Recombinant fusion protein targeting CD38 and CD47

Abstract

Provided is an antibody capable of specifically binding to CD38, or an antigen-binding portion thereof. Also provided is a recombinant fusion protein, comprising: i) a CD38 antibody or an antigen-binding portion thereof; and ii) a CD47 binding peptide, wherein the CD47 binding peptide is linked to an N-terminus of a heavy chain or light chain of the CD38 antibody or the antigen-binding portion thereof, and the CD47 binding peptide is an extracellular Ig-like domain of a signal regulatory protein (SIRP).

Description

Recombinant fusion protein targeting CD38 and CD47 Field of the Invention

[0001] The present application relates to an antibody or an antigen-binding portion thereof that can specifically bind to CD38, and a CD38 / CD47 bispecific antibody comprising the antibody or the antigen-binding portion thereof. Background Art

[0002] CD38

[0003] CD38 is a type II transmembrane glycoprotein with a molecular weight of 45 kDa, consisting of a long extracellular domain (258 aa) and a short N-terminal cytoplasmic tail (21 aa). It may also exist in body fluids in a soluble form of 39 kDa. Studies have shown that CD38 has an extracellular enzymatic effect, catalyzing the conversion of NAD+ to ADPR and cADPR, affecting cell growth and T cell activation by regulating cellular calcium ions, and exerting immunosuppressive activity by directly acting on various immune cell subsets, such as recruiting immunosuppressive cells such as Treg cells and MDSC cells. CD38 can also catalyze the conversion of NAD+ to cGDPR.

[0004] Under physiological conditions, CD38 is expressed at very low levels on lymphocytes, myeloid cells, and some non-blood tissues, and at higher levels on plasma cells, regulatory T cells (Tregs), regulatory B cells (Bregs), and myeloid-derived suppressor cells (MDSCs). CD38 is also highly expressed in many solid tumor cells, including hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, breast cancer, gastric cancer, esophageal cancer, and hematological tumors such as multiple myeloma (MM).

[0005] CD38 antibodies have pleiotropic mechanisms of action, such as cross-linking CD38 on the cell surface to induce CD38 + It improves the immunosuppressive environment by inhibiting the extracellular enzymatic action of CD38 and induces CD38 cell apoptosis through ADCC, ADCP and / or CDC. + Direct cell killing, etc. Many CD38 antibodies are currently in the clinical evaluation stage. Daratumumab is the first monoclonal antibody developed to target CD38 and can be used alone or in combination with standard of care for the treatment of MM. Isatuximab is also a CD38 antibody approved by the US Food and Drug Administration for the treatment of relapsed MM. In addition, daratumumab and isatuximab are undergoing preclinical and clinical trials for a variety of hematological and non-hematological malignancies, including plasma cell myeloma, lymphoma, pancreatic cancer, non-small cell lung cancer, triple-negative breast cancer and prostate cancer.

[0006] SIRP and CD47

[0007] Signal regulatory proteins (SIRPs) are transmembrane glycoproteins that include three family members, SIRPα (CD172a), SIRPβ (CD172b), and SIRPγ (CD172g). All three proteins contain similar extracellular regions, but have different intracellular domains. The extracellular region contains three immunoglobulin-like domains, one Ig-V and two Ig-C domains. The intracellular domain of SIRPα (CD172a) contains two inhibitory signal transduction regions, which can inhibit signal transduction and corresponding cellular functions. The intracellular regions of SIRPβ (CD172b) and SIRPγ (CD172g) are very short and do not contain signal transduction domains. However, SIRPβ (CD172b) can function as a signal transducer through adaptor proteins such as DAP12. SIRP is mainly expressed in macrophages (Mφ), dendritic cells (DC) and neurons.

[0008] CD47 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily and is expressed on the surface of all cell types, including red blood cells. Its ligands include integrins, thrombospondin-1, and SIRPs. CD47, by interacting with SIRPα, sends a "don't eat me" signal, inhibiting phagocytosis by dendritic cells and macrophages, thereby protecting blood cells, for example, from macrophage attack.

[0009] Studies have shown that many tumors or cancer cells that overexpress CD47 can inhibit the phagocytosis of cancer cells by macrophages. Cancer cells that overexpress CD47 include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, and pancreatic cancer cells. It has been reported that injecting CD47-specific antibodies that block the binding of CD47 to SIRPα into tumor-bearing mice can significantly inhibit tumor growth. When the same antibody is injected into mice carrying human leukemia cells, the tumor cells or cancer cells are completely eliminated. The biggest problem with CD47-binding molecules, such as the CD47 antibody magrolimab, is that they bind to normal cells in the body, especially red blood cells, causing side effects such as blood coagulation and anemia.

[0010] In order to overcome the serious side effects and therapy resistance in tumor treatment, new products need to be developed to meet the needs of patients.

[0011] The citation of any document in this application does not constitute an admission that these documents are prior art to this application. Summary of the Invention

[0012] The inventors of this application have combined a high-affinity CD38 antibody or its antigen-binding portion with a low-affinity CD47 binding peptide in the same molecule to more accurately target tumor cells or the tumor microenvironment, inhibiting immune escape and targeting CD38 through both CD38 and CD47 pathways. + CD47 + Directly kill tumor cells to achieve better anti-tumor effects and avoid damage to normal cells in the body such as red blood cells.

[0013] Specifically, the inventors of the present application screened monoclonal antibodies with strong CD38 binding ability, and used these monoclonal antibodies or their antigen-binding portions to construct CD38 / CD47 bispecific antibodies.

[0014] Thus, in a first aspect, the present application provides an isolated monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody), or an antigen-binding portion thereof, which can specifically bind to CD38 (e.g., human or monkey CD38), and can comprise i) a heavy chain variable region, which can comprise VH-CDR1, VH-CDR2, and VH-CDR3, and ii) a light chain variable region, which can comprise VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 can respectively comprise (1) GNTFTNYW (SEQ ID NO: 1), IDPSNGRT (SEQ ID NO: 2), AREEKATLVGMDY (SEQ ID NO: 3), QDIRNY (SEQ ID NO: 4), YTS, and RQGNTLPWT (SEQ ID NO: 5). NO: 5); (2) GYTFTSYG (SEQ ID NO: 8), INTYTGEP (SEQ ID NO: 9), ARKGFAY (SEQ ID NO: 10), RASESVDIYGNSF (SEQ ID NO: 11), RAS, and QQFNEDPYT (SEQ ID NO: 12); (3) GYTFTSYW (SEQ ID NO: 15), IFPGAGII (SEQ ID NO:16), VRRGKGPWFDY (SEQ ID NO:17), QNINVW (SEQ ID NO:18), KAS, and QQGQSYPRT (SEQ ID NO:19); (4) GYIFSTYW (SEQ ID NO:22), ILPGSGSI (SEQ ID NO:23), GRGWDYTMDY (SEQ ID NO:24), QSLLYSTNQKNY (SEQ ID NO: 25), WAS, and QNYYSYPRT (SEQ ID NO: 26); (5) GDFFSRYW (SEQ ID NO:29), INPDSSTI (SEQ ID NO:30), ARRWTTVYAMDY (SEQ ID NO:31), KSVSASGYSY (SEQ ID NO:32), LAS, and QHSRELPLT (SEQ ID NO:33); (6) GYTFTSYN (SEQ ID NO:36), IYPGNGGT (SEQ ID NO:37), ARGGELRRAYFSY (SEQ ID NO:38), ESVDSYGNTF (SEQ ID NO:39), LAS, and QQNKEDPWT (SEQ ID NO:40);(7) the amino acid sequence of GFSLTSYG (SEQ ID NO:43), IWRGGST (SEQ ID NO:44), AKSMITTGYAMDY (SEQ ID NO:45), EDIYKR (SEQ ID NO:46), GAT, and QQFWNTPPT (SEQ ID NO:47); (8) GFTFSYYW (SEQ ID NO:50), IRSKSNNYAT (SEQ ID NO:51), TRLWLSTRSMDY (SEQ ID NO:52), NSVSTSDFDF (SEQ ID NO:53), LAS, and QHSRELPRT (SEQ ID NO:54); or (9) GYTFTNHH (SEQ ID NO:57), INPYNDYT (SEQ ID NO:58), ARKWGEIYFDAMDH (SEQ ID NO:59), QDINNY (SEQ ID NO:60), YTS, and QQGHMLPYT (SEQ ID NO:61). Also provided are variants of the above antibodies or antigen-binding portions thereof, comprising up to about 3 amino acid residue substitutions, such as 1, 2, or 3 amino acid residue substitutions, in each CDR compared to the above antibodies or antigen-binding portions thereof.

[0015] The heavy chain variable region of the antibody or antigen-binding portion thereof of the present application can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55 or 62.

[0016] The light chain variable region of the antibodies or antigen-binding portions thereof of the present application may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 56, or 63.

[0017] The heavy chain variable region and light chain variable region of the antibodies or antigen-binding portions thereof of the present application can respectively comprise an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (1) SEQ ID NOs: 6 and 7; (2) SEQ ID NOs: 13 and 14; (3) SEQ ID NOs: 20 and 21; (4) SEQ ID NOs: 27 and 28; (5) SEQ ID NOs: 34 and 35; (6) SEQ ID NOs: 41 and 42; (7) SEQ ID NOs: 48 and 49; (8) SEQ ID NOs: 55 and 56; or (9) SEQ ID NOs: 62 and 63.

[0018] The isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain constant region and / or a light chain constant region, wherein the N-terminus of the heavy chain constant region is linked to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is linked to the C-terminus of the light chain variable region. The heavy chain constant region may be an IgG, IgD, IgA, IgM, or IgE heavy chain constant region, particularly a heavy chain constant region that naturally or after modification has binding affinity to Fc receptors and / or complement system proteins, particularly one with high binding affinity to Fc receptors and / or complement system proteins, or a functional fragment thereof, such as a fragment comprising the hinge region, CH2, and CH3 of the heavy chain constant region. In one embodiment, the heavy chain constant region may be an IgG1 heavy chain constant region, such as a human IgG1 heavy chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 64. The light chain constant region may be a kappa or lambda light chain constant region, such as a human kappa or lambda light chain constant region. In some embodiments, the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 65.

[0019] In some embodiments, the antibodies of the present application comprise two heavy chains and two light chains, or are composed of two heavy chains and two light chains, wherein each heavy chain comprises the aforementioned heavy chain constant region sequence, heavy chain variable region sequence, and / or CDR sequence, and each light chain comprises the aforementioned light chain constant region sequence, light chain variable region sequence, and / or CDR sequence. In some embodiments, the antibodies of the present application or their antigen-binding portions may be Fab, F(ab')2 fragments, Fv, scFv, or (scFv)2, etc.

[0020] The antibodies of the present application, or antigen-binding portions thereof, can be, for example, mouse, chimeric, or humanized.

[0021] The CD38 antibody or antigen-binding portion of the present invention has comparable or better (human or monkey) CD38 binding ability, comparable or better inhibitory effect on CD38 enzymatic action, comparable or higher CD38 induction, compared to existing CD38 antibodies such as daratumumab and isatuximab. + Apoptotic activity is comparable to or higher than that of CD38 + The cells induce ADCC, ADCP, and / or CDC activities and have in vivo anti-tumor activity.

[0022] The application also provides an immunoconjugate containing the present application antibody or its antigen binding portion, which can be connected to a therapeutic agent such as a cytotoxic molecule or an anticancer agent. The application also provides a bispecific molecule containing the present application antibody or its antigen binding portion, which can be connected to a second functional group, and the second functional group has a binding specificity different from the present application antibody or its binding portion. In some embodiments, the second functional group can specifically bind to CD47, such as signal regulatory protein α (SIRPα), particularly the extracellular Ig-like domain of signal regulatory protein α (SIRPα). On the other hand, the application provides a chimeric antigen receptor (CAR) or genetically modified T cell receptor (TCR) comprising the present application antibody or its antigen binding portion. The application also provides immune cells containing the above-mentioned CAR and / or TCR, including T cells, NK cells, etc. The application also provides an oncolytic virus that encodes or carries the present application antibody or its antigen binding portion.

[0023] The present application also includes nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, immunoconjugates, bispecific molecules, CARs, or TCRs of the present application. The present application may also provide an expression vector and a host cell. The expression vector may comprise the nucleic acid molecules of the present application. The host cell may comprise the expression vector of the present application, or have the nucleic acid molecules of the present application integrated into its genome.

[0024] The present application also provides a method for using the host cells of the present application to prepare the antibodies or their antigen-binding portions, immunoconjugates, bispecific molecules, CARs or TCRs of the present application, comprising: (i) expressing the antibodies or their antigen-binding portions, immunoconjugates, bispecific molecules, CARs, or TCRs in the host cells, and (ii) isolating the antibodies or their antigen-binding portions, immunoconjugates, bispecific molecules, CARs, or TCRs from the host cells or their cultures.

[0025] The present application also provides a composition comprising the antibody or antigen-binding portion thereof, immunoconjugate, bispecific molecule, CAR / TCR, immune cell carrying CAR / TCR, oncolytic virus, nucleic acid molecule, expression vector or host cell of the present application. In some embodiments, the composition can be a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding portion thereof, immunoconjugate, bispecific molecule, CAR / TCR, immune cell carrying CAR / TCR, oncolytic virus, nucleic acid molecule, expression vector or host cell, and may further comprise a pharmaceutically acceptable carrier.

[0026] The present application may provide a method for treating or alleviating a CD38-related disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present application to the subject.

[0027] CD38-related diseases can be CD38-related cancers, including solid tumors and hematologic tumors. Solid tumors can include hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, breast cancer, gastric cancer, esophageal cancer, etc. Hematologic tumors can include multiple myeloma, plasma cell myeloma, lymphoma, etc. In some embodiments, the CD38-related disease can be multiple myeloma.

[0028] The subject may be a mammal, particularly a human.

[0029] The present application also provides a method for inhibiting CD38 + Methods for growing cells, including CD38 + The cells are contacted with the composition of the present application. + The cells can be CD38 + Tumor cells, such as myeloma cells.

[0030] The present application also protects the use of the antibody or antigen-binding portion thereof in preparing bispecific antibodies, such as CD38 / CD47 bispecific antibodies, and the use of the composition of the present application in preparing a method for treating CD38-related diseases or inhibiting CD38. + Use in cell growth medicine.

