthesis.

[0026] Those skilled in the art are aware that the nucleotide sequences encoding the amino acid sequences of the antibodies or antigen-binding fragments thereof that bind to human CD38 can be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide homologues of the polynucleotide. The homologues of the polynucleotide in the present invention can be prepared by substituting, deleting or adding one or more bases of the gene encoding the antibody or antigen-binding fragment thereof that binds to human CD38 within the range of maintaining the antibody activity.

[0027] On the other hand, the present invention provides an expression vector, which contains the above-mentioned nucleotide molecule.

[0028] The expression vector herein is a conventional expression vector in the art, which refers to an expression vector containing appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and / or sequences, and other appropriate sequences. The expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid. For more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for nucleic acid manipulation can be found in Current Protocols in Molecular Biology, Second Edition, edited by Ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, pcDNA4, pDHFF, pGM-CSF or pCHO1.0.

[0029] In addition, the present invention provides a host cell, which contains the above-mentioned expression vector.

[0030] The host cells of the present invention are various conventional host cells in the art, as long as they can satisfy the stable self-replication of the above-mentioned recombinant expression vector and the effective expression of the carried nucleotide. The host cells include prokaryotic expression cells and eukaryotic expression cells. The host cells preferably include: COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5α, BL21(DE3) or TG1, more preferably E. coli TG1, BL21(DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies). Transforming the aforementioned expression vector into the host cell can obtain the preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the art, preferably chemical transformation, heat shock method or electroporation method.

[0031] On the other hand, the present invention provides a method for combining an antibody or an antigen-binding fragment thereof that binds to human CD38, the method comprising the following steps:

[0032] a) Culturing the above-mentioned host cells under expression conditions so as to express the antibody or an antigen-binding fragment thereof that binds to human CD38;

[0033] b) Separating and purifying the antibody or an antigen-binding fragment thereof that binds to human CD38 as described in a).

[0034] The method for culturing the host cells of the present invention and the methods for separating and purifying the antibody are conventional methods in the art. For specific operation methods, please refer to the corresponding cell culture technology manuals and antibody separation and purification technology manuals. The preparation method of the antibody or an antigen-binding fragment thereof that binds to human CD38 disclosed in the present invention includes: culturing the above-mentioned host cells under expression conditions so as to express the antibody or an antigen-binding fragment thereof that binds to human CD38; separating and purifying the antibody or an antigen-binding fragment thereof that binds to human CD38. By using the above method, the recombinant protein can be purified into a substantially homogeneous substance, for example, a single band on SDS-PAGE electrophoresis.

[0035] The antibody or an antigen-binding fragment thereof that binds to human CD38 disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer, pH change, etc. can be used to elute the antibody or an antigen-binding fragment thereof that binds to human CD38 bound to the affinity column. The inventors of the present invention conducted detection experiments on the obtained antibody or an antigen-binding fragment thereof that binds to human CD38. The experimental results show that the antibody or an antigen-binding fragment thereof that binds to human CD38 can bind well to the antigen and has a high affinity.

[0036] On the other hand, the present invention provides a composition containing the above-mentioned antibody or an antigen-binding fragment thereof that binds to human CD38 and a pharmaceutically acceptable carrier.

[0037] The antibody or an antigen-binding fragment thereof that binds to human CD38 provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert its efficacy more stably. These preparations can ensure the conformational integrity of the antibody or an antigen-binding fragment thereof that binds to human CD38 disclosed in the present invention, and at the same time protect the multiple functional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation). Usually, for liquid preparations, they can be stored stably at 2°C - 8°C for at least one year, and for freeze-dried preparations, they remain stable at 30°C for at least six months. The bispecific antibody preparation can be a suspension, aqueous injection, freeze-dried preparation, etc. commonly used in the pharmaceutical field.

[0038] For the aqueous injection or freeze-dried preparation of the antibody or its antigen-binding fragment that binds to human CD38 disclosed in the present invention, pharmaceutically acceptable carriers preferably include, but are not limited to, one or a combination of surfactants, solution stabilizers, isotonicity regulators, and buffers. Among them, surfactants preferably include, but are not limited to, non-ionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleyl, or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., and the addition amount should minimize the granulation tendency of the antibody or its antigen-binding fragment that binds to human CD38. Solution stabilizers preferably include, but are not limited to, one or a combination of the following: saccharides, for example, reducing sugars and non-reducing sugars; amino acids, for example, monosodium glutamate or histidine; alcohols, for example: trihydric alcohols, higher polyhydric alcohols, propylene glycol, polyethylene glycol, etc., and the addition amount of the solution stabilizer should keep the finally formed preparation stable within the time considered stable by those skilled in the art. Isotonicity regulators preferably include, but are not limited to, one or a combination of sodium chloride and mannitol. Buffers preferably include, but are not limited to, one or a combination of Tris, histidine buffer, and phosphate buffer.

[0039] On the other hand, the present invention provides the use of the above-mentioned antibody or pharmaceutical composition that binds to human CD38 in the preparation of drugs for treating multiple myeloma, leukemia, B-cell lymphoma, and autoimmune diseases.

[0040] As a preferred embodiment, the autoimmune diseases are selected from systemic lupus erythematosus, autoimmune hemolytic anemia, immune thrombocytopenic purpura, and myasthenia gravis.

[0041] When the antibody or its antigen-binding fragment that binds to human CD38 of the present invention and its composition are administered to animals including humans, the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the administration route, and can refer to the results of animal experiments and various situations, and the total dosage should not exceed a certain range. Specifically, the intravenous injection dosage is 1 - 1800 mg / day.

[0042] On the other hand, the present invention provides a CAR construct, the scFv segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to CD38, and the heavy chain variable region of the scFv includes:

[0043] Heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, wherein the amino acid sequence of H-CDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of H-CDR2 is as shown in SEQ ID NO: 2, the amino acid sequence of H-CDR3 is as shown in SEQ ID NO: 3, and

[0044] The light chain variable region of the scFv comprises:

[0045] Light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3, wherein the amino acid sequence of L-CDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of L-CDR2 is as shown in SEQ ID NO: 5, the amino acid sequence of L-CDR3 is as shown in SEQ ID NO: 6.

[0046] On the other hand, the present invention provides a recombinant immune cell that expresses an exogenous CAR construct as described above.

[0047] On the other hand, the present invention provides an antibody-drug conjugate that contains:

[0048] (a) an antibody moiety that comprises the antibody or an antigen-binding fragment thereof as described above; and

[0049] (b) a conjugate moiety conjugated to the antibody moiety, wherein the conjugate moiety is selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0050] On the other hand, the present invention provides a non-diagnostic in vitro method for detecting CD38 protein in a sample, the method comprising the steps of:

[0051] (1) contacting the sample with the antibody or an antigen-binding fragment thereof or the antibody-drug conjugate as described above in vitro;

[0052] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD38 protein in the sample.

[0053] On the other hand, the present invention provides a method for preventing and / or treating CD38-related diseases, the method comprising: administering to a subject in need the antibody or an antigen-binding fragment thereof, composition, antibody-drug conjugate, recombinant immune cell, or combination thereof that binds to human CD38 as described above.

[0054] In another preferred embodiment, the CD38-related diseases are selected from multiple myeloma, leukemia, B-lymphoma, autoimmune diseases, or a combination thereof.

[0055] On the basis of conforming to the common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0056] The reagents and raw materials used in the present invention are all commercially available.