[0031] In a second aspect, the present application provides a recombinant fusion protein, which may comprise i) a CD38 antibody or an antibody fragment thereof that specifically binds to CD38, and ii) a CD47-binding peptide that specifically binds to CD47, wherein the CD47-binding peptide may be linked to the CD38 antibody or its antibody fragment, wherein the CD38 antibody or its antibody fragment may comprise a heavy chain variable region, a heavy chain constant region, and a light chain variable region.

[0032] The heavy chain constant region can be linked to the C-terminus of the heavy chain variable region and has binding to Fc receptors and / or complement system proteins, particularly strong binding to Fc receptors and / or complement system proteins. The heavy chain constant region can be an IgG, IgD, IgA, IgM, or IgE heavy chain constant region, particularly a naturally or engineered heavy chain constant region that has binding to Fc receptors and / or complement system proteins, particularly strong binding to Fc receptors and / or complement system proteins, or a functional fragment thereof, such as a fragment comprising the hinge region, CH2, and CH3 of the heavy chain constant region. In one embodiment, the heavy chain constant region can be an IgG1 heavy chain constant region, such as a human IgG1 heavy chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:64.

[0033] The CD38 antibody or antibody fragment thereof that specifically binds to CD38 can be the CD38 antibody or antigen-binding portion thereof of the present application. In some embodiments, the heavy chain variable region may contain VH-CDR1, VH-CDR2 and VH-CDR3, and the light chain variable region may contain VL-CDR1, VL-CDR2 and VL-CDR3, wherein VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 may respectively comprise (1) GNTFTNYW (SEQ ID NO: 1), IDPSNGRT (SEQ ID NO: 2), AREEKATLVGMDY (SEQ ID NO: 3), QDIRNY (SEQ ID NO: 4), YTS, and RQGNTLPWT (SEQ ID NO: 5); (2) GYTFTSYG (SEQ ID NO: 8), INTYTGEP (SEQ ID NO: 9), ARKGFAY (SEQ ID NO: 10), RASESVDIYGNSF (SEQ ID NO: 11), RAS, and QQFNEDPYT (SEQ ID NO: 12). NO:12); (3) GYTFTSYW (SEQ ID NO:15), IFPGAGII (SEQ ID NO:16), VRRGKGPWFDY (SEQ ID NO:17), QNINVW (SEQ ID NO:18), KAS, and QQGQSYPRT (SEQ ID NO:19); (4) GYIFSTYW (SEQ ID NO:22), ILPGSGSI (SEQ ID NO:23), GRGWDYTMDY (SEQ ID NO:24), QSLLYSTNQKNY (SEQ ID NO:25), WAS, and QNYYSYPRT (SEQ ID NO:26); (5) GDFFSRYW (SEQ ID NO:29), INPDSSTI (SEQ ID NO:30), ARRWTTVYAMDY (SEQ ID NO:31), KSVSASGYSY (SEQ ID NO:32), LAS, and QHSRELPLT (SEQ ID NO:33); (6) GYTFTSYN (SEQ ID NO:36), IYPGNGGT (SEQ ID NO:37), ARGGELRRAYFSY (SEQ ID NO:38), ESVDSYGNTF (SEQ ID NO:39), LAS, and QQNKEDPWT (SEQ ID NO:40);(7) the amino acid sequence of GFSLTSYG (SEQ ID NO:43), IWRGGST (SEQ ID NO:44), AKSMITTGYAMDY (SEQ ID NO:45), EDIYKR (SEQ ID NO:46), GAT, and QQFWNTPPT (SEQ ID NO:47); (8) GFTFSYYW (SEQ ID NO:50), IRSKSNNYAT (SEQ ID NO:51), TRLWLSTRSMDY (SEQ ID NO:52), NSVSTSDFDF (SEQ ID NO:53), LAS, and QHSRELPRT (SEQ ID NO:54); or (9) GYTFTNHH (SEQ ID NO:57), INPYNDYT (SEQ ID NO:58), ARKWGEIYFDAMDH (SEQ ID NO:59), QDINNY (SEQ ID NO:60), YTS, and QQGHMLPYT (SEQ ID NO:61). The heavy chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55 or 62. The light chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 56, or 63. In some embodiments, the heavy chain variable region and the light chain variable region can each comprise an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (1) SEQ ID NOs: 6 and 7; (2) SEQ ID NOs: 13 and 14; (3) SEQ ID NOs: 20 and 21; (4) SEQ ID NOs: 27 and 28; (5) SEQ ID NOs: 34 and 35; (6) SEQ ID NOs: 41 and 42; (7) SEQ ID NOs: 48 and 49; (8) SEQ ID NOs: 55 and 56; or (9) SEQ ID NOs: 62 and 63.

[0034] In particular, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 may respectively comprise (1) GNTFTNYW (SEQ ID NO: 1), IDPSNGRT (SEQ ID NO: 2), AREEKATLVGMDY (SEQ ID NO: 3), QDIRNY (SEQ ID NO: 4), YTS, and RQGNTLPWT (SEQ ID NO: 5); (2) GYTFTSYG (SEQ ID NO: 8), INTYTGEP (SEQ ID NO: 9), ARKGFAY (SEQ ID NO: 10), RASESVDIYGNSF (SEQ ID NO: 11), RAS, and QQFNEDPYT (SEQ ID NO: 12); (3) GYTFTSYW (SEQ ID NO: 15), IFPGAGII (SEQ ID NO: 16), VRRGKGPWFDY (SEQ ID NO: 17), QNINVW (SEQ ID NO: 18), NO:18), KAS, and QQGQSYPRT (SEQ ID NO:19); (4) GYIFSTYW (SEQ ID NO:22), ILPGSGSI (SEQ ID NO:23), GRGWDYTMDY (SEQ ID NO:24), QSLLYSTNQKNY (SEQ ID NO:25), WAS, and QNYYSYPRT (SEQ ID NO:26); or (5) the amino acid sequence of GFDFSRYW (SEQ ID NO:29), INPDSSTI (SEQ ID NO:30), ARRWTTVYAMDY (SEQ ID NO:31), KSVSASGYSY (SEQ ID NO:32), LAS, and QHSRELPLT (SEQ ID NO:33). The heavy chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 6, 13, 20, 27, or 34. The light chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 7, 14, 21, 28, or 35.In some embodiments, the heavy chain variable region and the light chain variable region can respectively comprise an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (1) SEQ ID NOs: 6 and 7; (2) SEQ ID NOs: 13 and 14; (3) SEQ ID NOs: 20 and 21; (4) SEQ ID NOs: 27 and 28; or (5) SEQ ID NOs: 34 and 35.

[0035] The CD38 antibody or antibody fragment thereof may comprise a light chain constant region connected to the C-terminus of the light chain variable region. The light chain constant region may be a kappa or lambda light chain constant region, such as a human kappa or lambda light chain constant region. In some embodiments, the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 65.

[0036] The CD47 binding peptide can be a signal regulatory protein (SIRP) extracellular Ig-like domain, such as a signal regulatory protein α (SIRPα) extracellular Ig-like domain, particularly the first extracellular Ig-like domain of SIRPα (SIRPαD1). SIRPαD1 can comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 70.

[0037] The CD47-binding peptide can bind to the N-terminus of the heavy chain variable region or light chain variable region of a CD38 antibody or antibody fragment thereof. In some embodiments, the CD47-binding peptide can bind to the N-terminus of the heavy chain variable region of a CD38 antibody or antibody fragment thereof. In some embodiments, the CD47-binding peptide can bind to the N-terminus of the light chain variable region of a CD38 antibody or antibody fragment thereof.

[0038] The CD38 antibody or antibody fragment thereof of the present application can be connected to the CD47 binding peptide via a linker. The linker can be a peptide of 5-30, 10-30, 10-20, or 15 amino acids in length. The linker can be a GS linker, for example, GGGGSGGGGSGGGGS (SEQ ID NO: 73), GGGGSGGGGS (SEQ ID NO: 74), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75). In some embodiments, the linker can be GGGGSGGGGSGGGGS (SEQ ID NO: 73).

[0039] The recombinant fusion protein of the present application may comprise i) a CD47 binding peptide-linker-CD38 antibody heavy chain variable region-heavy chain constant region chain, and ii) a CD38 antibody light chain variable region-light chain constant region chain, or a CD38 antibody light chain variable region chain. In some embodiments, i) and ii) respectively comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (1) SEQ ID NOs: 78 and 79; (2) SEQ ID NOs: 80 and 81; (3) SEQ ID NOs: 82 and 83; (4) SEQ ID NOs: 84 and 85; (5) SEQ ID NOs: 86 and 87; or (6) SEQ ID NOs: 92 and 93. In some embodiments, i) and ii) comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (1) SEQ ID NOs: 78 and 7; (2) SEQ ID NOs: 80 and 14; (3) SEQ ID NOs: 82 and 21; (4) SEQ ID NOs: 84 and 28; or (5) SEQ ID NOs: 86 and 35. The amino acid sequences set forth in SEQ ID NOs: 78, 79, 80, 81, 82, 83, 84, 85, 86, and 87 can be encoded by the nucleotide sequences set forth in SEQ ID NOs: 94, 95, 96, 97, 98, 99, 100, 101, 102, and 103, respectively.

[0040] The recombinant fusion protein of the present application may comprise i) a CD38 antibody heavy chain variable region-a heavy chain constant region chain, and ii) a CD47 binding peptide-linker-CD38 antibody light chain variable region-a light chain constant region chain. In some embodiments, i) may comprise, from N-terminus to C-terminus, an amino acid sequence consisting of SEQ ID NO: 6 and SEQ ID NO: 64, or a sequence consisting thereof, and ii) may comprise, from N-terminus to C-terminus, an amino acid sequence consisting of SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 7, and SEQ ID NO: 65, or a sequence consisting thereof. In some embodiments, i) may comprise, from N-terminus to C-terminus, an amino acid sequence consisting of SEQ ID NO: 13 and SEQ ID NO: 64, or a sequence consisting thereof, and ii) may comprise, from N-terminus to C-terminus, an amino acid sequence consisting of SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 14, and SEQ ID NO: 65, or a sequence consisting thereof. In some embodiments, i) may comprise an amino acid sequence consisting of SEQ ID NO: 20 and SEQ ID NO: 64, or consisting thereof, from the N-terminus to the C-terminus, ii) may comprise an amino acid sequence consisting of SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 21, and SEQ ID NO: 65, or consisting thereof, from the N-terminus to the C-terminus. In some embodiments, i) may comprise an amino acid sequence consisting of SEQ ID NO: 27 and SEQ ID NO: 64, or consisting thereof, from the N-terminus to the C-terminus, ii) may comprise an amino acid sequence consisting of SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 28, and SEQ ID NO: 65, or consisting thereof, from the N-terminus to the C-terminus. In some embodiments, i) may comprise an amino acid sequence consisting of SEQ ID NO: 34 and SEQ ID NO: 64, or consisting thereof, from the N-terminus to the C-terminus, ii) may comprise an amino acid sequence consisting of SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 35, and SEQ ID NO: 65, or consisting thereof, from the N-terminus to the C-terminus.

[0041] The recombinant fusion protein of the present application has high CD38 binding affinity and low CD47 binding affinity, and can bind to both CD38 and CD47 simultaneously. Compared with the recombinant fusion protein constructed based on existing antibodies such as isatuximab, it has comparable or higher CD38 binding affinity, comparable CD47 binding affinity, and comparable or higher CD38 binding affinity. + CD47 + Cell binding ability, equivalent or better CD47-SIRPα blocking ability, equivalent or stronger CD38 + cells or CD38 + CD47+ Cells induce ADCC, ADCP, and / or CDC activity, comparable to or better than CD38 + cells or CD38 + CD47 + The activity of cell apoptosis, and / or equivalent or stronger in vivo anti-tumor effect. In addition, compared with cells that do not express or low express CD38, the recombinant fusion protein of the present application prefers to bind to cells that express CD38 or high express CD38. + CD47 + In particular, the recombinant fusion protein of the present application does not target CD47 + CD38 – More importantly, the recombinant fusion protein of the present application has comparable or better in vivo anti-tumor activity than its constituent components, CD38 antibody or its antigen-binding portion, CD47 binding peptide, or a combination of the two.

[0042] The present application also provides a nucleic acid molecule encoding the recombinant fusion protein of the present application, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector or having the nucleic acid molecule integrated into its genome. Also provided is a method for preparing a recombinant fusion protein using the host cell of the present application, comprising (i) expressing the recombinant fusion protein in the host cell, and (ii) isolating the recombinant fusion protein from the host cell or a cell culture thereof.

[0043] The present application also provides a composition, which may include the recombinant fusion protein, nucleic acid molecule, expression vector, or host cell of the present application. In some embodiments, the composition may be a pharmaceutical composition and may further include at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further includes at least one pharmaceutically acceptable adjuvant.

[0044] The recombinant fusion protein or composition of the present application can be used to treat diseases associated with overexpression of CD47 and / or CD38, or to prepare drugs for treating diseases associated with overexpression of CD47 and / or CD38.

[0045] The present application provides a method for treating or alleviating a disease associated with overexpression of CD47 and / or CD38 in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present application to the subject.

[0046] The disease associated with overexpression of CD47 and / or CD38 can be a cancer associated with overexpression of CD47 and / or CD38, including solid cancers and blood cancers, including but not limited to acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), lymphoma, multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, melanoma, glioma, esophageal cancer, plasma cell myeloma and prostate cancer.

[0047] The subject may be a mammal, particularly a human.

[0048] The present application also provides a method for inhibiting CD38 + cells or CD47 + CD38 + Methods for growing cells, including CD38 + cells or CD47 + CD38 + The cells are contacted with the composition of the present application. + cells or CD47 + CD38 + The cells can be CD38 + Tumor cells or CD47 + CD38 + Tumor cells, such as myeloma cells.

[0049] The present application also protects the use of the recombinant fusion protein or composition of the present application in preparing a drug for treating a disease associated with overexpression of CD47 and / or CD38, and in preparing a drug for inhibiting CD38. + cells or CD47 + CD38 + Use in cell growth medicine.

[0050] Based on the following detailed description and examples, other features and advantages of the present disclosure will be very clear, and the detailed description and examples should not be interpreted as being restrictive. All documents, Genbank registration numbers, patents and published patent applications cited in the specification are incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The detailed description given below is given by way of example but is not intended to limit the present application to the specific embodiments described, and can be better understood in conjunction with the accompanying drawings.

[0052] FIG1 shows the binding ability of the CD38 antibody of the present application to CHO cells (A) or CHO cells (B) overexpressing human CD38.

[0053] FIG2 shows the binding ability of the CD38 antibody of the present application to CHO cells overexpressing monkey CD38.

[0054] FIG3 shows the CD38 antibody of the present application and CD38 + Binding capacity of tumor cells Raji (A), Daudi (B), NCl-H929 (C), L-1236 (D), Reh (E), or MOLM-13 (F).

[0055] FIG4 shows the inhibitory effect of the CD38 antibodies of the present application on the enzymatic activity of CD38 on Daudi (A), L-1236 (B), Raji (C), or NCl-H929 (D).

[0056] FIG5 shows the activity of the CD38 antibody of the present application in inducing apoptosis in Daudi (A), L-1236 (B), Raji (C), or NCl-H929 (D) cells.

[0057] FIG6 shows the ADCC activity of the CD38 antibodies of the present application against Daudi (A), Raji (B), NCl-H929 (C), or L-1236 (D).

[0058] FIG7 shows the ADCP activity of the CD38 antibody of the present application against Raji (A), L-1236 (B), or NCl-H929 (C).

[0059] FIG8 shows the activity of the CD38 antibody of the present application on CDC induced by Raji (A), L-1236 (B), or Daudi (C).

[0060] FIG9 shows the average tumor volume change (A), survival curve (B), and individual tumor volume change (C) of each group of tumor-bearing mice after administration of the CD38 antibody of the present application.

[0061] FIG10 shows schematic structural diagrams of bispecific antibodies IMM5605 (C) and IMM5606 (D) composed of SIRPα (A) and CD38 antibody (B).

[0062] FIG11 shows the binding ability of the bispecific antibody of the present application to Daudi (A), Raji (B), NCl-H929 (C), Reh (D), L-1236 (E), human RBC (F), or platelet cells (G).

[0063] Figure 12 shows the activity of the bispecific antibody of the present application in simultaneously binding to CD38 and CD47 measured by BLI. The bispecific antibody was first exposed to CD38 protein and then to CD47+CD38 (A), or first exposed to CD47 protein and then to CD38+CD47 (B).

[0064] FIG13 shows the activity of the bispecific antibody of the present application in binding to CD38 and CD47 simultaneously as measured by flow cytometry. The bispecific antibody is first exposed to CD47 with a lower expression level of CD38. + Jurkat cells were then exposed to CD38-mFc protein (A), followed by CD38 - CD47 + NK92MI cells were then exposed to CD38-mFc protein (B), or first exposed to CD38 + CD47 - CHO-CD38 cells were then exposed to CD47-mFc protein (C).

[0065] FIG. 14 shows the bispecific antibody of the present application and D38 in the mixture + CD47 + Reh cells and CD38 - CD47 + Binding of NK92MI cells.

[0066] FIG15 shows the blocking ability of the bispecific antibody of the present application on CD47-SIRPα in the presence of Jurkat cells (A) or Reh cells (B).

[0067] FIG16 shows the inhibitory effect of the bispecific antibody of the present application on the enzymatic activity of CD38 on Daudi (A) and L1236 (B) cells.

[0068] FIG17 shows the ADCC activity of the bispecific antibodies of the present application against Raji (A), NCl-H929 (B) or HL60 (C).

[0069] FIG18 shows the ADCP-inducing activity of the bispecific antibody of the present application against Raji.

[0070] FIG19 shows the activity of the bispecific antibody of the present application in inducing CDC against Daudi.

[0071] FIG20 shows the activity of the bispecific antibody of the present application in inducing apoptosis in Daudi cells.

[0072] FIG21 shows the mean tumor volume change (A) and individual tumor volume change (B) of tumor-bearing mice after administration of IMM01, isatuximab, IMM5601, and isatuximab+IMM01.

[0073] FIG22 shows the average tumor volume change (A) and individual tumor volume change (B) in tumor-bearing mice after administration of the bispecific antibody of the present application. DETAILED DESCRIPTION

[0074] Over the past 30 years, the incidence of blood cancer has increased year by year, accounting for about 9% of all cancers. Chemotherapy and immunotherapy are still the two main treatments for blood cancer. Although chemotherapy can achieve a certain therapeutic effect, it cannot achieve complete remission (CR), and recurrence and drug resistance problems continue to occur. The administration of antibodies subverts CD38 + Treatment of hematological malignancies, especially when chemotherapy is ineffective. CD38 is an important target for the treatment of multiple myeloma (MM) because plasma cells in patients with multiple myeloma have elevated CD38 expression compared to normal cells. Multiple myeloma is incurable, despite the availability of various types of drugs on the market, such as proteasome inhibitors, immunomodulators, and monoclonal antibodies. Multiple myeloma is the second most common blood cancer after non-Hodgkin's lymphoma, with approximately 100,000 new cases each year. CD38 is also expressed in high amounts on some cells in diseases other than MM, such as chronic lymphocytic leukemia, acute leukemia, and various lymphomas (follicular cell lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma).

[0075] Daratumumab is a human IgG1 monoclonal antibody developed by Janssen for the treatment of MM as well as lymphoma, leukemia and systemic amyloidosis. Its mechanism of action includes ADCC, direct apoptosis / cytotoxicity, CDC and ADCP. Daratumumab can induce CD38 through Fc + Cross-linking of CD38 on the cell surface leads to CD38 + It can induce cell apoptosis and kill CD38+ cells through ADCC, CDC and ADCP, including immunosuppressive cells such as regulatory B cells, regulatory T cells and myeloid-derived suppressor cells.

[0076] Isatuximab is a humanized IgG1 CD38 antibody developed by Sanofi and has been approved for the treatment of relapsed refractory multiple myeloma (RRMM). It also has Fc-dependent and Fc-independent mechanisms of action. Fc-dependent mechanisms include ADCP, ADCC, and CDC, while Fc-independent mechanisms include lysosome-dependent cell death and cysteine-containing aspartate protease-dependent apoptosis. Isatuximab can be activated by NK or CD8 + Cell activation and CD38 + Treg cell suppression leads to lysis of multiple myeloma and improves the tumor microenvironment (TME).

[0077] Macrophages express SIRPα, which binds to CD47 expressed on cells and receives CD47 +The "don't eat me" signal released by cells maintains the immune system's immune tolerance to its own normal cells. This mechanism can be exploited by cancer cells, which evade the immune system's attack by upregulating the expression of CD47 on their surface. CD47 is associated with blood cancers such as non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), lymphoblastic lymphoma / acute lymphoblastic leukemia (LBL / ALL), and multiple myeloma (MM). Therefore, inhibiting the interaction of CD47-SIRPα can enhance the immune system's phagocytic clearance of tumor cells. A variety of therapeutic agents targeting CD47 have been developed, including fusion proteins containing SIRPα, CD47 antibodies, and other molecules that bind to CD47. The difficulty of using CD47 binding molecules in treatment is that CD47 is expressed on almost all cells, which can easily cause off-target effects. In particular, when CD47 monoclonal antibodies bind to red blood cells, they can cause blood coagulation and anemia.

[0078] Despite significant advances in the treatment of lymphomas, leukemias, and multiple myeloma, therapy resistance and related disease relapse are common. Treatment of relapsed or refractory disease is complex, and uncontrolled disease progression at this stage is highly likely to result in death. New compounds and combination therapies are needed to improve disease control, prevent myeloma-related complications, delay progression, and ultimately improve survival. Therapeutic agents with improved target recognition specificity and combination therapies that elicit multiple cytotoxic mechanisms are needed to overcome resistance to existing therapies and improve efficacy.

[0079] In this context, the inventors of the present application have proposed a CD38 / CD47 targeted therapy that can promote tumor killing induced by innate immune cells and overcome CD38 antibody tolerance. The bispecific antibody of the present application comprises two CD38 binding domains and two CD47 binding domains, and the heavy chain constant region is designed to enhance ADCC, ADCP, CDC and apoptosis induction. The bispecific antibody can regulate the activity of CD38 by inhibiting the ribose cyclase activity of CD38. Moreover, due to the lower CD47 binding affinity and higher CD38 binding affinity, the bispecific antibody of the present application hardly binds to RBCs, thus does not cause hemagglutination, and acts on CD38 with higher selectivity. + Tumor cells.

[0080] Unless otherwise specified, the terms used herein have the ordinary meanings in dictionaries, textbooks, technical reference books, or as generally understood by those skilled in the art. The following descriptions of certain terms are intended only to facilitate understanding of this application and are not intended to be limiting of these terms unless otherwise specified.

[0081] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0082] The term "or" refers to a single element of the listed alternative elements unless the context clearly dictates otherwise.

[0083] The term "comprise" or "include" means that the elements, integers or steps are included, but does not exclude the addition of any other elements, integers or steps. In this article, when the term "comprise" or "include" is used, unless otherwise indicated, combinations of the elements, integers or steps mentioned are also covered.

[0084] "CD38" refers to cluster of differentiation 38. The term includes variants, homologs, orthologs, and paralogs. For example, an antibody specific for human CD38 may, in some cases, cross-react with CD38 protein from another species, such as monkey.

[0085] "Human CD38" refers to a CD38 protein having a human amino acid sequence, for example, a CD38 protein having the amino acid sequence of GEnBank Accession No. BAA18966.1 (Nata, K. et al., Human gene encoding CD38 (ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase): organization, nucleotide sequence and alternative splicing. Gene 186(2), 285-292 (1997)). "Monkey CD38" refers to a CD38 protein having a monkey amino acid sequence.

[0086] The term "antibody" as used herein is intended to include full-length IgG, IgA, IgD, IgE, and IgM antibodies and any antigen-binding fragments thereof (i.e., antigen-binding portions). Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V H The heavy chain constant region is composed of three domains, namely C H1 、C H2 and C H3 Each light chain consists of a light chain variable region (V L or VL) and the light chain constant region. The light chain constant region consists of a domain C L Composition. H and V L The V domains can be further divided into regions of hypervariability, termed complementarity determining regions (CDRs), separated by more conserved framework regions (FRs).H and V L It is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The heavy chain constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including binding to a variety of immune system cells (e.g., effector cells) and the first component (C1q) of the traditional complement system. A "functional fragment" of an antibody constant region refers to a fragment that retains certain desired functions in the constant region, such as a fragment that retains FcR / complement system component binding activity in the heavy chain constant region, such as an Fc fragment.

[0087] The crystallizable region (Fc region) is the tail region of an antibody and is the domain that determines the antibody's effector function (i.e., how the antibody establishes a relationship with specific cell receptors or other defense proteins). Fc receptors (FcRs) are proteins found on the surface of certain cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. These cells contribute to the protective function of the immune system. The Fc region can interact with Fc receptors and some proteins of the complement system to activate the immune system.

[0088] As used herein, the "antigen-binding portion" of an antibody (or simply the antibody portion) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD38 protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) a Fab fragment, which consists of a V L 、V H 、C L and C H1 (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) V H and C H1 (iv) an Fd fragment consisting of an antibody single-arm V L and V H Fv fragment composed of V H (vi) isolated complementarity determining regions (CDRs); and (vii) dAb-V L , a fragment comprising a single variable domain and a heavy chain constant domain. In addition, although the two domains V L and V HEncoded by different genes, they can be recombinantly linked via a synthetic linker that makes the two into a single protein chain, where V L and V H The regions are paired to form monovalent molecules. These single-chain antibodies are also intended to be included in the meaning of the term. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0089] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to CD38 protein is substantially free of antibodies that specifically bind to antigens other than GPRC5D protein. However, an isolated antibody that specifically binds to human CD38 protein may have cross-binding properties to other antigens, such as CD38 proteins from other species. Furthermore, an isolated antibody is substantially free of other cellular material and / or chemical substances.

[0090] "Monoclonal antibody" or "mAb" or "monoclonal antibody composition" refers to an antibody molecule prepared from a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0091] "Mouse-derived antibodies" refer to antibodies whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. The mouse-derived antibodies of the present application may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by random or point mutations in vitro or by somatic mutations in vivo. However, the term "mouse-derived antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into mouse framework sequences.

[0092] A "chimeric antibody" is an antibody that is derived from a combination of non-human (e.g., mouse) genetic material and human genetic material. More generally, a chimeric antibody is an antibody that combines genetic material from one species with that from another.

[0093] A "humanized antibody" is an antibody derived from a non-human (eg, mouse) species whose protein sequence has been altered to increase similarity to naturally occurring antibodies in humans.

[0094] As used herein, "specifically recognize" or "specifically bind" to a target, such as human CD38, means that an antibody or antigen-binding fragment can distinguish the target biomolecule from one or more reference molecules, and has a binding affinity or activity to the target biomolecule that is, for example, 1-fold, 5-fold, 10-fold, etc. higher than that of the other reference molecules. Specificity determination methods include, but are not limited to, SPR, BLI, Western blotting, ELISA, RIA, ECL, IRMA testing, and peptide scanning.

[0095] “EC50 ", also known as half-maximal effect concentration, refers to the concentration of antibody or recombinant fusion protein that causes 50% of the maximum effect.

[0096] “IC 50 ", refers to the half-inhibitory concentration, which is the concentration of the antibody or recombinant fusion protein required to inhibit the specified biological process by half.

[0097] "Antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refers to a cell-mediated immune defense in which immune system effector cells actively lyse target cells to which cell membrane surface antigens are bound by the CD38 antibody or antigen-binding portion thereof, or the recombinant fusion protein of the present application.

[0098] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to an immune elimination mechanism in which the CD38 antibody or antigen-binding portion thereof, or the recombinant fusion protein of the present application, binds to target cells and recruits immune effector cells, such as phagocytes, via the constant region, such as Fc, thereby promoting phagocytosis of target cells by the immune effector cells.