[0057] The positive and progressive effects of the present invention are as follows:

[0058] At present, there is an urgent need to develop new, specific and highly effective therapeutic drugs for diseases with strong CD38 expression in clinical practice, so as to improve the quality of life of people suffering from such diseases and provide more and more effective treatment options for patients. 50G12-Humanized of the present invention has a high affinity for human CD38, can effectively neutralize human CD38, induce apoptosis after binding to the corresponding antigen on the cell membrane surface, prolong the survival time of the test animals, and has good clinical application prospects. Brief Description of the Drawings

[0059] Figure 1 : Binding activity of murine antibody to target antigen human CD38-Fc

[0060] Figure 2 : Blocking activity of murine antibody on CD38 cyclase activity

[0061] Figure 3A : Binding activity of murine antibody to recombinant high-expression cell line CHOS-CD38 cells

[0062] Figure 3B : Binding activity of murine antibody to tumor cell line DND-41 cells

[0063] Figure 4A : CDC activity of murine antibody on CHOS-CD38 cells

[0064] Figure 4B : CDC activity of murine antibody on Raji cells

[0065] Figure 4C : CDC activity of murine antibody on DND-41 cells

[0066] Figure 4D : CDC activity of murine antibody on Ramos cells

[0067] Figure 4E : CDC activity of murine antibody on Daudi cells

[0068] Figure 5 : Binding activity of humanized antibody 50G12-Humanized to Daudi cells

[0069] Figure 6: Humanized antibody 50G12-Humanized inhibits the cyclase activity of human CD38

[0070] Figure 7 : The ADCC activity of humanized antibody 50G12-Humanized

[0071] Figure 8A : The CDC activity of humanized antibody 50G12-Humanized on Daudi cells

[0072] Figure 8B : The CDC activity of humanized antibody 50G12-Humanized on DND-41 cells

[0073] Figure 9 : The ability of humanized antibody 50G12-Humanized to induce apoptosis

[0074] Figure 10 : The anti-tumor activity of humanized antibody 50G12-Humanized in a Ramos lymphoma animal model Detailed implementation manners

[0075] After extensive and in-depth research and a large number of screenings, the inventor of the present invention obtained a CD38 antibody with high affinity and good biological activity, especially excellent ADCC and CDC activities. The humanized CD38 antibody of the present invention has equivalent or even better activity than commercially available CD38 antibodies. In particular, in a mouse lymphoma model, the humanized CD38 antibody of the present invention can significantly extend the survival time of the test animals. Therefore, the CD38 antibody of the present invention can be developed into an anti-tumor drug with superior efficacy. On this basis, the inventor of the present invention completed the present invention.

[0076] Terms

[0077] In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also have regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. One end of each light chain has a variable region (VL), and the other end has a constant region. The light chain constant region includes a domain CL; the constant region of the light chain pairs with the CH1 domain of the heavy chain constant region, and the variable region of the light chain pairs with the variable region of the heavy chain. The constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC), etc. The heavy chain constant region includes IgG1, IgG2, IgG3, IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy chain and light chain of the antibody are covalently linked together by a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by an inter-polypeptide disulfide bond formed between the hinge regions.

[0078] In the present invention, the terms "Fab" and "Fc" refer to that papain can cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment is composed of the VH and CH1 of the heavy chain of the antibody and the VL and CL domains of the light chain. The Fc fragment, i.e., the fragment crystallizable (Fc), is composed of the CH2 and CH3 domains of the antibody. The Fc fragment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells.

[0079] In the present invention, the term "scFv" is a single chain antibody (scFv), which is usually formed by linking the variable region of the heavy chain of the antibody and the variable region of the light chain with a linker peptide of 15-25 amino acids.

[0080] In the present invention, the term "variable" means that certain portions of the variable regions in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy-chain variable region and the light-chain variable region. The more conserved portions in the variable regions are called framework regions (FRs). The variable regions of the native heavy chain and light chain each contain four FR regions, which are generally in a β-sheet configuration and are connected by three CDRs forming connecting loops, and in some cases can form partial β-sheet structures. The CDRs in each chain are brought closely together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)).

[0081] As used herein, the term "framework region" (FR) refers to the amino acid sequences inserted between CDRs, i.e., those portions of the variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species. The light chain and heavy chain of an immunoglobulin each have four FRs, which are respectively designated as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H. Accordingly, the light-chain variable domain can thus be designated as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy-chain variable domain can thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FRs of the present invention are human antibody FRs or derivatives thereof, and the derivatives of the human antibody FRs are substantially the same as the naturally occurring human antibody FRs, i.e., having a sequence identity of 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0082] Given the amino acid sequences of the CDRs, those skilled in the art can readily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0083] As used herein, the term "human framework region" is a framework region that is substantially the same as the framework region of a naturally occurring human antibody (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%).

[0084] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the domains of the light and heavy chains to fold into an exchanged dual variable region immunoglobulin. In the present invention, preferred linkers refer to Linker1 and Linker2, where Linker1 connects VH and VL of a single-chain antibody (scFv), and Linker2 is used to connect the scFv to the heavy chain of another antibody.

[0085] Examples of suitable linkers include single glycine (Gly) or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker can vary with the type of secondary structural elements to be achieved in the linker.

[0086] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids having similar or close properties compared to the amino acid sequence of the bispecific antibody of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0087] Table A

[0088] Initial residue Representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu

[0089] Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0090] In the present invention, the terms "anti-", "bind", and "specifically bind" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. Generally, an antibody binds to the antigen with an equilibrium dissociation constant (KD) of less than about 10 -7 M, for example less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or less. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between an antibody and an antigen is measured using surface plasmon resonance (SPR) in a BIACORE instrument or the relative binding affinity between an antibody and an antigen is measured using ELISA.

[0091] In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is the region of an antigen that is bound by an antibody.

[0092] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0093] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually to clone it into a vector, then transfer it into cells, and then isolate the relevant sequence from the proliferated host cells by conventional methods.

[0094] The present invention also relates to a vector comprising the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0095] Pharmaceutical compositions and uses

[0096] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the substances to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, or intracavitary injection. In the present invention, the term "pharmaceutical composition" means that the bispecific antibody of the present invention can form a pharmaceutical preparation composition together with a pharmaceutically acceptable carrier to exert its efficacy more stably. These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, and at the same time protect the multi-functional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation). The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned bispecific antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should match the administration route. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 10 micrograms per kilogram of body weight per day - about 50 milligrams per kilogram of body weight. In addition, the bispecific antibody of the present invention can also be used together with other therapeutic agents.

[0097] When using the pharmaceutical composition, a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight - about 10 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are within the scope of the skills of a skilled physician.

[0098] Antibody-drug conjugate (ADC)

[0099] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0100] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, which is conjugated to the antibody, preferably by chemical conjugation. Among them, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule can be one or more of a toxin protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0101] The antibody of the present invention and the effector molecule can be conjugated through a linker. Examples of the linker can be any one or several of a non-selective linker, a linker using a carboxyl group, a peptide chain, and a linker using a disulfide bond. The non-selective linker is a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The linker using a carboxyl group can be any one or several of cis-aconitic anhydride-based linkers (such as cis-aconitic anhydride) and acylhydrazone-based linkers (the coupling site is acylhydrazone).

[0102] Certain residues on the antibody (such as Cys or Lys, etc.) are used to connect with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (such as a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be directly or indirectly connected to the antibody through a linker.

[0103] The antibody can be conjugated with a drug to form an antibody-drug conjugate (ADCs). Typically, the ADC contains a linker located between the drug and the antibody. The linker can be a degradable or non-degradable linker. The degradable linker typically degrades easily in the intracellular environment. For example, the linker degrades at the target site, so that the drug is released from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-based linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptide-based linker can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide bond linkers). The non-degradable linker typically releases the drug under the condition that the antibody is hydrolyzed by protease.