[0099] "Complement-dependent cytotoxicity" or "CDC" refers to the effector function of the CD38 antibody, or antigen-binding portion thereof, or recombinant fusion protein of the present application, which, upon binding to a surface antigen, triggers the classic complement pathway, including formation of the membrane attack complex and target cell lysis. The CD38 antibody, or antigen-binding portion thereof, or recombinant fusion protein of the present application, upon binding to a target cell, triggers CDC against the target cell.

[0100] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows and horses, are preferred.

[0101] The term "therapeutically effective amount" refers to an amount of the CD38 antibody or antigen-binding portion thereof, or the recombinant fusion protein of the present application, sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). The therapeutically effective amount is related to the disease being treated, and those skilled in the art can readily determine the actual effective amount.

[0102] "Sequence identity" herein refers to the percentage of nucleotides / amino acids in a sequence that are identical to the nucleotides / amino acid residues in a reference sequence after sequence alignment, with spaces introduced, if necessary, to achieve the maximum percentage of sequence identity between the two sequences. Those skilled in the art can perform pairwise sequence alignment or multiple sequence alignment to determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences by various methods, such as using computer software such as Clustal Omega, T-coffee, Kalign, and MAFFT.

[0103] The heavy chain variable region and light chain variable region sequences or sequence numbers of the antibodies or antigen-binding portions thereof of the present application are listed in Table 1. The heavy chain variable region CDRs and light chain variable region CDRs are identified using the IMGT numbering system, and the CDR sequences or sequence numbers identified thereby are also listed in Table 1. The heavy chain variable region CDRs and light chain variable region CDRs of the antibodies or antigen-binding portions thereof of the present application can also be identified using numbering systems such as Chothia, Kabat, AbM, or Contact based on the full-length variable region sequence.

[0104] The antibodies of the present application may have a heavy chain constant region, such as a natural or modified one with FcR and / or complement system protein binding, particularly one with high FcR and / or complement system protein binding. In some embodiments, the heavy chain constant region may be an IgG1 constant region. The light chain constant region may be a kappa constant region, such as a human kappa constant region.

[0105] The antibodies or antigen-binding portions thereof of the present application may comprise heavy and / or light chain variable region sequences or CDR1, CDR2, and CDR3 sequences that have one or more conservative modifications compared to the CD38 antibodies or antigen-binding portions thereof of the present application. It is known in the art that some conservative sequence modifications do not abolish antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8.

[0106] Thus, in one embodiment, the antibody or antigen-binding portion thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region and the light chain variable region comprising CDR1, CDR2 and CDR3, respectively, wherein:

[0107] (a) the heavy chain variable region CDR1 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0108] (b) the heavy chain variable region CDR2 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0109] (c) the heavy chain variable region CDR3 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0110] (d) the light chain variable region CDR1, and / or CDR2, and / or CDR3 comprise the sequences listed in Table 1, and / or conservative modifications thereof; and

[0111] (e) The antibody or antigen-binding portion thereof specifically binds to human CD38.

[0112] The term "conservative sequence modification" as used herein refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid replacements, additions, and deletions. Modifications can be introduced into the present application's antibodies or their antigen-binding portion thereof by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid replacements are amino acid residues that are replaced with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR region of the antibodies of the present application, or antigen-binding portions thereof, can be replaced with other amino acid residues from the same side chain group, and the resulting antibodies can be tested for retention of function (i.e., the function described above) using the functional assays described herein.

[0113] The antibody of the present application or its antigen-binding portion thereof can be coupled with therapeutic agent to form immunoconjugate, such as antibody-drug conjugate (ADC). Suitable therapeutic agent includes cytotoxic molecules, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In ADC, antibody and therapeutic agent can be cross-linked by a joint, and the joint can be cut, such as peptide joints, disulfide joints or hydrazone joints. More preferably, the joint is a peptide joint, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu. ADCs can be prepared as described in U.S. Patents 7,087,600, 6,989,452, and 7,129,261, PCT Publications WO 02 / 096910, WO 07 / 038,658, WO 07 / 051,081, WO 07 / 059,404, WO 08 / 083,312, and WO 08 / 103,693, and U.S. Patent Publications 20060024317, 20060004081, and 20060247295. ADCs only have a basis for application when the antibody is internalized. Internalizing antibodies can be conjugated to cytotoxic molecules, such that the cytotoxic molecules specifically damage cells that internalize the antibody. In particular, the cytotoxic molecules can be internalized by the antibody and enter the target cell. Cytotoxic molecules can be any small molecule compound or protein molecule that causes damage to target cells, such as microtubule polymerization inhibitors, DNA damaging agents, etc.

[0114] On the other hand, the present application relates to a bispecific molecule comprising an antibody of the present application or its antigen-binding portion thereof connected to at least one other functional molecule such as another peptide or protein (e.g., another antibody or receptor ligand) to generate a bispecific molecule that binds to at least two different binding sites or targeting molecules. The term "bispecific molecule" includes molecules with three or more specificities. Bispecific molecules can appear in a variety of forms and sizes. At one end of the size spectrum, bispecific molecules maintain the traditional antibody format, except that they have two binding arms and each arm has a different specificity, instead of having two binding arms with the same specificity. At the other extreme is a bispecific molecule, which is composed of two single-chain antibody fragments (scFv) connected by peptide chains, referred to as a Bs(scFv)2 construct. Bispecific molecules of intermediate size include two different F(ab) fragments connected by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic modification, somatic cell hybridization or chemical methods.

[0115] The present application also provides a chimeric antigen receptor comprising a CD38 single-chain antibody scFv, wherein the scFv comprises the heavy and light chain CDRs, or heavy and light chain variable regions, described herein. The CD38 chimeric antigen receptor may comprise (a) an extracellular antigen-binding domain comprising a CD38 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain. The present application also provides an immune cell, such as a T cell or NK cell, comprising the chimeric antigen receptor of the present application.

[0116] Oncolytic viruses preferentially infect and kill cancer cells. The antibodies or antigen-binding portions thereof of the present application can be used together with oncolytic viruses. In addition, oncolytic viruses encoding the antibodies or antigen-binding portions thereof of the present application can be introduced into the human body.

[0117] The recombinant multifunctional fusion protein of the present application can attack tumors through multiple mechanisms of action, including i) reducing the adenosine produced by CD38 catalysis, thereby reducing the recruitment of immunosuppressive cells and relieving the activation of inhibitory signaling pathways in immune cells such as NK cells, dendritic cells, cytotoxic T cells, etc., ii) relieving the SIRP-mediated inhibitory signal on macrophages, iii) activating NK cells and / or macrophages to kill cancer cells, etc.

[0118] The recombinant fusion protein of the present application comprises a CD38 antibody or an antigen-binding portion thereof, wherein at least one paratope of the CD38 antibody or its antigen-binding portion is connected to the extracellular Ig-like domain of the signal regulatory protein (SIRP) via a linker at the N-terminus of the heavy chain or light chain constituting the paratope. The recombinant protein can simultaneously bind to CD47, CD38, and FcR, i) reducing adenosine produced by CD38 on cancer cells, thereby reducing the recruitment of immunosuppressive cells and relieving the activation of inhibitory signal pathways in immune cells such as NK cells, dendritic cells, and cytotoxic T cells; ii) blocking the interaction between CD47 on cancer cells and SIRP on macrophages, relieving the SIRP-mediated inhibitory signal on macrophages; and iii) the antibody Fc region binds to FcR on NK cells or macrophages, activating NK cells or macrophages to kill cancer cells.

[0119] In one embodiment, a paratope of a CD38 antibody or its antigen-binding portion is connected to the extracellular Ig-like domain of a signal regulatory protein (SIRP) via a linker at the N-terminus of the heavy or light chain constituting the paratope. In another embodiment, each paratope of a CD38 antibody or its antigen-binding portion is connected to the extracellular Ig-like domain of a signal regulatory protein (SIRP) via a linker at the N-terminus of the heavy or light chain constituting the paratope. In one embodiment, each paratope of a CD38 antibody or its antigen-binding portion is connected to the extracellular Ig-like domain of a signal regulatory protein (SIRP) via a linker at the N-terminus of the heavy chain constituting the paratope. In one embodiment, each paratope of a CD38 antibody or its antigen-binding portion is connected to the extracellular Ig-like domain of a signal regulatory protein (SIRP) via a linker at the N-terminus of the light chain constituting the paratope. Although the present application only constructed and tested an exemplary recombinant fusion protein in which the extracellular Ig-like domain of a regulatory protein (SIRP) is linked to the N-terminus of the heavy chain of a CD38 antibody or its antigen-binding portion, the inventors of the present application believe that a recombinant fusion protein in which the extracellular Ig-like domain of a regulatory protein (SIRP) is linked to the N-terminus of the light chain of a CD38 antibody or its antigen-binding portion can also achieve comparable effects, especially comparable in vivo anti-tumor activity.

[0120] The three main components included in the fusion protein of the present application are the extracellular Ig-like domain of signal regulatory protein (SIRP), a linker, and a CD38 antibody or its antigen binding portion. Those skilled in the art will appreciate that there are many design options for the above three components. Preferably, human sequences are used in the treatment of human cancer because the strong immunogenicity of non-human animal proteins or peptides may cause allergic reactions and other adverse reactions. However, based on different application purposes, other animal proteins or peptides can also be used in the present application and can be humanized.

[0121] Any extracellular Ig-like domain of any SIRP (SIRPα, SIRPβ and SIRPγ) that can bind to CD47 can be selected for the construction of the fusion protein. In one embodiment, the signal regulatory protein in the recombinant fusion protein is SIRPα, and the extracellular Ig-like domain of the signal regulatory protein is the first extracellular Ig-like domain of SIRPα (SIRPαD1). In one embodiment, SIRPαD1 is a mutant SIRPαD1, which has an N80A mutation at position 80 of SEQ ID NO: 70 compared to the wild-type SIRPαD1. The mutation at this site can achieve the effect of deglycosylation. In one embodiment, the recombinant fusion protein comprises a SIRPαD1 having an amino acid sequence as shown in SEQ ID NO: 70. In another embodiment, SIRPαD1 may comprise an amino acid sequence having at least 95%, 98% or 99% sequence identity to SEQ ID NO: 70, wherein SIRPαD1 is capable of binding to CD47 on the surface of cancer / tumor cells and blocking the interaction between CD47 and SIRP on the surface of macrophages.

[0122] The linker mainly acts as a spacer between the extracellular Ig-like domain of SIRP and the N-terminus of the heavy or light chain of the CD38 antibody. The linker can be composed of amino acids connected by peptide bonds, preferably 5-30, 10-30, 10-20, or 15 amino acids connected by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. One or more of these amino acids can be glycosylated or deglycosylated, as known to those skilled in the art. In one embodiment, 5-30 10-30, 10-20, or 15 amino acids can be selected from glycine, alanine, proline, asparagine, glutamine, serine and lysine. In one embodiment, the linker is composed of amino acids that have a large number of empty bond steric hindrances, such as glycine and alanine. Exemplary linkers are polyglycine (particularly Gly, poly (Gly-Ala)) and polyalanine. Exemplary suitable linkers shown in the examples below are GS linkers, such as GGGGSGGGGSGGGGS (SEQ ID NO: 73), GGGGSGGGGS (SEQ ID NO: 74), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75). In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 73).

[0123] The linker can also be a non-peptide linker. For example, an alkyl linker such as -NH-, -(CH2)sC(O)-, where s = 2-20, can be used. These alkyl linkers can also be connected via any non-sterically hindered group such as a lower alkyl group (e.g., C 1-4lower acyl), halogen (such as Cl, Br), CN, NH2, phenyl, etc.

[0124] In some embodiments, the CD38 antibody can be an isolated monoclonal antibody comprising two heavy chains and two light chains, or composed of two heavy chains and two light chains. The Fab portion (or paratope) of the CD38 antibody can bind to CD38 on the surface of cancer / tumor cells to reduce the adenosine produced by CD38 on cancer cells, thereby reducing the recruitment of immunosuppressive cells and relieving the activation of inhibitory signaling pathways in immune cells such as NK cells, dendritic cells, and cytotoxic T cells. The Fc portion of the CD38 antibody can bind to FcRs on the surface of NK cells and / or macrophages to stimulate NK cells or macrophages to kill cancer cells.

[0125] Compared with cells that do not express or low express CD38, the recombinant fusion protein of the present application prefers to bind to cells that express or high express CD38, thereby better targeting CD38. + Disease cells, such as CD38 + The invention can inhibit tumor cells without affecting normal cells in the body, such as red blood cells, or can minimize the impact on normal cells in the body. Therefore, side effects such as hemagglutination or anemia that occur in monospecific CD47 antibodies will hardly occur when the recombinant fusion protein of the present invention is administered.

[0126] The present application provides polynucleotides encoding CD38 antibodies or antigen-binding portions thereof, or recombinant fusion proteins, and expression vectors for expressing the recombinant fusion proteins. Examples of vectors include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transformable artificial chromosomes (TACs), mammalian artificial chromosomes (MACs), and artificial episomal chromosomes (HAECs).

[0127] The application provides host cells comprising the above-mentioned expression vectors. Host cells can be transformed or transfected with the expression vectors. Suitable host cells include Escherichia coli (E. coli), yeast, and other eukaryotic organisms. In particular, E. coli, yeast, or mammalian cell lines (e.g., COS or CHO) are used.

[0128] In another aspect, the present application provides a pharmaceutical composition comprising the CD38 antibody or antigen-binding portion thereof, a recombinant fusion protein, a nucleic acid molecule expressing the above or a nucleic acid molecule, an expression vector or host cell containing the nucleic acid molecule, formulated together with a pharmaceutically acceptable adjuvant. The composition may optionally contain one or more other pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present application may also be administered in combination therapy with, for example, another immunostimulant, an anticancer drug, or a vaccine.

[0129] The pharmaceutical composition may contain any number of excipients. Useful excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, stabilizers, colorants, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0130] The main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection, physiological saline or artificial cerebrospinal fluid, which can be supplemented with other materials common in injection. For example, the vehicle or carrier can be a neutral buffered saline solution or a saline solution mixed with serum albumin. Other exemplary pharmaceutical compositions include Tris buffer or acetate buffer, which can also include sorbitol or its suitable substitute. In one embodiment of the application, the composition can be prepared for storage in the form of lyophilized or aqueous solution by mixing the selected components with the required purity with any formulation (Remington's Pharmaceutical Sciences, as above). In addition, the therapeutic composition can be formulated as a lyophilized agent using a suitable excipient such as sucrose.

[0131] In some embodiments, the pharmaceutical composition can be administered intravenously, intramuscularly, subcutaneously, parenterally, spinally, or epidermally (e.g., by injection or bolus). Depending on the different routes of administration, the active molecule can be encased in a material to protect it from acid and other natural conditions that may inactivate it. As used herein, the term "parenteral administration" refers to a mode of administration other than the intestinal tract and topical administration typically performed by injection, including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, tracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, dura mater, and intrasternal injection and infusion. Alternatively, the antibody of the present application can be administered by non-injection routes, such as local, epidermal, or mucosal modes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration.

[0132] Pharmaceutical compositions can be in the form of sterile aqueous solutions or suspensions. They can also be formulated as microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0133] The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form varies depending on the subject to be treated and the specific route of administration, and is generally that amount of the composition that produces a therapeutic effect. Generally speaking, in terms of percentage, this amount is about 0.01% to about 99% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0134] The dosage regimen can be adjusted to achieve the optimal desired response (e.g., therapeutic response). For example, multiple divided doses are administered over time, or the dose is proportionally reduced or increased according to the severity of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage units for ease of administration and for uniformity of dosage. The dosage unit type used herein refers to a physically separate unit suitable for a single administration to a subject to be treated; each unit contains the amount of active compound calculated in advance to produce the desired therapeutic effect together with the drug carrier. Alternatively, the fusion protein can be administered in a sustained release dosage form, in which case the frequency of administration is reduced.

[0135] For administration of CD38 antibodies, or antigen-binding portions thereof, or recombinant fusion proteins, the dosage range is about 0.0001-100 mg / kg of recipient body weight.

[0136] A "therapeutically effective amount" of the CD38 antibody, or antigen-binding portion thereof, or recombinant fusion protein of the present application preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevents damage or disability caused by the disease. For example, for the treatment of tumor-bearing subjects, a "therapeutically effective amount" means that tumor growth is inhibited by at least about 40%, at least about 60%, at least about 80%, or even at least about 99% relative to untreated subjects. A therapeutically effective amount of the CD38 antibody, or antigen-binding portion thereof, or recombinant fusion protein of the present application can reduce tumor volume or alleviate symptoms in a subject (typically a human, or another mammal).

[0137] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0138] The pharmaceutical compositions can be administered via medical devices such as (1) needle-free subcutaneous injection devices (e.g., U.S. Patents 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556); (2) microinfusion pumps (U.S. Patent 4,487,603); (3) transdermal devices (U.S. Patent 4,486,194); (4) infusion devices (U.S. Patents 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patents 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0139] In certain embodiments, the CD38 antibodies, or antigen-binding portions thereof, or recombinant fusion proteins of the present application can be formulated to ensure appropriate in vivo distribution. For example, to ensure that the therapeutic fusion proteins of the present application cross the blood-brain barrier, the fusion proteins can be formulated in liposomes and can additionally include targeting groups to enhance selective delivery to specific cells or organs.

[0140] The present application also relates to in vivo gene therapy, in which nucleic acid molecules encoding the present application's CD38 antibody or its antigen-binding portion thereof, or recombinant fusion protein are directly introduced into a subject. For example, the nucleic acid sequence encoding the present application's CD38 antibody or its antigen-binding portion thereof, or recombinant fusion protein is introduced into the target cell via local injection of a nucleic acid construct with or without a suitable delivery vector, such as an adeno-associated viral vector. Other alternative viral vectors include, but are not limited to, retroviruses, adenoviruses, herpes simplex viruses, and papillomavirus vectors. The in vivo physical transfer of viral vectors can be achieved by local injection of the desired nucleic acid construct or other suitable delivery vectors containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or microparticle bombardment (gene gun).

[0141] The compositions of the present disclosure can be used alone or in combination with other therapeutic agents to enhance their therapeutic efficacy or reduce potential side effects.

[0142] Another object of the present application is to provide a method for preparing the above-mentioned CD38 antibody or its antigen-binding portion, recombinant fusion protein, or pharmaceutical composition. In one embodiment, the preparation method comprises the following steps: (1) providing a nucleic acid molecule encoding a CD38 antibody or its antigen-binding portion, or recombinant fusion protein; (2) constructing an expression vector comprising the nucleic acid molecule of (1); (3) transfecting or transforming suitable host cells with the expression vector in (2) and culturing these host cells to express the protein; and (4) purifying the protein. Preparation can be performed by a person of ordinary skill using well-known techniques.

[0143] Another object of the present application is to provide a method for treating cancer using the pharmaceutical composition of the present application, comprising administering an effective amount of the above-mentioned pharmaceutical composition to a patient or subject in need. In one embodiment, the pharmaceutical composition is used to treat tumors or cancers that overexpress CD47 and / or CD38, including but not limited to acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), lymphoma, multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, melanoma, glioma, esophageal cancer, plasma cell myeloma and prostate cancer.

[0144] In one embodiment, diseases associated with overexpression of CD47 and / or CD38 include, but are not limited to, Crohn's disease, allergic asthma, and rheumatoid arthritis.

[0145] The present application will be further described with reference to the following non-limiting examples.

[0146] Example

[0147] Example 1. Preparation of mouse CD38 antibody

[0148] BALB / c mice were immunized with CHO cells expressing human CD38 (CHO-CD38) and a fusion protein of the full-length extracellular domain of human CD38 and mouse IgG1-Fc. Splenocytes from these immunized mice were fused with Sp2 / 0 myeloma cells to identify hybridomas expressing antibodies that specifically bind to CHO-CD38 cells but not to CHO cells. Hybridomas producing highly potent antibodies were isolated by limiting dilution to obtain single-cell clones.

[0149] Finally, 9 CD38 antibodies were obtained, and their heavy chain and light chain variable regions were sequenced. The obtained sequences are listed in Table 1.

[0150] Table 1. Heavy and light chain variable region sequences of CD38 antibodies

[0151] Example 2. Preparation, purification and characterization of chimeric CD38 antibodies

[0152] Vectors expressing the heavy chain variable region and heavy chain constant region of each antibody (amino acid sequence as shown in SEQ ID NO: 64) and vectors expressing the light chain variable region and light chain constant region of each antibody (amino acid sequence as shown in SEQ ID NO: 65) were constructed and co-transfected into CHO-S cells (Gibco, Cat# A29127) in the presence of polyethyleneimine (PEI, Polysciences, Cat# 24765). CHO-S cells were cultured in TransFx-C CHO transfection medium (Hyclone, Cat# SH30942.02), and cell supernatants were collected after 8-10 days and applied to a Protein A agarose column (Bestchrom, Cat# AA0273). Buffer (NaCl (140 mM) + phosphate buffer (PB, 20 mM), pH = 7.4 ± 0.1) was added to the column, and the antibody on the column was eluted with elution buffer (NaAc (25 mM) + NaCl (100 mM), pH = 3.5 ± 0.1). The pH of the collected fractions was adjusted to 5.2 ± 0.2 with 2 M Tris. The purity of the obtained antibody was evaluated by size exclusion high performance liquid chromatography (SEC-HPLC).

[0153] Example 3. CD38 Binding Affinity Test of Chimeric CD38 Antibodies

[0154] The chimeric CD38 antibody prepared in Example 2 was tested for binding affinity using the BLI method and a Gator™ label-free bioanalyzer (Gator Bio).

[0155] Specifically, an anti-human IgG probe (Gator Bio, Cat#20-5036) was preincubated in Q buffer (10 mM PBS + 0.02% Tween + 0.2% BSA, pH = 7.4) for 300 s. The probe was immersed in Q buffer for 20 s for baseline detection. Afterwards, the probe was immersed in 10 μg / mL of each antibody solution for 60 s, washed in Q buffer for 20 s, incubated in a well containing 5 μg / mL human CD38 antigen (Kactus Biosystems, Cat#CD3-HM138) for 60 s, and immersed in Q buffer for 60 s. Daratumumab and esatuximab were used as positive controls. The heavy and light chain variable regions of daratumumab comprise the amino acid sequences set forth in SEQ ID NOs: 66 and 67, and the heavy and light chain constant regions comprise the amino acid sequences set forth in SEQ ID NOs: 64 and 65. The heavy and light chain variable regions of esatuximab comprise the amino acid sequences set forth in SEQ ID NOs: 68 and 69, and the heavy and light chain constant regions comprise the amino acid sequences set forth in SEQ ID NOs: 64 and 65. The blank control followed the same procedures, except that the antibody incubation step was replaced with a 60-second immersion in Q buffer.

[0156] In Gator software, the background value was subtracted and a 1:1 fitting was performed to obtain the binding curve and calculate the K D value.

[0157] The results are shown in Table 2. As can be seen, the CD38 antibodies of the present application have comparable or better binding affinities to human CD38 than the positive control. In particular, except for 65A7, the binding affinities of the antibodies of the present application to human CD38 protein are higher than that of the positive control.

[0158] Table 2. CD38 binding affinity of various CD38 antibodies in this application

[0159] Example 4. Epitope Binding Competition and Sequence Homology Analysis of Chimeric CD38 Antibodies

[0160] The binding epitopes of each CD38 antibody of the present application were analyzed using a BLI-based tandem method. Specifically, the probe with an anti-His tag was immersed in Q buffer for 15 seconds, incubated with 10 μg / mL of His-tagged CD38 antigen (Kactus Biosystems, Cat#CD3-HM138) for 120 seconds, incubated with 10 μg / mL of the first CD38 antibody for 120 seconds, and then incubated with 10 μg / mL of the second CD38 antibody for 60 seconds. Whether there is epitope binding between the two antibodies is determined based on the binding of the second antibody to the CD38 antigen. If the second antibody can bind to the CD38 antigen, there is no epitope binding competition between the first CD38 antibody and the second CD38 antibody. Otherwise, it indicates that there is epitope binding competition between the two CD38 antibodies.

[0161] The results showed that the 9 antibodies of the present application can be divided into 4 categories, 35G5, 26B4 and 25B5 are in one category, 65A7 and 82G10 are in one category, 12C10 and 32D6 are in one category, and 4A8 and 16C2 are in one category.

[0162] In addition, DNAMAN software was used to compare the variable region sequences of the CD38 antibodies of the present application to analyze the sequence homology of each antibody. The results showed that the heavy chain / light chain amino acid sequence homology between these antibodies was low, especially the heavy chain sequence, indicating the sequence diversity of the prepared antibodies.

[0163] Example 5. Chimeric CD38 Antibody and CD38 + Cell binding

[0164] Flow cytometry was used to detect the CD38 antibody of the present application and CHO cells overexpressing human CD38, CHO cells overexpressing monkey CD38, CD38 + The binding capacity of tumor cells, human red blood cells (RBC, provided by healthy volunteers) and human platelet cells (provided by healthy volunteers) was measured using daratumumab and isatuximab prepared as positive controls and hIgG1-Fc (SEQ ID NO: 71) as an isotype control.

[0165] Among them, CHO cells overexpressing human CD38 (Uiprot, P28907) were prepared by introducing a human CD38 expression vector into CHO cells by electroporation, obtaining stable polyclones through pressure screening using the glutamine synthetase screening system (GS), and obtaining stable high-expressing subclonal cell lines using the limiting dilution method. CHO cells overexpressing monkey CD38 (Uiprot, Q5VAN0) were obtained using a similar method as described above. +Tumor cells included the MM cell line NCI-H929 (ATCC, Cat#CRL-3580), non-Hodgkin's lymphoma cell lines Daudi (Cell Bank of the Chinese Academy of Sciences, Cat#TCHu140) and Raji (Cell Bank of the Chinese Academy of Sciences, Cat#TCHu 44), Hodgkin's lymphoma cell line L-1236 (Shanghai Qiansi Biotechnology Co., Ltd.), acute lymphoblastic leukemia cell line Reh (Cell Bank of the Chinese Academy of Sciences, Cat#TCHu131), and acute myeloid leukemia cell line MOLM-13 (Nanjing Kebai Biotechnology Co., Ltd.). In addition, anticoagulated blood collected from volunteers was separated by low-speed centrifugation using lymphocyte separation solution (Sigma, Cat#10771) according to the manufacturer's instructions, and the red blood cells in the bottom layer and the plasma layer in the top layer were centrifuged at high speed to obtain platelets.

[0166] Specifically, in a 96-well plate, 50 μl of serially diluted antibodies were added to 50 μl of a 1×10 5 CHO cells overexpressing human CD38, CHO cells overexpressing monkey CD38, each CD38 + Tumor cells, red blood cells, or platelets were incubated in 0.5% BSA-PBS buffer at 4°C for 45 min. 150 μl of 0.5% BSA-PBS was added to the plate to wash away unbound antibodies. 100 μl of FITC-anti-human IgG Fc secondary antibody (1:500 dilution, Sigma, Cat#F9512) was then added to the plate and incubated at 4°C in the dark for 45 min. After washing the cells with 0.5% BSA-PBS, the cells were analyzed by flow cytometry (Luminex, The FITC fluorescence signal on the cells was detected by easyCyte™ 8HT Base System. The data were analyzed using guavaSoft_33_x64 software.

[0167] The results of the antibody binding test to CHO cells overexpressing human CD38 and CHO cells are shown in Figure 1 (AB). It can be seen that the CD38 antibody of the present application binds to CHO cells overexpressing human CD38 in a concentration-dependent manner, but does not bind to CHO cells, indicating that the binding of the antibody to CD38 is specific. The binding EC values ​​of daratumumab, isatuximab, 4A8, 12C10, 16C2, 25B5, 26B4, 65A7, 35G5, 82G10, and 32D6 are shown in Figure 1 (AB). 50The values ​​were 327.4ng / ml, 312.7ng / ml, 866.7ng / ml, 264.4ng / ml, 483.3ng / ml, 135.0ng / ml, 245.0ng / ml, 242.1ng / ml, 188.5ng / ml, 667.9ng / ml and 194.9ng / ml, respectively. Among them, 12C10, 25B5, 26B4, 65A7, 35G5, and 32D6 had higher binding affinity to cell surface CD38, which was better than the positive control.