[0104] Before attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, and the attachment is effected through the reactive groups. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodo-, bromo- or chloro-), haloesters (e.g., iodo-, bromo- or chloro-), halomethyl ketones (e.g., iodo-, bromo- or chloro-), benzyl halides (e.g., iodo-, bromo- or chloro-), vinyl sulfones, pyridyl disulfides, mercury derivatives such as 3,6-di-(mercurimethyl) dioxane, and the counterions are acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonates. The linker can include, for example, a maleimide attached to the antibody through a succinimide.

[0105] The drug can be any cytotoxic, cell growth inhibitory or immunosuppressive drug. In an embodiment, the linker attaches the antibody and the drug, and the drug has a functional group capable of bonding to the linker. For example, the drug can have an amino, carboxyl, thiol, hydroxyl, or keto group capable of bonding to the linker. In the case where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0106] Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, the drug-linker can be used to form an ADC in a single step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0107] Typically, functional groups on the linker are selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkyne group on the drug moiety. The drug is covalently attached to the linker through a 1,3-dipolar cycloaddition between the azide and the alkyne. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazines and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other conjugation strategies, such as those described in Bioconjugate Techniques, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for the selective reaction of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used either for the linker or for the drug.

[0108] The present invention also provides a method for preparing an ADC, which may further include: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0109] In certain embodiments, the method of the present invention includes: combining an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further includes: combining the antibody-linker conjugate with a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.

[0110] In some embodiments, the antibody-drug conjugate ADC is represented by the following formula:

[0111]

[0112] Wherein:

[0113] Ab is an antibody,

[0114] LU is a linker;

[0115] D is a drug;

[0116] And the subscript p is a value selected from 1 to 8.

[0117] The following examples are for further illustration of the present invention and should not be construed as limitations on the present invention. The examples do not include detailed descriptions of conventional methods, such as those for constructing vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press. Unless otherwise stated, percentages and parts are by weight.

[0118] The experimental materials used in the following examples, their sources, and the preparation methods of the experimental reagents are specifically described as follows.

[0119] Experimental materials:

[0120] CHO-S cells: Purchased from Thermo Fisher Scientific.

[0121] Recombinant cell line CHOS-CD38: Human full-length CD38 was stably transfected into CHO-S cells, and monoclonal cell lines stably expressing CD38 were obtained through cloning and screening.

[0122] Raji cells: Purchased from ATCC, CCL-86.

[0123] Mouse myeloma cell line SP2 / 0: Purchased from ATCC, catalog number CRL-1581.

[0124] Balb / c mice: Purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0125] CB-17 SCID mice: Purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0126] Ramos cells: Purchased from ATCC, catalog number CRL-1596.

[0127] Daudi cells: Purchased from ATCC, catalog number CCL-213.

[0128] DND-41 cells: Purchased from Fenghui Biology.

[0129] Human peripheral blood mononuclear cells PBMC: Purchased from Ausbian Biotechnology (Shanghai) Co., Ltd.

[0130] Reverse transcription kit: Purchased from Takara.

[0131] Goat anti-mouse secondary antibody: purchased from Millipore, product number AP181P.

[0132] Donkey anti-mouse PE-fluorescent secondary antibody: purchased from Jackson, product number 715-116-150.

[0133] Goat anti-human PE-fluorescent secondary antibody: purchased from Jackson, product number 109-115-098

[0134] F16 Black Maxisorp Plate: purchased from Nunc, product number 475515.

[0135] Experimental reagents:

[0136] PBS buffer: Shanghai Sangon Biotech Co., Ltd., product number B548117-0500.

[0137] SFM medium: purchased from Thermo Fisher Scientific, product number 12045-076.

[0138] TMB: purchased from BD, product number 555214.

[0139] NGD: purchased from sigma, product number N5131-25MG.

[0140] Bovine serum albumin (BSA): purchased from Fetal Bovine Serum.

[0141] β-mercaptoethanol, fetal bovine serum, glutamine, sodium pyruvate, MEM-NEAA, 1% Penicillin-streptomycin were all purchased from Gibco.

[0142] Phenol red-free RPMI-1640: purchased from Gibco, product number 11835055.

[0143] CytoTox 96 Non-Radioactive Cytotoxicity Assay reaction solution: purchased from Promega, product number: G1780.

[0144] HAT: purchased from Sigma-Alhrich, product number H0262-10VL.

[0145] CCK-8: purchased from Dojindo, product number CK04.

[0146] Trizol: purchased from Thermo Fisher Scientific, product number 15596018.

[0147] Experimental instruments:

[0148] Electrofusion instrument: Purchased from BTX.

[0149] Flow cytometer (CytoFLEX Cytometer System): Purchased from Beckman Coulter).

[0150] The antibody sequences of the present invention are as shown

[0151]

[0152]

[0153]

[0154]

[0155] Preparation of the positive control antibody in Example 1

[0156] The amino acid sequences of the heavy and light chain variable regions of Daratumumab described in the examples of the present invention are from "WHO Drug Information, Vol. 24, No. 1, 2010", namely SEQ ID NO 19 and 20 of the present invention. The amino acid sequences of the heavy and light chain variable regions of Isatuximab described in the examples of the present invention are from "WHO Drug Information, Vol. 29, No. 3, 2015", namely SEQ ID NO 21 and 22 of the present invention.

[0157] The DNA of the above-mentioned heavy chain variable region and light chain variable region was synthesized by Shanghai Sangon Biotech Co., Ltd. The synthesized Daratumumab heavy chain variable region gene was linked to the human IgG1 heavy chain constant region gene to obtain the full-length heavy chain gene, named Daratumumab-HC-IgG1; the Daratumumab light chain variable region gene was linked to the human Kappa chain constant region gene to obtain the full-length light chain gene, named Daratumumab-LC. The Daratumumab-HC-IgG1 and Daratumumab-LC genes were respectively constructed into the pcDNA3.4 expression vector, and the obtained heavy chain and light chain expression vectors were co-transferred into HEK293F cells by the PEI transfection method to express the antibody. The HEK293F cells were cultured with Free Style 293 Expression Medium. The transfected HEK293F cells were cultured in a CO2 shaking incubator for 5 days, and the cell supernatant was collected by centrifugation. The antibody in the supernatant was purified by Protein A affinity chromatography, and the obtained antibody was named Daratumumab. In addition, the antibody Isatuximab was obtained by a similar experimental method.

[0158] The sequence information of the extracellular domain of human CD38 was obtained from https: / / www.uniprot.org / uniprot / P28907. The DNA of the extracellular domain of CD38 was synthesized by Shanghai Sangon Biotech Co., Ltd., and the recombinant gene was constructed into the pcDNA3.4 expression vector. The recombinant protein expressed with His-tag was purified in one step from the culture supernatant using a metal chelating affinity chromatography column; the recombinant protein expressed with Fc-tag was purified in one step using a Protein A / G affinity chromatography column. The finally obtained protein was named CD38-His / CD38-Fc.

[0159] Example 2 Immunization of Animals with Antigen and Preparation and Screening of Hybridomas

[0160] Step 1: Immunization of Mice with Antigen

[0161] Balb / c mice were immunized intraperitoneally with the recombinant overexpressing cell line CHOS-CD38 or the tumor cell line Raji cells routinely. On the first day, 100 μl of Freund's complete adjuvant was intraperitoneally injected into Balb / c mice; on the second day, Balb / c mice were immunized intraperitoneally with the recombinant cell line CHOS-CD38 or Raji cells, 5*10 6 cells / mouse; on the fourteenth day, Balb / c mice were boost-immunized intraperitoneally with the recombinant cell line CHOS-CD38 or Raji cells, 5*10 6Cells / mouse. On the 36th day, the mice were boosted with the recombinant cell line CHOS-CD38 or Raji cells as last time. Three weeks later, they were challenged by intraperitoneal injection of CD38-His antigen protein. 3-4 days later, the spleens of the mice were taken for cell fusion experiments.