[0168] The results of the antibody binding test with CHO cells overexpressing monkey CD38 and CHO cells are shown in Figure 2. It can be seen that the antibodies 65A7, 12C10, 32D6, 82G10, 4A8 and 16C2 of the present application can bind to CHO cells overexpressing monkey CD38, while the positive control daratumumab does not bind to CHO cells overexpressing monkey CD38.

[0169] Antibodies to CD38 + The combination of tumor cells is shown in Figure 3 (AF), EC 50 The values ​​are summarized in Table 3. The results showed that each antibody bound to CD38 in a concentration-dependent manner. + The binding affinity of 35G5 and 25B5 was higher than that of tumor cells.

[0170] Table 3. The CD38 antibodies of this application and the CD38 + Tumor cell binding to EC 50 value

[0171] The results of antibody binding to human RBCs showed that the CD38 antibody of the present application did not bind to RBCs, even at a concentration of 100 nM. In addition, the CD38 antibody of the present application did not bind to platelets, even at a concentration of 100 nM.

[0172] Example 6. Inhibitory effect of chimeric CD38 antibody on CD38 enzyme activity

[0173] Take 10 μg / mL of the CD38 antibody of this application and mix with 1×10 6 / mL CD38 +Tumor cells, including Daudi, L1236, Raji, and NCl-H929, were mixed in equal volumes and added to a final volume of 100 μL per well in a 96-well flat-bottom plate (BeyoGold, Cat#FCP968). The cells were incubated in the dark for 15 minutes at room temperature. Nicotinamide guanine dinucleotide (NGD) substrate solution (Sigma Aldrich, Cat#N5131) was diluted to 120 μM, and 50 μL was added to each well of the 96-well plate. The plate was then incubated in the dark for 2 hours at room temperature. cGDPR production was quantified using a multi-function microplate reader (Molecular Devices, SpectraMax M3) with an excitation wavelength of 300 nm (EX300) and an emission wavelength of 410 nm (EM400). The inhibition rate of cGDPR production by the antibody was calculated as [1 - (antibody-treated wells - blank control wells) / (negative control-treated wells - blank control wells)] × 100%. Blank control wells contained 50 μL of CD38 + cells and 100 μL PBS, and the negative control group contained 50 μL CD38 + cells, 50 μL PBS, and 50 μL NGD.

[0174] The results are shown in Figure 4 (AD). + The strongest inhibitory effect was observed on CD38 cyclase activity on tumor cells, whereas 4A8, 12C10, 65A7, and 32D6 had no effect on CD38 cyclase activity.

[0175] Example 7. Ability of chimeric CD38 antibodies to induce tumor cell apoptosis

[0176] 50 μl containing 5 × 10 4 The culture medium of each tumor cell, including Daudi, L-1236, Raji, and NCI-I929, was mixed with 50 μl of 10 μg / ml of each CD38 antibody, seeded into a 96-well plate, and incubated at 37°C in 5% CO2 for 24 hours. Subsequently, 1 mg / ml propidium iodide (PI, Sigma, Cat# P4170) was added at 0.2 μl / well, mixed, and incubated at room temperature in the dark for 10 minutes. PI was detected by flow cytometry. + The proportion of cells.

[0177] The results are shown in Figure 5 (AD). Antibodies 12C10 and 82G10 induced strong apoptosis in Daudi and L-1236 cells, with activity higher than the two positive controls. Interestingly, each antibody barely induced apoptosis in NCI-H929 cells.

[0178] Example 8. Chimeric CD38 Antibody to CD38+ Cell-induced ADCC, ADCP, and CDC activities

[0179] The CD38 antibody of the present application was tested for its ADCC, ADCP and CDC activity on hematological cancer cell lines, including the MM cell line NCI-H929, the non-Hodgkin's lymphoma cell lines Daudi and Raji, the Hodgkin's lymphoma cell line L-1236, the acute lymphoblastic leukemia cell line Reh, and the acute myeloid leukemia cell line MOLM-13.

[0180] To 10 ml containing 1 × 10 6 / ml CD38 + 2 μl of 200 nM carboxyfluorescein succinimidyl ester (CSFE, Sigma, Cat#21888) was added to the culture medium of tumor cells and incubated at 37°C in 5% CO2 for 30 minutes, mixing by inverting every 10 minutes. 4 CSFE-labeled CD38 + Tumor cells, including Daudi, Raji, NCI-H9292 and L-1236 culture medium, were plated on 96-well plates, 50 μl of serially diluted CD38 antibodies were added, and incubated at 37°C in 5% CO2 for 30 min. 100 μl of 5 × 10 4 The culture medium of the NK cell line (YMAK-C004) overexpressing FcgRIIIA (158V) was incubated at 37°C in 5% CO2 for 4 hours. After the incubation, 1 mg / ml PI solution was added at 0.4 μl / well, mixed and incubated at room temperature in the dark for 10 minutes. CSFE was detected by flow cytometry. + PI in cells + The percentage of cells.

[0181] The results of ADCC are shown in Figure 6 (AD), EC 50 The values ​​are summarized in Table 4. The antibodies of the present application inhibit CD38 in a concentration-dependent manner. + Tumor cells elicited robust ADCC, with activity superior to daratumumab and comparable to that of isatuximab.

[0182] Table 4. The CD38 antibodies of this application + ECs that induce ADCC 50 value

[0183] THP-1 cells (Cell Bank of the Chinese Academy of Sciences, Cat# SCSP-567) were collected and washed with RPMI-1640 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum. PMA (Sigma, Cat# P-050) with a working concentration of 200 ng / mL was added to 100 μL of 4 × 10 5 / mL THP-1 cells were cultured in 96-well flat-bottom plates at 37°C and 5% CO2 for 48 h. 6 / ml each CD38 + Tumor cells, including Raji, L-1236, and NCI-H929, were cultured with 2 μl of 200 nM CSFE and incubated at 37°C for 30 min, mixing every 10 min. + After the tumor cells were washed twice with complete medium, the cell density was adjusted to 2 × 10 6 / mL. Take 50μL CD38 + Tumor cells were added to the 96-well plate containing the THP-1 cells, and 50 μL of serially diluted antibodies were added to each well. The cells were incubated at 37°C, 5% CO2 for 2 h. The plate was washed 5 times with PBS to remove the suspended cells (unphagocytosed CD38 + Tumor cells), adherent macrophages were pipetted and resuspended in PBS, and CSFE was detected by flow cytometry. + cells (i.e., phagocytic CD38 + The percentage of THP-1 cells was measured.

[0184] The results are shown in Figure 7 (AC), EC 50 The values ​​are summarized in Table 5. The antibodies of the present application can be directed against CD38 + Tumor cells elicited strong ADCP in a concentration-dependent manner.

[0185] Table 5. The CD38 antibodies of this application + EC cells that trigger ADCP 50 value

[0186] 50 μl of serially diluted CD38 antibodies were added to 50 μl of 25-fold diluted normal human serum standard complement (Quidel, Cat#A113), and 50 μl of 3×10 4 CD38 + Tumor cells, including Raji, L-1236, NCI-H929, and Daudi, were cultured at 37°C and 5% CO2 for 4 h. Then, 1 mg / ml PI solution was added at 0.2 μl / well, mixed, and incubated at room temperature for 10 min. PI was detected by flow cytometry.+ The CDC intensity triggered by CD38 antibody was calculated as: PI + Cell % = PI of antibody-treated cells + %-PI of blank control cells + %.

[0187] The results are shown in Figure 8 (AC), EC 50 The values ​​are summarized in Table 6. Antibodies 12C10, 35G5, 82G10 and 26B4 showed no significant difference in the expression of these antibodies against CD38 + Tumor cells triggered strong CDC. Interestingly, none of the antibodies triggered CDC on NCI-H929 cells.

[0188] Table 6. EC of CDC induced by CD38 antibodies of the present application on cancer cells 50 value

[0189] Example 9. In vivo anti-tumor efficacy of chimeric CD38 antibodies

[0190] The in vivo anti-tumor effects of CD38 antibody and CD38 antibody + SIRPα-Fc combination were tested in the NCI-H929 multiple myeloma xenograft model.

[0191] Specifically, 200 μL containing 5×10 6 The culture medium of NCI-H929 cells was resuspended in 100 μl of cold PBS-Matrigel (the volume ratio of PBS to Matrigel was 1:1) and injected into the back of the right side of CB17-SCID mice near the axilla. 3At 4 hr, mice were randomly divided into 6 groups of 6 mice each based on tumor size and body weight, and this day was designated as D0. On D0, D7, D14, and D21, each group of mice was intraperitoneally administered with PBS, IMM01 (0.23 mg / kg), IMM0120 (3 mg / kg), 26B4 (0.42 mg / kg), IMM01+26B4 (0.23 mg / kg+0.42 mg / kg), and IMM0120+26B4 (0.23 mg / kg+0.42 mg / kg), respectively. Among them, IMM01 is SIRPαD1-IgG1 Fc, which contains the mutated SIRPα first extracellular Ig-like domain (SIRPαD1, SEQ ID NO: 70) and the human IgG1 Fc region (SEQ ID NO: 71) from the N-terminus to the C-terminus; IMM0120 is SIRPαD1-IgG4 Fc, which contains the mutated SIRPα first extracellular Ig-like domain (SIRPαD1, SEQ ID NO: 70) and the human IgG4 Fc region (SEQ ID NO: 72) from the N-terminus to the C-terminus.

[0192] The mouse body weight and tumor size were measured twice a week. 3 The mice were euthanized at D100.

[0193] Statistical analysis was performed using GraphPad Prism 8.0. Differences between two or more groups were tested using one-way analysis of variance combined with the Holm-Sidak test, and differences within groups were tested using the Student's t-test. P ≤ 0.05 indicated a significant difference.

[0194] The mean and individual tumor volumes of mice in each group are shown in Figure 9 (A and C). The CD38 antibody 26B4 demonstrated significantly superior tumor inhibition compared to the vehicle control group and the IMM0120 group. When 26B4 was combined with IMM0120, the tumor inhibition effect was significantly better than that achieved by IMM0120 and 26B4 alone, as well as by IMM01 alone. The tumor inhibition effect was maximized when 26B4 was combined with IMM01. Complete remission (CR) was achieved in 1, 0, 1, 2, and 1 mice in the IMM01, IMM0120, 26B4, IMM01+26B4, and IMM0120+26B4 groups, respectively. Survival curves for each group are shown in Figure 9 (B), and the results are generally consistent with the tumor inhibition observed in the tumor volume change graph. Furthermore, it can be seen that the tumor inhibition effect of the combination of 26B4 and IMM01 is more sustained.

[0195] Example 10. Preparation of bispecific antibodies

[0196] Based on the above results, five CD38 antibodies, namely 4A8, 12C10, 26B4, 35G5, and 65A7, were selected to construct CD38 / CD47 bispecific antibodies in a 2+2 format. The structures are shown in Figure 10 (CD), where SD1 represents the mutated first extracellular Ig-like domain of SIRPα (SIRPαD1, SEQ ID NO: 70). Each bispecific antibody comprises a high-affinity IgG-type CD38 antibody with a low-affinity CD47 binding domain (i.e., SIRPαD1) linked to the N-terminus of its heavy chain variable region or light chain variable region via a GS linker (SEQ ID NO: 73). The CD38 heavy chain constant region is an engineered human IgG1 Fc (SEQ ID NO: 64), which can enhance Fc-dependent effector functions, including ADCC, ADCP, and CDC. At the same time, daratumumab and isatuximab are used to replace the CD38 antibody of the present application, and bispecific antibodies are constructed with the same structure. The heavy chain constant region and the light chain constant region are consistent with the bispecific antibody of the present application. The bispecific antibody in which SIRPαD1 is connected to the N-terminus of the heavy chain of the CD38 antibody of the present application is called IMM5605, and the bispecific antibody in which SIRPαD1 is connected to the N-terminus of the light chain of the CD38 antibody of the present application is called IMM5606. The bispecific antibody in which SIRPαD1 is connected to the N-terminus of the heavy chain of isatuximab is called IMM5601, and the bispecific antibody in which SIRPαD1 is connected to the N-terminus of the heavy chain of daratumumab is called IMM5603.

[0197] For the long chain in IMM5601, IMM5603, and IMM5605, i.e., the SIRPαD1-linker-CD38 antibody heavy chain, 57 nucleotides encoding the mouse IgG1 heavy chain signal peptide (SEQ ID NO: 76) were added to the 5' end of the SIRPαD1-linker-CD38 antibody heavy chain coding sequence (SEQ ID NO: 88, 90, 94, 96, 98, 100, 102), and a Kozak sequence (GCCGCCACC) was added to the 5' end of the signal peptide sequence. Finally, HindIII and NheI restriction enzyme cleavage sites were added to the 5' and 3' ends of the resulting sequence, respectively. For the short chains in IMM5601, IMM5603, and IMM5605, namely the CD38 antibody light chains, the same signal peptide sequence and Kozak sequence were added to the 5' end of the antibody light chain coding sequences (SEQ ID NOs: 89, 91, 95, 97, 99, 101, 103). HindIII and XbaI restriction enzyme sites were added to the 5' and 3' ends of the resulting sequences, respectively. The resulting sequences were synthesized and cloned into the pMac-H and pMac-L vectors, respectively.

[0198] For the long chain in IMM5606, i.e., the CD38 antibody heavy chain, 57 nucleotides encoding the mouse IgG1 heavy chain signal peptide (SEQ ID NO: 76) were added to the 5' end of the CD38 antibody heavy chain coding sequence, and a Kozak sequence (SEQ ID NO: 77) was added to the 5' end of the signal peptide sequence. For the short chain in IMM5606, i.e., the SIRPαD1-linker-CD38 antibody light chain, the same signal peptide sequence and Kozak sequence were added to the 5' end of the SIRPαD1-linker-CD38 antibody light chain coding sequence. The resulting sequences were synthesized and cloned into the pMac-H and pMac-L vectors, respectively.