[0162] Step 2: Preparation and screening of hybridomas

[0163] 3-4 days after the last immunization of the mice, using a conventional hybridoma technology protocol, mouse spleen cells and mouse myeloma cells SP2 / 0 were electrofused by an electrofusion apparatus. The fused cells were evenly suspended in a complete medium, which was a medium composed of mixing RPMI1640 and DMEM F12 media at a ratio of 1:1 and adding 1% Glutmine, 1% Sodium pyruvate, 1% MEM-NEAA (Minimum Essential Medium - Non-Essential Amino Acid Solution), 1% Penicillin-streptomycin, 50 μM β-mercaptoethanol and 20% FBS (Fetal Bovine Serum). The fused cells were seeded at 10 5 cells / 100 μl / well into a total of 36 96-well culture plates and cultured overnight. The next day, 100 μl of the complete medium containing 2×HAT was added to each well, so that the culture solution in the 96-well plate was 200 μl / well (containing 1×HAT). 7-12 days later, the supernatant was harvested, and hybridoma wells positive for human CD38 binding activity were screened by the Cell based ELISA method.

[0164] Among them, the method for screening hybridoma wells positive for human CD38 binding activity by the Cell based ELISA method is as follows: The recombinant cell line CHOS-CD38 was diluted to 2*10 6Cells / ml. Add 100 μl per well into a cell culture plate and incubate overnight at 37°C. The next day, discard the supernatant, add 100 μl per well of cell fixative and fix at room temperature for one hour. Then discard the supernatant, add 5% skim milk powder and block at 37°C for 2 hours. Wash the plate once with PBST for later use. Add the collected hybridoma supernatant to the blocked plate in sequence, 100 μl per well, and incubate at 37°C for 1 h. Wash the plate 3 times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody, and incubate at 37°C for 30 min; after washing the plate 5 times with PBST, pat dry the residual liquid droplets as much as possible on absorbent paper, add 100 μl of TMB to each well, and incubate at room temperature (20 ± 5°C) in the dark for 5 min; add 50 μl of 2M H2SO4 termination solution to each well to terminate the substrate reaction, and read the OD value at 450 nm with an enzyme-linked immunosorbent assay reader to analyze the binding ability of the antibody to be tested with the target antigen CD38. A total of 30 hybridoma cell lines were obtained through screening. Amplify the 30 hybridoma cell lines obtained by screening in a complete medium containing serum, centrifuge and change the medium to serum-free culture medium SFM, so that the cell density is 1 - 2×10 7 / ml, culture for 1 week under the conditions of 8% CO2 and 37°C, centrifuge to obtain the culture supernatant, and purify it by Protein G affinity chromatography to obtain 3 anti-human CD38 monoclonal antibody proteins, named 50G12, 279D11, and 153F11 respectively, among which 50G12 is the antibody with the best activity.

[0165] Example 3 Binding ability of murine antibody 50G12 to human CD38-Fc protein

[0166] The indirect enzyme-linked immunosorbent assay (ELISA) was used to determine the binding ability of the murine antibody to human CD38-Fc protein. The specific method is as follows:

[0167] Dilute CD38-Fc protein to 1 μg / ml with coating solution (50 mM carbonate coating buffer, pH 9.6), coat the plate at 4°C overnight; then block with 5% skim milk powder and incubate at 37°C for 2 hours; after washing the plate 3 times with PBST, serially dilute the prepared anti-human CD38 antibody 50G12 protein with 1% BSA buffer, add 100 μl per well to the pre-coated CD38-Fc plate, and incubate at 37°C for one hour; wash the plate 3 times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody, and incubate at 37°C for 30 min; after washing the plate 3 times with PBST, pat dry the residual liquid droplets as much as possible on absorbent paper, add 100 μl of TMB to each well, and incubate at room temperature (20 ± 5°C) in the dark for 5 min. Add 50 μl of 2M H2SO4 termination solution to each well to terminate the substrate reaction, and read the OD value at 450 nm with an enzyme-linked immunosorbent assay reader to analyze the binding ability of the antibody to be tested with the target antigen human CD38-Fc. The results are as Figure 1 shown.

[0168] FromFigure 1 It can be seen that the murine antibodies 50G12, 279D11, and 153F11 all have good binding activity with the target antigen CD38-Fc, and 50G12 has the optimal binding activity, with an EC 50 of 0.1727 nM.

[0169] Example 4 Ability of Murine Antibodies to Inhibit CD38 Enzyme Activity

[0170] CD38 is an enzyme that can catalyze the conversion of nicotinamide guanine dinucleotide (NGD) into cyclic GDP-ribose, which can emit fluorescence. Here, the inhibitory effects of murine antibodies 50G12, 279D11, 153F11 and control antibodies Daratumumab, Isatuximab, IgG control on CD38 enzyme activity were measured by fluorescence method. The specific method is as follows:

[0171] The murine antibodies 50G12, 279D11, 153F11 to be tested and the control antibodies Daratumumab, Isatuximab, IgG control were diluted to 600 μg / ml with Tris-Hcl and serially diluted 3-fold for a total of 10 wells; the CD38-His antigen was diluted to 5 μg / ml with Tris-Hcl, and the antigen and the sample antibodies were added to the reaction plate in equal volumes, 50 μl each, shaken for 10 min, and incubated at 37 °C for 30 min. Background wells were set: (1) Diluent well: 200 μl of diluent; (2) NGD well: 100 μl of diluent; (3) Antigen well: 50 μl of antigen and 150 μl of diluent. After incubation, NGD was diluted to 250 μg / ml with Tris-Hcl, and 100 μl was added to each well except the diluent well and the antigen well, shaken for 5 min, and incubated at 37 °C for 90 min. Reading the plate with a multifunctional microplate reader at Ex: 300, EM: 410, collecting and processing the data, the results are as Figure 2 shown.

[0172] As Figure 2 can be seen, only the murine antibody 50G12 and Isatuximab can effectively block CD38 cyclase activity.

[0173] Example 5 Binding Ability of Murine Antibodies to Recombinant High-Expression Cell Line CHOS-CD38 Cells and Tumor Cell Line DND-41 Cells

[0174] Flow cytometry was used to detect the binding activity of murine antibodies to the recombinant high-expression cell line CHOS-CD38 cells and the tumor cell line DND-41 cells. The specific method is as follows:

[0175] Collect CHOS-CD38 cells and DND-41 cells separately, centrifuge to remove the cell culture medium, and wash twice with PBS buffer; count the cells and dilute them to 2*10 6 cells / ml with 1% BSA FACS buffer, and plate the cells in a 96-well round bottom plate for later use; dilute the antibody to be tested in 8 gradients with 1% BSA buffer and add it to the above-mentioned cell round bottom plate, incubate at 4°C for 1 hour; after centrifugation, discard the supernatant, wash 3 times with 1% BSA FACS buffer, add 100 μl of donkey anti-mouse PE fluorescent secondary antibody or goat anti-human PE fluorescent secondary antibody to each well at a ratio of 1:300 (see the fluorescent secondary antibody instruction manual for details), incubate at 4°C for 1 hour; wash 3 times with 1% BSA FACS buffer, then resuspend with 1% BSA FACS buffer, 200 μl / well, and use FACScalibur BD to measure and analyze the samples. The results of antibody binding to cells are shown in Figure 3A 、 Figure 3B 。

[0176] From Figure 3A 、 Figure 3B it can be seen that the murine antibodies 50G12, 279D11, and 153F11 have good binding activities on both CHOS-CD38 and DND-41 cells. On CHOS-CD38 cells, the EC 50 values are 686.9 ng / ml, 84.9 ng / ml, and 488.9 ng / ml respectively; on DND-41 cells, they are 133.8 ng / ml, 84.44 ng / ml, and 96.89 ng / ml respectively.

[0177] Example 6 Determination of CDC Activity of Murine Antibodies

[0178] After a specific antibody binds to the corresponding antigen on the cell membrane surface, it can activate the classical complement pathway, and the formed membrane attack complex lyses the target cells, which is called the CDC effect. We measured the CDC activities on CHOS-CD38, Raji, DND-41, Ramos, and Daudi cells respectively. The specific method is as follows:

[0179] Dilute the anti-human CD38 monoclonal antibody to an initial concentration of 20 μg / ml with cell culture medium as the buffer, and then dilute it at a 3-fold concentration gradient to obtain a total of 8 dilutions. After counting the target cells (such as Daudi cells) expressing CD38, resuspend them to 3*10 5cells / ml. 100 μl of various concentration dilutions of anti-human CD38 monoclonal antibody and 80 μl of target cells with high CD38 expression were pre-incubated for 15 min, and then 20 μl of 50% fresh human serum (donated by volunteers) was added and mixed well. The positive control wells were target cells alone plus serum, and the negative control wells were cell-free medium. Incubate in an incubator for 12 - 18 h, add 20 μl of CCK-8, and measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader after 4 h. Calculate the killing rate based on the readings at 450 nm. The formula for calculating the killing rate is:

[0180] Killing rate (%) = (Absorbance of positive control - Absorbance of experimental group) / (Absorbance of positive control - Absorbance of negative control) * 100.

[0181] The results are as Figure 4A 、 Figure 4B 、 Figure 4C 、 Figure 4D 、 Figure 4E shown. On different cell models, murine antibodies 50G12 and 279D11 both have strong CDC activity, while 153F11 is relatively weak.

[0182] Example 7 Humanization of Murine Anti-Human CD38 Monoclonal Antibody

[0183] Step 1: Determination of the Variable Region Sequences of Murine Anti-Human CD38 Monoclonal Antibody

[0184] Total RNA was extracted from the 50G12 hybridoma monoclonal cell line using Trizol, and mRNA was reverse transcribed into cDNA using a reverse transcription kit. The light chain variable region and heavy chain variable region genes of 50G12 were amplified by PCR using the combination primers reported in the literature (《Antibody Engineering》Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequences of the combination primers are from page 323), and then the PCR products were cloned into the pMD18-T vector, sequenced, and the variable region gene sequences were analyzed. The variable region sequence information of murine antibody 50G12 is as follows: The full length of the heavy chain variable region gene sequence is 357 bp, encoding 119 amino acid residues. The nucleotide sequence is as shown in SEQ ID NO: 13, and the amino acid sequence is as shown in SEQ ID NO: 7; The full length of the light chain variable region gene sequence is 321 bp, encoding 107 amino acid residues. The nucleotide sequence is as shown in SEQ ID NO: 14, and the amino acid sequence is as shown in SEQ ID NO: 8.

[0185] Step 2: Humanization of Murine Anti-Human CD38 Monoclonal Antibody

[0186] By analyzing the amino acid sequences of the heavy chain variable region and the light chain variable region of the murine antibody 50G12, the antigen - complementary determining regions (CDRs) and framework regions (FRs) of the heavy chain and light chain of the murine antibody 50G12 monoclonal antibody were determined respectively according to the Kabat rules. The amino acid sequences of the heavy chain CDRs of the murine antibody 50G12 are H - CDR1: SEQ ID NO: 1, H - CDR2: SEQ ID NO: 2, and H - CDR3: SEQ ID NO: 3, and the amino acid sequences of the light chain CDRs are L - CDR1: SEQ ID NO: 4, L - CDR2: SEQ ID NO: 5, and L - CDR3: SEQ ID NO: 6.

[0187] At https: / / www.ncbi.nlm.nih.gov / igblast / , the homology between the heavy chain variable region of the murine 50G12 monoclonal antibody and the human IgG germline sequence was compared. IGHV1 - 46*01 was selected as the template for heavy chain CDR transplantation. The heavy chain CDRs of the murine antibody 50G12 were transplanted into the IGHV1 - 46*01 framework region, and WGQGTLVTVSS was added after H - CDR3 as the fourth framework region to obtain the CDR - transplanted heavy chain variable region sequence. Similarly, the homology between the light chain variable region of the murine antibody 50G12 and the human IgG germline sequence was compared. IGKV1 - 39*01 was selected as the template for light chain CDR transplantation. The light chain CDRs of the murine antibody 50G12 were transplanted into the framework region of IGKV1 - 39*01, and FGQGTKVEIK was added after L - CDR3 as the fourth framework region to obtain the CDR - transplanted light chain variable region sequence. Based on the CDR - transplanted variable regions, some amino acid sites in the framework regions were subjected to back - mutation. Back - mutation means mutating some amino acids (amino acids important for maintaining antibody structure and affinity) in the framework region of the CDR - transplanted variable region into the amino acids at the corresponding positions in the murine framework region.

[0188] When performing the mutation, the amino acid sequences were encoded by Kabat, and the positions of the sites were indicated by Kabat codes. Preferably, for the CDR - transplanted heavy chain variable region, according to the Kabat encoding, the T at position 30 was mutated to N, the M at position 69 was mutated to L, the R at position 71 was mutated to A, and the T at position 73 was mutated to K. For the CDR - transplanted light chain variable region, the L at position 47 was mutated to W, the I at position 48 was mutated to M, and the F at position 71 was mutated to Y. The heavy chain variable region and the light chain variable region with the above - mentioned mutation sites were respectively defined as the humanized heavy chain variable region and the humanized light chain variable region, named 50G12 - Hu - VH and 50G12 - Hu - VL respectively. The amino acid sequence of 50G12 - Hu - VH is SEQ ID NO: 9, and the amino acid sequence of 50G12 - Hu - VL is SEQ ID NO: 10.

[0189] The DNA encoding the above-mentioned humanized heavy and light chain variable regions was synthesized by Shanghai Sangon Biotech Co., Ltd. The synthesized humanized heavy chain variable region DNA was ligated with the human IgG1 heavy chain constant region DNA to obtain the full-length humanized heavy chain DNA, named 50G12-Hu-HC. The DNA sequence of the humanized heavy chain variable region is shown in SEQ ID NO: 15, and the DNA sequence of the full-length humanized heavy chain is shown in SEQ ID NO: 17. The humanized light chain variable region DNA was ligated with the human Kappa chain constant region DNA to obtain the full-length humanized light chain DNA, named 50G12-Hu-LC. The DNA sequence of the humanized light chain variable region is shown in SEQ ID NO: 16, and the DNA sequence of the full-length humanized light chain is shown in SEQ ID NO: 18. The 50G12-Hu-HC and 50G12-Hu-LC genes were respectively constructed into the pcDNA3.4 expression vector, and the antibodies were expressed and purified by the method described in the above examples. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain is shown in SEQ ID NO: 12. The obtained antibody was named 50G12-Humanized.

[0190] In addition, the heavy chain variable region of the murine antibody 50G12 was ligated with the human IgG1 heavy chain constant region to obtain a chimeric heavy chain gene, named 50G12-Chi-HC. The light chain variable region of murine 50G12 was ligated with the human Kappa chain constant region to obtain a chimeric light chain gene, named 50G12-Chi-LC. The 50G12-Chi-HC and 50G12-Chi-LC genes were respectively constructed into the pcDNA3.4 expression vector, and the antibodies were expressed and purified by the method described in the above examples. The obtained antibody was named 50G12-Chimeric.