[0199] The constructed expression vector was used to transiently express the protein in CHO-S cells. The general process is as follows: 1) The day before transient transfection, CHO-S cells were cultured at a rate of 1×10 6 1) Prepare the DNA at a density of 1 μg / ml for each heavy / light (long / short) chain expression vector in a 1:1 ratio and add it to 1 / 20 the volume of OPTI-MEM medium (Gibco). 2) Prepare the DNA at a density of 1 μg / ml for each heavy / light (long / short) chain expression vector in a 1:1 ratio and add it to 1 / 20 the volume of OPTI-MEM medium (Gibco). 3) Prepare PEI (MW 40,000 polyethyleneimine hydrochloride, Polysciences) at a 1 mg / ml ratio of 4:1 for each PEI:DNA and add it to 1 / 20 the volume of OPTI-MEM medium (Gibco). 4) Slowly add the PEI dilution to the DNA dilution, mix well, and incubate at room temperature for 20 minutes. 5) Add the PEI / DNA mixture to the cell suspension and incubate the cells in a 37°C, 5% CO2, 110 rpm incubator with shaking. 6) Add a transfection enhancer (1 mM sodium butyrate, 0.25% v / v DMSO), and simultaneously lower the culture temperature to 33 degrees; 7) When the cell viability dropped below 50%, centrifuge at 3000 rpm for 5 minutes, collect the supernatant, and perform affinity purification using Protein A filler.

[0200] The resulting bispecific antibodies were tested for purity by HPLC-SEC and SDS-PAGE, and the results showed that the purities of the resulting bispecific antibodies were all greater than 90%. In particular, the HPLC-SEC results showed that the purities of IMM5601, IMM5603, IMM5605-4A8, IMM5605-12C10, IMM5605-26B4, and IMM5605-35G5 were 97.05%, 95.64%, 98.52%, 92.98%, 97.66%, and 98.64%, respectively.

[0201] Example 11. Binding affinity test of bispecific antibodies to CD38 or CD47

[0202] The bispecific antibodies prepared in Example 10 were tested for CD38 or CD47 binding affinity using the BLI method according to the procedures of Example 3. Specifically, human CD38 antigen (Kactus Biosystems, Cat#CD3-HM138) or CD47 antigen (Sino Biological, Cat#12283-HCCH) was used as the binding target.

[0203] The results of the bispecific antibody binding to CD38 are shown in Table 7, and the results of the bispecific antibody binding to CD47 are shown in Table 8.

[0204] It can be seen that, except for the bispecific antibody constructed based on 65A7, whose CD38 binding affinity is comparable to that of IMM5601 and IMM5603, the CD38 binding affinity of the bispecific antibodies of the present application is higher than that of IMM5601 and IMM5603, while the binding affinity to CD47 is roughly the same.

[0205] Moreover, the binding affinity of these bispecific antibodies to CD38 is significantly higher than that to CD47. + cells, the bispecific antibody of the present application is more inclined to bind to CD38 + CD47 + cells, which can reduce off-target effects.

[0206] Table 7. CD38 binding affinity of the bispecific antibodies of the present application

[0207] Table 8. CD47 binding affinity of the bispecific antibodies of the present application

[0208] Example 12. Bispecific Antibody and CD38 + CD47 + Tumor cell binding

[0209] According to the operation of Example 5, the bispecific antibody of the present application was tested by flow cytometry for binding to CD38. + CD47 + Binding capacity of tumor cells, human RBCs, and platelets, CD38 + CD47 + Tumor cells included hematological cancer cells Daudi, Raji, NCI-H929, Reh, and L-1236. IMM5601, IMM5603, IMM01, hIgG1-Fc, hu5F9 (CD47 antibody), etc. were used as controls.

[0210] In addition, the hemagglutination activity of the bispecific antibodies of the present application was tested. Specifically, each bispecific antibody at a working concentration of 40 μg / ml was added to 200 μl of whole blood, mixed, and incubated for 2 hours. The incubated whole blood was diluted 2000 times, that is, 1 μl was added to 199 μl PBS buffer, gently mixed, and 20 μl of the mixture was added to 180 μl PBS. After standing in a 96-well flat-bottom plate for 15 minutes, the red blood cell agglutination results were recorded under a microscope.

[0211] Bispecific antibodies and CD38+CD47 + The results of tumor cell binding are shown in Figure 11 (AE), EC 50 The values ​​are summarized in Table 9. Compared with IMM01, the bispecific antibody of the present application binds more CD38+CD47 + The binding affinity was found to be concentration-dependent. The bispecific antibody constructed with 35G5 and 12C10 showed the highest binding affinity.

[0212] Table 9. Bispecific antibodies of the present application and CD38+CD47 + Tumor cell-bound EC 50 value

[0213] The results of the bispecific antibody binding to human RBCs are shown in Figure 11(F). The bispecific antibody of the present application barely binds to RBCs, exhibiting only very low binding affinity even at a concentration of 100 nM. Furthermore, the bispecific antibody of the present application does not induce hemagglutination (results not shown).

[0214] The results of the bispecific antibody binding to platelet cells are shown in Figure 11(G). It can be seen that the bispecific antibody of the present application binds to platelet cells in a concentration-dependent manner, but the binding force is significantly weaker than that of the CD47 antibody Hu5F9.

[0215] Example 13. Ability of bispecific antibodies to bind to CD47 and CD38 simultaneously

[0216] The ability of the bispecific antibody of the present application to simultaneously bind to two targets was evaluated using a BLI-based randomized assay.

[0217] Specifically, an anti-human IgG probe was used and immersed in Q buffer for 20 seconds for baseline detection. Afterwards, the probe was incubated in 10 μg / mL of each bispecific antibody for 120 seconds, rinsed with Q buffer for 20 seconds, incubated in the first antigen well at 10 μg / mL for 60 seconds, and then incubated in the well containing 10 μg / mL of the first antigen and 10 μg / mL of the second antigen for 30 seconds. The first antigen was human CD38 protein (Kactus Biosystems, Cat#CD3-HM138), and the second antigen was CD47 protein (Sino Biological, Cat#12283-HCCH); or the first antigen was CD47 protein and the second antigen was CD38 protein.

[0218] The results are shown in Figure 12 (AB). Regardless of whether it is exposed to CD38 or CD47 first, the bispecific antibody of the present application can bind to two antigens simultaneously.

[0219] In addition, flow cytometry was used to test the ability of the bispecific antibody of the present application to bind to two targets simultaneously.

[0220] Specifically, 50 μl of serially diluted bispecific antibody (initial concentration 60 μg / ml, 2-fold serial dilution to 12 concentrations) and 50 μl of 1×10 5 CD38 低 / CD47 + Jurkat cells (Cell Bank of the Chinese Academy of Sciences, Cat# SCSP-653S) or CD38 - / CD47 + NK92MI cells (ATCC, Cat#CRL-2408) were mixed and incubated at 2-8 degrees for 45 minutes. After incubation, 0.5% BSA-PBS buffer was added to wash once, 50 μl of 5 μg / ml homemade recombinant protein CD38-mFc (CD38 Val 43-Ile 300, NP_001766) was added, and incubated at 2-8 degrees for 45 minutes. After incubation, 0.5% BSA-PBS buffer was washed once, 100 μl of 500-fold diluted PE-labeled goat anti-mouse IgG secondary antibody (Biolegend, Cat#405307) was added, and incubated at 2-8 degrees for 45 minutes. After incubation, 0.5% BSA-PBS buffer was washed once, resuspended and analyzed by flow cytometry.

[0221] 50 μl of serially diluted bispecific antibody (starting concentration 10 μg / ml, 2-fold serial dilutions to 12 concentrations) and 50 μl of 1×10 5 CD38 + CD47 -CHO-CD38 cells (i.e., CHO cells overexpressing human CD38 in Example 5) were mixed and incubated at 2-8 degrees for 45 minutes. After the incubation, 0.5% BSA-PBS buffer was added to wash once, 50 μl of 1 μg / ml homemade recombinant protein CD47-mFc (CD47 Gln 19-Pro 139, Q08722-3) was added, and incubated at 2-8 degrees for 45 minutes. After the incubation, 0.5% BSA-PBS buffer was washed once, 100 μl of 500-fold diluted PE-labeled goat anti-mouse IgG secondary antibody (Biolegend, Cat#405307) was added, and incubated at 2-8 degrees for 45 minutes. After the incubation, 0.5% BSA-PBS buffer was washed once, and the cells were resuspended and analyzed by flow cytometry.

[0222] The results are shown in FIG13 (AC), which show that the bispecific antibodies of the present application can bind to two antigens simultaneously, among which 35G5 and 12C10 show the strongest ability to bind to two targets simultaneously.

[0223] Example 14. Bispecific Antibodies Preferentially Bind to CD38 + CD47 + cell

[0224] The cell binding preference of the bispecific antibody of the present application was tested by flow cytometry.

[0225] Specifically, CD38 + CD47 + CD24 + Reh cells (1×10 6 / ml) and CD38 - CD47 + CD24 - NK92MI cells (1×10 6 / ml) were mixed at a 1:1 ratio, 50 μl of the above mixed cells were taken, 50 μl of each bispecific antibody at different concentrations and 50 μl of 5 μg / ml mouse anti-human CD24 antibody (produced by Yiming Onco) were added, and incubated at 2-8 degrees for 45 minutes. After the incubation, 0.5% BSA-PBS buffer was added to wash once, 50 μl of 500-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Sigma, Cat#F9512) and 50 μl of 500-fold diluted PE-labeled goat anti-mouse IgG secondary antibody (Biolegend, Cat#405307) were added, and incubated at 2-8 degrees for 45 minutes. After the incubation, 0.5% BSA-PBS buffer was washed once, and the binding results of the bispecific antibodies and the two cells were analyzed by flow cytometry after resuspension.

[0226] The results are shown in Figure 14. Compared with CD38 - CD47+ NK92MI, the bispecific antibody of the present application binds significantly more CD38 + CD47 + Reh cells, indicating that the bispecific antibody is effective against CD38 + CD47 + Cell binding preference.

[0227] Example 15. Ability of bispecific antibodies to block CD47-SIRPα

[0228] The blocking ability of the bispecific antibody of the present application on CD47-SIRPα was tested.

[0229] Specifically, 1×10 5 / CD47 + Jurkat cells (low expression of CD38) or CD47 + CD38 + Reh cells were added with 50 μl of serially diluted bispecific antibodies and incubated at 2-8°C for 30 min. Subsequently, 50 μl of 1 μg / mL SIRPα-mFc (SEQ ID NO: 77) was added and incubated for 45 min. Cells were washed with PBS + 0.5% BSA and then incubated with PE-anti-mouse IgG (1:500 dilution, Biolegend, Cat#405307) at 2-8°C for 45 min. The PE fluorescence signal of the cells was then measured using a flow cytometer.

[0230] The results are shown in Figure 15 (AB). In the presence of Jurkat cells, the activity of the bispecific antibody of the present application in blocking CD47-SIRPα is significantly lower than that of IMM01, while in the presence of Reh cells, the activity of the bispecific antibody of the present application in blocking CD47-SIRPα is 200-500 times higher than that of IMM01. This shows that the bispecific antibody of the present application is effective in blocking CD47 + CD38 + The cells have strong activity in binding to SIRPα, indicating that the CD38 antibody may also be involved in the blocking of CD47-SIRPα. In the presence of Reh cells, IMM5601, IMM5603, IMM5605-4A8, IMM5605-12C10, IMM5605-26B4, IMM5605-35G5, IMM5605-65A7, and IMM01 blocked the IC of CD47-SIRPα. 50 The values ​​were 0.605, 0.835, 1.136, 0.428, 1.463, 0.255, 0.564, and 215.6 ng / ml, respectively.

[0231] Example 16. Inhibitory effect of bispecific antibodies on CD38 enzyme activity

[0232] According to the procedure of Example 6, the inhibitory effect of the bispecific antibody of the present application on the ADP-ribosyl cyclase activity of CD38 on cells was tested.

[0233] The results are shown in Figure 16 (AB). The bispecific antibody constructed based on 26B4 had the strongest inhibitory effect on cellular CD38 cyclase activity, followed by the bispecific antibody constructed based on 35G5. The other bispecific antibodies showed no inhibitory effect.

[0234] Example 17. Bispecific Antibody to CD38 + CD47 + Cell-induced ADCC, ADCP, and CDC activities

[0235] According to the operation of Example 8, the bispecific antibody of the present application was tested for CD38 + CD47 + Raji cells, CD38 + CD47 + NCI-H929, and CD47 + CD38 – HL-60 cells elicited ADCC, ADCP, and CDC activities.

[0236] The results of ADCC are shown in Figure 17 (AC). The bispecific antibody of the present application induces ADCC on Raji and NCI-H929 cells in a concentration-dependent manner. Except for the bispecific antibody constructed based on 26B4, the difference in ADCC activity of other bispecific antibodies is about 2-3 times. The bispecific antibody of the present application does not induce ADCC on HL-60 cells, that is, it does not induce ADCC on CD47 + Single positive cells induce ADCC. Accordingly, it is speculated that high levels of ADCC will not occur in normal cells expressing CD47.

[0237] The results of ADCP are shown in Figure 18. The bispecific antibody of the present application elicited strong ADCP against Raji and NCI-H929 in a concentration-dependent manner, and its activity was comparable to that of IMM5601 and IMM5603.

[0238] The results of CDC are shown in FIG19 . The bispecific antibodies constructed based on 12C10, 35G5, and 26B4 showed relatively strong CDC eliciting activity.

[0239] EC for the above ADCC, ADCP and CDC tests 50 The values ​​are summarized in Table 10.

[0240] Table 10. EC of the bispecific antibodies of the present application for inducing ADCC, ADCP, and CDC on tumors 50 value

[0241] Example 18. Ability of bispecific antibodies to induce apoptosis in tumor cells

[0242] According to the procedure of Example 7, the ability of the bispecific antibody of the present application to induce tumor cell apoptosis was tested by flow cytometry.

[0243] As shown in FIG20 , the bispecific antibody constructed based on 12C10 showed the strongest apoptosis-inducing ability, which was significantly higher than IMM5601 and IMM5603.

[0244] Example 19. In vivo anti-tumor efficacy of bispecific antibodies

[0245] The in vivo anti-tumor effect of the bispecific antibody of the present application was tested in the NCI-H929 multiple myeloma xenograft model.