[0191] Example 8 Binding ability of 50G12-Humanized to CD38

[0192] The binding abilities of 50G12-Chimeric and 50G12-Humanized to CD38 on the surface of Daudi cells were detected by flow cytometry. The specific method is as follows:

[0193] After counting the Daudi cells, they were inoculated into a 96-well round-bottom culture plate with PBS solution containing 1% BSA, 2×10 per well 5cells; add 50 μl of anti-CD38 antibody diluted in gradient with PBS solution to the above 96-well plate; incubate at room temperature for 1 hour, then centrifuge and discard the supernatant, and then wash the cells twice with PBS; add FITC-labeled goat anti-human (Fc-Speicific) antibody (diluted 1:1000 with PBS containing 1% BSA), incubate at room temperature for half an hour; centrifuge and wash the cells, and then detect the mean fluorescence intensity (MFI) of the FITC channel on a flow cytometer; use the software provided by the flow cytometer to process the experimental data and calculate the mean fluorescence intensity; use GraphPad Prism6 for data analysis and plotting, and calculate EC 50 .

[0194] The results are as follows Figure 5 As shown in Figure 2, 50G12-Chimeric, 50G12-Humanized, Daratumumab, and Isatuximab can effectively bind to Daudi cells. 50 The results above show that the ability of 50G12-Humanized to bind to Daudi cells is basically the same. Among them, Isotype Control is a human IgG1 antibody that does not bind to Daudi cells.

[0195] Example 9 50G12-Humanized inhibits CD38 cyclase activity

[0196] The inhibitory effect of 50G12-Humanized on CD38 cyclase activity was determined by fluorescence method. The specific method is as follows:

[0197] Prepare 50mM MES buffer at pH 6.5, and use MES buffer to prepare 200μM nicotinamide guanine dinucleotide (NGD) solution; dilute CD38-His to 2μg / ml with MES buffer, and then add anti-CD38 antibody with a final concentration of 10μg / ml; add 50μL NGD solution to F16 Black Maxisorp Plate, and then add 50μL solution containing CD38-His and anti-CD38 antibody; read the fluorescence value (Relative Fluorescence Unit, RFU) in kinetic mode using a multi-function microplate reader SpectraMax M5, with the excitation and emission wavelengths set at 300nm and 410nm, respectively; use GraphPad Prism6 for data analysis and graphing.

[0198] The results are as followsFigure 6 As shown in the figure, both 50G12-Humanized, Daratumumab and Isatuximab can effectively inhibit the enzyme activity of CD38-His, and their slopes are 155931, 331046 and 93316 respectively. The smaller the slope, the stronger the inhibitory effect. Therefore, the order of the three inhibitors of CD38 cyclase activity from strong to weak is Isatuximab, 50G12-Humanized and Daratumumab.

[0199] Example 10 Determination of the ADCC Activity of 50G12-Humanized

[0200] The Fab fragment of the antibody binds to the antigenic epitope on the cell surface, and its Fc fragment binds to the Fc receptor on the surface of effector cells (NK cells, macrophages, etc.), which can mediate the direct killing of target cells by effector cells. This is the ADCC effect. The ADCC activity of anti-CD38 antibodies was measured as follows:

[0201] Add 2% fetal bovine serum to phenol red-free RPMI-1640; mix the target cells, Daudi cells and human peripheral blood mononuclear cells (PBMC) at a ratio of 1:25 with this medium, and inoculate them into round-bottom 96-well plates, 150 μL per well, and finally make each well contain 2×10 4 Daudi cells and 5×10 5 PBMC; add 50 μL of serially diluted anti-CD38 antibody; incubate overnight in a cell culture incubator at 37 °C and 5% CO2; take 50 μL of cell culture supernatant, add 50 μL of CytoTox 96 Non-Radioactive Cytotoxicity Assay reaction solution, add the stop solution to terminate the reaction after 30 min, and read OD490 with an enzyme-linked immunosorbent assay reader; use GraphPad Prism6 for data analysis and graphing, and calculate EC 50 .

[0202] The results are as Figure 7 shown, both 50G12-Humanized, Daratumumab and Isatuximab can effectively kill target cells, and their EC 50 are 0.1058 nM, 0.08876 nM and 0.0694 nM respectively, and the ADCC activities of the three are basically equivalent.

[0203] Example 11 Determination of the CDC Activity of 50G12-Humanized

[0204] The CDC activity of humanized antibody 50G12-Humanized was determined in Daudi and DND-41 cell models. The detailed experimental methods are as follows: Dilute the anti-human CD38 monoclonal antibody to an initial concentration of 20 μg / ml with cell culture medium as the buffer, and then dilute it at a 3-fold concentration gradient to obtain a total of 8 dilutions. After counting the target cells (such as Daudi cells) expressing CD38, resuspend them to 3*10 5 cells / ml. Incubate 100 μl of various dilutions of the anti-human CD38 monoclonal antibody and 80 μl of target cells with high expression of CD38 for 15 min in advance, and then add 20 μl of 50% fresh human serum (donated by volunteers) and mix well. The positive control wells are target cells plus serum alone, and the negative control wells are cell-free culture medium. Incubate in an incubator for 12-18 h, add 20 μl of CCK-8, and measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader after 4 h. Calculate the killing rate according to the reading at 450 nm. The formula for calculating the killing rate is:

[0205] Killing rate (%) = (Absorbance of positive control - Absorbance of experimental group) / (Absorbance of positive control - Absorbance of negative control) * 100.

[0206] The experimental results are as shown in Figure 8A and 8B : In the Daudi cell model, the CDC activity of humanized antibody 50G12-Humanized is comparable to that of Daratumumab and Isatuximab; in the DND-41 cell model, the CDC activity of humanized antibody 50G12-Humanized is superior to that of Daratumumab and Isatuximab.

[0207] Example 12 Ability of 50G12-Humanized to Induce Apoptosis

[0208] Anti-CD38 antibodies can induce cell apoptosis after binding to the corresponding antigen on the cell membrane surface (Deckert J, Wetzel M, Bartle LM, et al. SAR650984, A Novel Humanized CD38-Targeting Antibody, Demonstrates Potent Antitumor Activity in Models of Multiple Myeloma and Other CD38+Hematologic Malignancies [J]. Clinical Cancer Research, 2014, 20 (17): 4574-4583.). In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is transferred from the inner side of the cell membrane to the outer side, thereby exposing PS to the external cell environment. Annexin V is a 35-36 kDa calcium ion-dependent phospholipid binding protein that has a high affinity for PS and can bind to exposed PS. Here, the activity of anti-CD38 antibodies in inducing cell apoptosis was determined using the FITC-labeled Annexin V apoptosis detection kit. The specific method is as follows:

[0209] RPMI-1640 was supplemented with 2% fetal bovine serum; Daudi cells were seeded into 96-well plates with 1×10 cells per well. 5 cells / 150μL; add 50μL of gradient diluted anti-CD38 antibody; incubate in a 37℃, 5% CO2 cell culture incubator for 24h; stain apoptotic cells with FITC-labeled Annexin V apoptosis detection kit; centrifuge and wash the cells, and then detect the average fluorescence intensity of the FITC channel on a flow cytometer; process the experimental data with the software provided by the flow cytometer and calculate the proportion of stained cells to total cells; use GraphPad Prism6 for data analysis and plotting, and calculate EC 50 .

[0210] The results are as follows Figure 9 As shown in Figure 2, 50G12-Humanized, Daratumumab, and Isatuximab can effectively induce cell apoptosis. 50 The EC values ​​of the three are 0.5675nM, 0.3059nM and 0.302nM respectively. 50Basically equivalent, but the highest proportions of apoptotic cells induced by 50G12-Humanized, Daratumumab, and Isatuximab are 28.4%, 13.4%, and 49.0% respectively. Therefore, the order of the ability of the three to induce apoptosis from strong to weak is Isatuximab, 50G12-Humanized, and Daratumumab.

[0211] Example 13 In Vivo Efficacy Evaluation of 50G12-Humanized

[0212] The in vivo antitumor activity of the anti-CD38 humanized antibody 50G12-Humanized was verified on a xenograft tumor model of human Ramos lymphoma cell line in CB-17 SCID mice. The specific method is as follows:

[0213] Ramos cells were cultured in vitro. After harvesting, the cell concentration was adjusted to 5×10 7 cells / ml. By the method of tail vein injection, 200 μl / animal of cell suspension was inoculated into female CB-17 SCID mice to establish a xenograft tumor model. On the 7th day after inoculation, the mice were randomly divided into a control group, a Daratumumab treatment group, an Isatuximab treatment group, and a 50G12-Humanized treatment group, with 10 mice in each group. Treatment was started at a dose of 40 mg / kg of the antibody, administered twice a week for three consecutive weeks. The survival time of the tumor-bearing mice was observed. The humane endpoint for the animals was defined as unilateral or bilateral hind limb paralysis of the tumor-bearing mice, or a weight loss of more than 20%, or a severely poor physical condition resulting in the inability to freely eat and drink. The test animals were euthanized, and the survival time was recorded.

[0214] The results are as Figure 10 shown. The median survival time of the control group was 25 days. Compared with the control group, both 50G12-Humanized and the positive control antibodies Daratumumab and Isatuximab could significantly prolong the survival time of the test animals, with median survival times of 38 days, 35.5 days, and 38.5 days respectively. Sequence Listing <110> 3SBio Inc. <120> Antibody Binding to Human CD38, Its Preparation Method and Use <130> P2021-1794 <150> 2020108054202 <151> 2020-08-12 <160> 22 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Mus musculus <400> 1 Thr Tyr Trp Met Gln 1 5 <210> 2 <211> 17 <212> PRT <213> mus musculus <400> 2 Ala Ile Tyr Pro Gly Asp Gly Asp Ile Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 3 <211> 11 <212> PRT <213> mus musculus <400> 3 Glu Gly Tyr Tyr Tyr Gly Gly Ala Leu Asp Tyr 1 5 10 <210> 4 <211> 12 <212> PRT <213> mus musculus <400> 4 Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His 1 5 10 <210> 5 <211> 7 <212> PRT <213> mus musculus <400> 5 Gly Thr Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> mus musculus <400> 6 His Arg Tyr His Arg Ser Pro Trp Thr 1 5 <210> 7 <211> 120 <212> PRT <213> mus musculus <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Thr Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Ile Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Tyr Tyr Tyr Gly Gly Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 8 <211> 108 <212> PRT <213> mus musculus <400> 8 Gln Ile Phe Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Lys Leu Trp 35 40 45 Met Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Arg Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> CD38 Humanized Antibody Heavy Chain Variable Region 50G12-Hu-VH <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Thr Tyr 20 25 30 Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Ile Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Tyr Tyr Tyr Gly Gly Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> CD38 Humanized Antibody Light Chain Variable Region 50G12-Hu-VL <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Met Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Arg Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> CD38 Humanized Antibody Heavy Chain 50G12-Hu-HC <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Thr Tyr 20 25 30 Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Ile Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Tyr Tyr Tyr Gly Gly Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 12 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CD38 humanized antibody light chain 50G12-Hu-LC <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Met Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Arg Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 13 <211> 360 <212> DNA <213> mus musculus <400> 13 caggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcctc agtgaagttg 60 tcctgcaagg cttctggcta cacctttaat acctattgga tgcagtgggt aaaacagagg 120 cctggacagg gtctggaatg gattggggct atttatcctg gagatggtga tattacatat 180 aatcagaagt ttaagggcaa ggccacattg actgcagata aatcttccaa cacagcctac 240 atgcacctca gcagcttggc atctgaggac tcagcggtct attactgtgc aagagaggga 300 tattattacg gcggggcttt ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360 <210> 14 <211> 324 <212> DNA <213> mus musculus <400> 14 caaatttttc tcacccagtc tccagcaatc atgtctgcat ctctagggga acgggtcacc 60 atgacctgca ctgccagctc aagtgtgagt tcaagctact tgcactggta ccagcagaag 120 ccaggatccc cccccaaact ctggatgtat ggcacatcca acctggcttc tggagtccca 180 cctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 240 gctgaagatg ctgccactta ttactgccac cggtatcatc gttccccgtg gacgttcggt 300 ggaggcacca agctggaaat caaa 324 <210> 15 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> 50G12-Hu-VH heavy chain variable region nucleotide sequence <400> 15 caggtgcagc tcgtgcagtc cggcgctgag gtgaagaagc ccggcgcctc cgtgaaggtg 60 tcctgcaagg cctccggcta caccttcaac acctattgga tgcaatgggt gaggcaggcc 120 cccggccagg gcctggagtg gatgggcgcc atctaccccg gcgatggcga catcacctac 180 aaccagaagt ttaagggcag ggtgaccctg acagctgata aatctacatc tactgtgtac 240 atggagttat cttctctgag atctgaggat acagctgtgt actattgtgc tagagaggga 300 tactattatg gcggagccct ggattattgg ggacagggaa cactggtgac agtgtcttct 360 <210> 16 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> 50G12-Hu-LC light chain variable region nucleotide sequence <400> 16 gatatccaga tgacccagtc tccttcttcc ctgtccgctt ctgtgggaga tagagtgaca 60 attacatgta ccgcttcttc ttctgtgtct tcttcttacc tgcattggta tcagcagaag 120 cctggcaagg ctcctaaact gtggatgtat ggaacatcta atctggcttc tggcgtgcct 180 tctagatttt ctggctctgg atctggcacc gattacacac tgaccatctc tagcctgcag 240 cctgaggatt ttgccacata ctactgtcac agatatcaca gatctccttg gacctttggc 300 cagggcacca aggtggagat caag 324 <210> 17 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> 50G12-Hu-HC heavy chain nucleotide sequence <400> 17 caggtgcagc tcgtgcagtc cggcgctgag gtgaagaagc ccggcgcctc cgtgaaggtg 60 tcctgcaagg cctccggcta caccttcaac acctattgga tgcaatgggt gaggcaggcc 120 tcctgcaagg cctccggcta caccttcaac acctattgga tgcaatgggt gaggcaggcc 120 cccggccagg gcctggagtg gatgggcgcc atctaccccg gcgatggcga catcacctac 180 cccggccagg gcctggagtg gatgggcgcc atctaccccg gcgatggcga catcacctac 180 aaccagaagt ttaagggcag ggtgaccctg acagctgata aatctacatc tactgtgtac 240 aaccagaagt ttaagggcag ggtgaccctg acagctgata aatctacatc tactgtgtac 240 atggagttat cttctctgag atctgaggat acagctgtgt actattgtgc tagagaggga 300 atggagttat cttctctgag atctgaggat acagctgtgt actattgtgc tagagaggga 300 tactattatg gcggagccct ggattattgg ggacagggaa cactggtgac agtgtcttct 360 tactattatg gcggagccct ggattattgg ggacagggaa cactggtgac agtgtcttct 360 gcgagcacca agggaccttc cgtgtttccc ctcgccccca gctccaaaag caccagcggc 420 gcgagcacca agggaccttc cgtgtttccc ctcgccccca gctccaaaag caccagcggc 420 ggaacagctg ctctcggctg tctcgtcaag gattacttcc ccgagcccgt gaccgtgagc 480 ggaacagctg ctctcggctg tctcgtcaag gattacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gagccctgac aagcggcgtc cacaccttcc ctgctgtcct acagtcctcc 540 tggaacagcg gagccctgac aagcggcgtc cacaccttcc ctgctgtcct acagtcctcc 540 ggactgtaca gcctgagcag cgtggtgaca gtccctagca gctccctggg cacccagaca 600 ggactgtaca gcctgagcag cgtggtgaca gtccctagca gctccctggg cacccagaca 600 tatatttgca acgtgaatca caagcccagc aacaccaagg tcgataagaa ggtggagcct 660 tatatttgca acgtgaatca caagcccagc aacaccaagg tcgataagaa ggtggagcct 660 aagtcctgcg acaagaccca cacatgtccc ccctgtcccg ctcctgaact gctgggaggc 720 aagtcctgcg acaagaccca cacatgtccc ccctgtcccg ctcctgaact gctgggaggc 720 ccttccgtgt tcctgttccc ccctaagccc aaggacaccc tgatgatttc caggacaccc 780 ccttccgtgt tcctgttccc ccctaagccc aaggacaccc tgatgatttc caggacaccc 780 gaggtgacct gtgtggtggt ggacgtcagc cacgaggacc ccgaggtgaa attcaactgg 840 tacgtcgatg gcgtggaggt gcacaacgct aagaccaagc ccagggagga gcagtacaat 900 tccacctaca gggtggtgtc cgtgctgacc gtcctccatc aggactggct gaacggcaaa 960 gagtataagt gcaaggtgag caacaaggcc ctccctgctc ccatcgagaa gaccatcagc 1020 aaagccaagg gccagcccag ggaacctcaa gtctataccc tgcctcccag cagggaggag 1080 atgaccaaga accaagtgag cctcacatgc ctcgtcaagg gcttctatcc ttccgatatt 1140 gccgtcgagt gggagtccaa cggacagccc gagaacaact acaagacaac accccccgtg 1200 ctcgattccg atggcagctt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 1260 caacaaggca acgtcttcag ttgcagcgtc atgcatgagg ccctccacaa ccactacacc 1320 cagaagagcc tctccctgag ccctggaaag 1350 <210> 18 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of 50G12-Hu-LC light chain <400> 18 gatatccaga tgacccagtc tccttcttcc ctgtccgctt ctgtgggaga tagagtgaca 60 attacatgta ccgcttcttc ttctgtgtct tcttcttacc tgcattggta tcagcagaag 120 cctggcaagg ctcctaaact gtggatgtat ggaacatcta atctggcttc tggcgtgcct 180 tctagatttt ctggctctgg atctggcacc gattacacac tgaccatctc tagcctgcag 240 cctgaggatt ttgccacata ctactgtcac agatatcaca gatctccttg gacctttggc 300 cagggcacca aggtggagat caagagaacc gtcgccgctc ccagcgtctt catcttcccc 360 cccagcgatg agcagctgaa gagcggaacc gccagcgtgg tgtgcctgct gaacaacttc 420 taccccaggg aggccaaggt gcaatggaag gtggacaacg ccctacagag cggcaactcc 480 caggagagcg tgaccgagca ggacagcaag gatagcacct acagcctgag cagcaccctc 540 accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccatcag 600 ggcctgagca gccctgtgac caagagcttc aacaggggcg agtgc 645 <210> 19 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Amino acids of the heavy chain variable region of Daratumumab <400> 19 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acids of the light chain variable region of Daratumumab <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 21 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Amino acids of the heavy chain variable region of Isatuximab <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr 65 70 75 80 Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acids of the light chain variable region of Isatuximab <400> 22 Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly 1 5 10 15 Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105