[0246] First, the tumor suppression effects of IMM5601 and isatuximab + IMM01 were compared. 6 The culture medium of NCI-H929 cells was resuspended in 100 μl of cold PBS-Matrigel (the volume ratio of PBS to Matrigel was 1:1) and injected into the back of the right side of CB17-SCID mice near the axilla. 3 At 4 hr, mice were randomly divided into 5 groups based on tumor size and body weight, with 6 mice in each group. This day was designated as D0. On D0, D7, D14, and D21, mice in each group were intraperitoneally administered with DPBS, IMM01 (0.3 mg / kg), isatuximab (1.0 mg / kg), IMM5601 (1.2 mg / kg), and IMM01 + isatuximab (0.3 mg / kg + 1.0 mg / kg). The tumor volume of mice was measured twice a week. When the volume of the mice reached 3,000 mm, the tumor volume was measured twice a week. 3 Mice were euthanized at 4 hr. The experiment ended on D31. The heavy chain constant region of isatuximab is identical to that of the CD38 monoclonal antibody and the bispecific antibody.

[0247] Tumor volume (V) was calculated as V = (length × width 2 ) / 2. The relative tumor volume (RTV) of each mouse was calculated as RTV=V t / V0, where V t is the tumor volume on day t, and V0 is the tumor volume on D0.

[0248] The relative tumor inhibition rate (TGI) was calculated as TGI% = (1-T / C) × 100%, where T and C were the relative tumor volumes (RTV) of the experimental group and the solvent control group at a specific time point, respectively, and T / C% was the relative tumor proliferation rate, that is, the percentage of the relative tumor volumes (RTV) of the experimental group and the solvent control group at a certain time point, calculated as T / C% = T RTV / C RTV ×100%,T RTV is the average RTV of the experimental group, C RTV is the average RTV of the solvent control group, and the definition of RTV is the same as above.

[0249] The average tumor volume of mice in each group is shown in Figure 21(A). At D21, the TGIs for the IMM01, isatuximab, IMM5601, and isatuximab + IMM01 groups were 72.12%, 86.52%, 94.07%, and 86.88%, respectively. P values ​​compared to the vehicle control group were 0.0057, 0.0014, 0.0007, and 0.0013, respectively. It can be seen that isatuximab and isatuximab + IMM01 had comparable tumor inhibition effects, while IMM5601 had a greater tumor inhibition effect than either isatuximab alone or isatuximab + IMM01. Changes in individual tumor size in mice in each group are shown in Figure 21(B). By the end of the experiment, tumors in two mice in the IMM5601 group had completely resolved, while complete tumor suppression was not observed in the other groups.

[0250] Afterwards, similar procedures were followed to test the anti-tumor efficacy of the bispecific antibody of the present application in vivo. Specifically, 200 μL containing 5×10 6 The culture medium of NCI-H929 cells was resuspended in 100 μl of cold PBS-Matrigel (the volume ratio of PBS to Matrigel was 1:1) and injected into the back of the right side of CB17-SCID mice near the axilla. 3 At 4 hr, mice were randomly divided into 8 groups based on tumor size and body weight, with 6 mice in each group. This day was designated as D0. On D0 and D3, PBS, IMM5601 (1.0 mg / kg), IMM5603 (1.0 mg / kg), IMM5605-35G5 (1.0 mg / kg), IMM5605-4A8 (1.0 mg / kg), IMM5605-12C10 (1.0 mg / kg), IMM5605-26B4 (1.0 mg / kg), and IMM5605-65A7 (1.0 mg / kg) were administered to each group of mice intraperitoneally. The tumor volume of the mice was measured 2-4 times per week. When the volume of the mice reached 3,000 mm, the tumor volume was measured 2-4 times per week. 3The experiment ended on D28, and all remaining mice were euthanized, and their tumors were weighed and photographed.

[0251] The average tumor volume of each group of mice is shown in Figure 22 (A). Compared to the solvent control group, the bispecific antibodies of the present application all showed a strong tumor inhibition effect, with P values ​​less than 0.0001. The changes in individual tumor size of each group of mice are shown in Figure 22 (B). Before the end of the experiment, the tumors of 4, 3, 3, 3, 6, 4 and 3 mice in the IMM5601, IMM5603, IMM5605-35G5, IMM5605-4A8, IMM5605-12C10, IMM5605-26B4, and IMM5605-65A7 groups, respectively, completely disappeared.

[0252] The following is a partial sequence of this application.

[0253] Although the present application has been described in conjunction with one or more embodiments, it should be understood that the present application is not limited to these embodiments. The description in this application is intended to cover all variants and equivalents, all of which are included in the subject matter and scope of the appended claims. All documents cited in this article are incorporated herein by reference in their entirety.

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Claims

1. An isolated monoclonal antibody, or an antigen-binding portion thereof, capable of specifically binding to CD38, comprising i) a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3, and ii) a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3, Among them, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 respectively contain (1) GNTFTNYW (SEQ ID NO: 1), IDPSNGRT (SEQ ID NO: 2), AREEKATLVGMDY (SEQ ID NO: 3), QDIRNY (SEQ ID NO: 4), YTS, and RQGNTLPWT (SEQ ID NO: 5); (2) GYTFTSYG (SEQ ID NO:8), INTYTGEP (SEQ ID NO:9), ARKGFAY (SEQ ID NO:10), RASESVDIYGNSF (SEQ ID NO:11), RAS, and QQFNEDPYT (SEQ ID NO:12); (3) GYTFTSYW (SEQ ID NO:15), IFPGAGII (SEQ ID NO:16), VRRGKGPWFDY (SEQ ID NO:17), QNINVW (SEQ ID NO:18), KAS, and QQGQSYPRT (SEQ ID NO:19); (4) GYIFSTYW (SEQ ID NO:22), ILPGSGSI (SEQ ID NO:23), GRGWDYTMDY (SEQ ID NO:24), QSLLYSTNQKNY (SEQ ID NO:25), WAS, and QNYYSYPRT (SEQ ID NO:26); (5) GDFFSRYW (SEQ ID NO:29), INPDSSTI (SEQ ID NO:30), ARRWTTVYAMDY (SEQ ID NO:31), KSVSASGYSY (SEQ ID NO:32), LAS, and QHSRELPLT (SEQ ID NO:33); (6) GYTFTSYN (SEQ ID NO:36), IYPGNGGT (SEQ ID NO:37), ARGGELRRAYFSY (SEQ ID NO:38), ESVDSYGNTF (SEQ ID NO:39), LAS, and QQNKEDPWT (SEQ ID NO:40); (7) GFSLTSYG (SEQ ID NO:43), IWRGGST (SEQ ID NO:44), AKSMITTGYAMDY (SEQ ID NO:45), EDIYKR (SEQ ID NO:46), GAT, and QQFWNTPPT (SEQ ID NO:47); (8) GFTFSYYW (SEQ ID NO:50), IRSKSNNYAT (SEQ ID NO:51), TRLWLSTRSMDY (SEQ ID NO:52), NSVSTSDFDF (SEQ ID NO:53), LAS, and QHSRELPRT (SEQ ID NO:54); or (9) Amino acid sequences of GYTFTNHH (SEQ ID NO: 57), INPYNDYT (SEQ ID NO: 58), ARKWGEIYFDAMDH (SEQ ID NO: 59), QDINNY (SEQ ID NO: 60), YTS, and QQGHMLPYT (SEQ ID NO: 61).

2. The antibody or antigen-binding portion thereof of claim 1 , wherein the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55 or 62.

3. The antibody or antigen-binding portion thereof of claim 1 , wherein the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 56, or 63.

4. The antibody or antigen-binding portion thereof of claim 2, wherein the heavy chain variable region and the light chain variable region comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to (1) SEQ ID NOs: 6 and 7; (2) SEQ ID NOs: 13 and 14; (3) SEQ ID NOs: 20 and 21; (4) SEQ ID NOs: 27 and 28; (5) SEQ ID NOs: 34 and 35; (6) SEQ ID NOs: 41 and 42; (7) SEQ ID NOs: 48 and 49; (8) SEQ ID NOs: 55 and 56; or (9) SEQ ID NOs: 62 and 63. 5 . The antibody or antigen-binding portion thereof according to claim 1 , further comprising a heavy chain constant region linked to the heavy chain variable region, and / or a light chain constant region linked to the light chain variable region. 6 . The antibody or antigen-binding portion thereof according to claim 5 , wherein the heavy chain constant region is a heavy chain constant region having binding ability to Fc receptors and / or complement system proteins.

7. A recombinant fusion protein comprising i) a CD38 antibody or an antigen-binding portion thereof, and ii) a CD47 binding peptide, The CD38 antibody or its antigen-binding portion comprises a heavy chain variable region, a heavy chain constant region, and a light chain variable region, wherein the heavy chain variable region comprises HV-CDR1, HV-CDR2, and HV-CDR3, and the light chain variable region comprises LV-CDR1, LV-CDR2, and LV-CDR3, wherein VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 respectively comprise (1) GNTFTNYW (SEQ ID NO: 1), IDPSNGRT (SEQ ID NO: 2), AREEKATLVGMDY (SEQ ID NO: 3), QDIRNY (SEQ ID NO: 4), YTS, and RQGNTLPWT (SEQ ID NO: 5); (2) GYTFTSYG (SEQ ID NO:8), INTYTGEP (SEQ ID NO:9), ARKGFAY (SEQ ID NO:10), RASESVDIYGNSF (SEQ ID NO:11), RAS, and QQFNEDPYT (SEQ ID NO:12); (3) GYTFTSYW (SEQ ID NO:15), IFPGAGII (SEQ ID NO:16), VRRGKGPWFDY (SEQ ID NO:17), QNINVW (SEQ ID NO:18), KAS, and QQGQSYPRT (SEQ ID NO:19); (4) GYIFSTYW (SEQ ID NO:22), ILPGSGSI (SEQ ID NO:23), GRGWDYTMDY (SEQ ID NO:24), QSLLYSTNQKNY (SEQ ID NO:25), WAS, and QNYYSYPRT (SEQ ID NO:26); (5) GDFFSRYW (SEQ ID NO:29), INPDSSTI (SEQ ID NO:30), ARRWTTVYAMDY (SEQ ID NO:31), KSVSASGYSY (SEQ ID NO:32), LAS, and QHSRELPLT (SEQ ID NO:33); (6) GYTFTSYN (SEQ ID NO:36), IYPGNGGT (SEQ ID NO:37), ARGGELRRAYFSY (SEQ ID NO:38), ESVDSYGNTF (SEQ ID NO:39), LAS, and QQNKEDPWT (SEQ ID NO:40); (7) GFSLTSYG (SEQ ID NO:43), IWRGGST (SEQ ID NO:44), AKSMITTGYAMDY (SEQ ID NO:45), EDIYKR (SEQ ID NO:46), GAT, and QQFWNTPPT (SEQ ID NO:47); (8) GFTFSYYW (SEQ ID NO:50), IRSKSNNYAT (SEQ ID NO:51), TRLWLSTRSMDY (SEQ ID NO:52), NSVSTSDFDF (SEQ ID NO:53), LAS, and QHSRELPRT (SEQ ID NO:54); or (9) the amino acid sequences of GYTFTNHH (SEQ ID NO: 57), INPYNDYT (SEQ ID NO: 58), ARKWGEIYFDAMDH (SEQ ID NO: 59), QDINNY (SEQ ID NO: 60), YTS, and QQGHMLPYT (SEQ ID NO: 61), The heavy chain constant region has Fc receptor and complement system protein binding ability and is connected to the C-terminus of the heavy chain variable region. The CD47 binding peptide comprises the first extracellular Ig-like domain of signal regulatory protein α (SIRPα) (SIRPαD1), wherein SIRPαD1 comprises the amino acid sequence shown in SEQ ID NO: 70, The CD47 binding peptide is linked to the N-terminus of the heavy chain variable region or the light chain variable region of the CD38 antibody or antigen binding portion thereof.

8. The recombinant fusion protein of claim 7, wherein the CD47 binding peptide is linked to the N-terminus of the heavy chain variable region of the CD38 antibody or antigen-binding portion thereof.

9. The recombinant fusion protein of claim 7, wherein the CD47 binding peptide is linked to the N-terminus of the light chain variable region of the CD38 antibody or antigen-binding portion thereof.

10. The recombinant fusion protein of claim 7, wherein the heavy chain variable region and the light chain variable region comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to (1) SEQ ID NOs: 6 and 7; (2) SEQ ID NOs: 13 and 14; (3) SEQ ID NOs: 20 and 21; (4) SEQ ID NOs: 27 and 28; (5) SEQ ID NOs: 34 and 35; (6) SEQ ID NOs: 41 and 42; (7) SEQ ID NOs: 48 and 49; (8) SEQ ID NOs: 55 and 56; or (9) SEQ ID NOs: 62 and 63, respectively. The recombinant fusion protein according to claim 7 , wherein the CD47 binding peptide is linked to the CD38 antibody or the antigen-binding portion thereof via a linker. 12 . The recombinant fusion protein according to claim 11 , wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 73, 74 or 75.

13. The recombinant fusion protein according to claim 7, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

64.

14. The recombinant fusion protein of claim 7, wherein the CD38 antibody or antigen-binding portion thereof further comprises a light chain constant region connected to the C-terminus of the light chain variable region.

15. The recombinant fusion protein according to claim 7, comprising i) CD47 binding peptide-linker-CD38 antibody heavy chain variable region-heavy chain constant region chain, and ii) CD38 antibody light chain variable region-light chain constant region chain, wherein i) and ii) comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (1) SEQ ID NOs: 78 and 79; (2) SEQ ID NOs: 80 and 81; (3) SEQ ID NOs: 82 and 83; (4) SEQ ID NOs: 84 and 85; (5) SEQ ID NOs: 86 and 88; or (6) SEQ ID NOs: 92 and 93, respectively.

16. A nucleic acid molecule encoding the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 6, or the recombinant fusion protein according to any one of claims 7 to 15. An expression vector comprising the nucleic acid molecule of claim 16 .

18. A host cell comprising the expression vector according to claim 17, or having the nucleic acid molecule according to claim 16 integrated into its genome.

19. A composition comprising the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-6, the recombinant fusion protein of any one of claims 7-15, the nucleic acid molecule of claim 16, the expression vector of claim 17, or the host cell of claim 18.

20. Use of the composition of claim 19 in the preparation of a medicament for treating or alleviating a disease associated with overexpression of CD47 and / or CD38, wherein the disease is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), lymphoma, multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, melanoma, glioma, esophageal cancer, plasma cell myeloma and prostate cancer.