Claims

1. An antibody or antigen-binding fragment thereof that binds to human CD38, characterized in that, Comprising: (a) Heavy chain complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of H-CDR2 is as shown in SEQ ID NO: 2, the amino acid sequence of H-CDR3 is as shown in SEQ ID NO: 3, and (b) Light chain complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of L-CDR2 is as shown in SEQ ID NO: 5, the amino acid sequence of L-CDR3 is as shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof that binds to human CD38 according to claim 1, wherein The antibody is a murine antibody, a chimeric antibody or a humanized antibody.

3. The antibody or antigen-binding fragment thereof that binds to human CD38 according to claim 1, wherein The antigen-binding fragment includes a Fab fragment, an F(ab’)2 fragment, an Fv fragment.

4. The antibody or antigen-binding fragment thereof that binds to human CD38 according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment that binds to human CD38 is as shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

8.

5. The antibody or antigen-binding fragment thereof that binds to human CD38 according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment that binds to human CD38 is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

10.

6. The antibody or antigen-binding fragment thereof that binds to human CD38 according to claim 1, wherein The amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment that binds to human CD38 is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain is as shown in SEQ ID NO:

12.

7. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the antibody or its antigen-binding fragment that binds to human CD38 as described in any one of claims 1-6.

8. The nucleotide molecule according to claim 7, wherein, The nucleotide sequence of the nucleotide molecule encoding the heavy chain variable region is as shown in SEQ ID NO: 13, and the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO:

14.

9. The nucleotide molecule according to claim 7, wherein The nucleotide sequence of the nucleotide molecule encoding the heavy chain variable region is as shown in SEQ ID NO: 15, and the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO:

16.

10. The nucleotide molecule according to claim 7, wherein The nucleotide sequence of the nucleotide molecule encoding the heavy chain is as shown in SEQ ID NO: 17, and the nucleotide sequence encoding the light chain is as shown in SEQ ID NO:

18.

11. An expression vector, characterized in that, The expression vector contains the nucleotide molecule as described in any one of claims 7-10.

12. A host cell, characterized in that, The host cell contains the expression vector as described in claim 11.

13. A method for preparing an antibody or an antigen-binding fragment thereof that binds to human CD38 as described in any one of claims 1-6, characterized in that, The method comprises the following steps: a) Culturing the host cell as described in claim 12 under expression conditions to express the antibody or its antigen-binding fragment that binds to human CD38; b) Separating and purifying the antibody or its antigen-binding fragment that binds to human CD38 described in a).

14. A composition, characterized in that, The composition contains the antibody or its antigen-binding fragment that binds to human CD38 as described in any one of claims 1-6 and a pharmaceutically acceptable carrier.

15. Use of an antibody or antigen-binding fragment thereof that binds to human CD38 as described in any one of claims 1-6, or the composition as described in claim 14, in the preparation of a medicament for treating multiple myeloma, T-cell acute lymphoblastic leukemia, Burkitt's lymphoma, or an autoimmune disease; the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, autoimmune hemolytic anemia, and immune thrombocytopenic purpura.

16. A CAR construct, characterized in that, The scFv segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to CD38, and the heavy-chain variable region of the scFv comprises: Heavy-chain complementarity-determining regions H-CDR1, H-CDR2, and H-CDR3, the amino acid sequence of H-CDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of H-CDR2 is as shown in SEQ ID NO: 2, the amino acid sequence of H-CDR3 is as shown in SEQ ID NO: 3, and The light-chain variable region of the scFv comprises: Light-chain complementarity-determining regions L-CDR1, L-CDR2, and L-CDR3, the amino acid sequence of L-CDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of L-CDR2 is as shown in SEQ ID NO: 5, the amino acid sequence of L-CDR3 is as shown in SEQ ID NO:

6.

17. A recombinant immune cell, characterized in that, The immune cell expresses an exogenous CAR construct as described in claim 16.

18. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains: (a) An antibody moiety, the antibody moiety comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-6; and (b) A conjugate moiety conjugated to the antibody moiety, the conjugate moiety being selected from the group consisting of a detectable label, a drug, a toxin, or a combination thereof.

19. The antibody-drug conjugate according to claim 18, wherein, The detectable label is a radionuclide.

20. The antibody-drug conjugate according to claim 18, wherein, The drug is a cytokine, an enzyme, or a combination thereof.

21. A method for in vitro detecting CD38 protein in a sample, characterized in that, The method comprises the steps of: (1) In vitro, contacting the sample with an antibody or antigen-binding fragment thereof as described in any one of claims 1-6, or an antibody-drug conjugate as described in claim 18; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD38 protein in the sample.

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