Bispecific recombinant protein and application thereof

Abstract

The invention discloses a bispecific recombinant protein which comprises a high-affinity tumor targeting arm and a low-affinity fusion protein for blocking the interaction of CD47 and SIRP alpha, and an antibody corresponding to the high-affinity tumor targeting arm is not combined with CD47. The binding affinity of the fusion protein to a target antigen on a tumor cell is at least 6 times of the binding affinity of a monomeric fusion protein and a dimer corresponding to the fusion protein for blocking the interaction of the CD47 and the SIRP alpha on the CD47 on the tumor cell, and the fusion protein for blocking the interaction of the CD47 and the SIRP alpha comprises an extracellular truncation of the SIRP alpha. The invention also discloses a nucleic acid molecule for coding the recombinant protein and application of the recombinant protein and the nucleic acid molecule in preparation of medicines for treating tumors. According to the bispecific recombinant protein disclosed by the invention, the tumor targeting saturation binding abundance of the recombinant protein capable of regulating the macrophage function is remarkably improved, the side effect of non-tumor targeting is reduced, and the bispecific recombinant protein has a great clinical application value.

Description

[0001] This application is a divisional application of the application number CN202111362860.6 filed on May 8, 2018, and the invention name is “Bispeci?c Recombinant Proteins and Their Applications”. Technical Field

[0002] The present invention belongs to the field of biomedicine, and in particular relates to a recombinant protein and a use thereof. Background Art

[0003] With the deepening of research in the field of tumor treatment, the development and application of tumor molecular targeted therapeutic drugs have received more and more attention. Based on the advantages of strong targeting, small side effects and significant efficacy, antibody drugs have quickly become hot drugs in the field of tumor targeted therapy. Currently, dozens of tumor-targeting antibody drugs have been approved for clinical use and have achieved significant therapeutic effects, such as Rituximab targeting CD20 target molecules. (Rituximab, the first monoclonal antibody approved for cancer treatment in the United States, originally used to treat non-Hodgkin's lymphoma, produced by Roche) (ibritumomab tiuxetan, originally approved by the U.S. FDA for the treatment of poorly differentiated non-Hodgkin's lymphoma resistant to rituximab, produced by IDEC Pharmaceuticals), (tositumomab and iodine I131tositumomab, produced by GSK), (ofatumumab, produced by GSK), etc., Herceptin targeting the Her2 target molecule (Trastuzumab, a famous drug for treating breast cancer, produced by Genentech), (Pertuzumab, produced by Roche), (ado-trastuzumab emtansine, produced by Roche), etc., Avastin targeting VEGF or its receptor target molecules (bevacizumab, produced by Genentech / Roche), (ramucirumab, produced by Eli Lilly), etc., Erbitux targeting EGFR (Cetuximab, one of the world's top ten best-selling anti-cancer drugs, originally approved for the treatment of colorectal cancer, produced by Eli Lilly), (panitumumab, a drug for the treatment of colorectal cancer, produced by Amgen), etc., targeting the PD-L1 target molecule (atezolizumab, produced by Roche) etc. (Cao Rui et al., Research progress of antibody drugs for tumor targeted therapy, "Chinese Journal of Biochemical Pharmacology", 2016, 36(6): 15-18).

[0004] The anti-tumor mechanism of the anti-tumor antibody drugs (uncoupled naked antibodies) targeting tumor cells used clinically is mainly achieved through two functions of antibodies. First, the first function of antibodies is affinity, that is, the antibody exerts its effector function after specifically binding to the target antigen on the surface of the tumor and kills tumor cells. By binding to the target antigen, the antibody molecule can block the tumor growth factor signaling pathway, induce cell apoptosis or inhibit the formation of new blood vessels in the tumor microenvironment. Secondly, the second function of antibodies is achieved with the help of the immune system, that is, the killing effect on tumor cells can also rely on the immune system to mediate cell death, such as antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by the constant region of the antibody, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP). More and more data show that antibody-mediated immune killing activity is an important mechanism for antibodies to exert anti-tumor effects (for a review, see Barnhart BC, el al. Role of Fc-FcγR interactions in the antitumor activity of therapeutic antibodies. Immunology and Cell Biology, 2017, 95: 340–346)

[0005] In recent years, studies have confirmed that tumor cells can evade the monitoring, identification and attack of the body's immune system by modifying their own surface antigens and changing the microenvironment around the tumor tissue, and continue to divide and grow, which is the so-called tumor immune escape. For example, tumor cells highly express CD47, which binds to the inhibitory receptor signal regulatory protein α (signal regulatory protein α, SIRPα) on the surface of macrophages to inhibit the immune function of macrophages and significantly inhibit the activity of immune cells. At the same time, tumor cells highly express CD47 to inhibit Fc receptor-mediated phagocytosis, ultimately affecting the tumor targeted therapy effect of antibody drugs (Willingham SB, et al. The CD47-signal regulatory protein Alpha (SIRPα) interaction is a therapeutic target for human solid tumors. Proceedings of the National Academy of Sciences of the United States of America. 2012, 109 (17): 6662-6667).

[0006] CD47 is a widely expressed membrane glycoprotein. It is a receptor and ligand for SIRPα and can form a CD47-SIRPα signaling complex to mediate a series of reactions such as apoptosis, proliferation, and immunity. In 2000, Oldenborg et al. confirmed that CD47 is an important signal on the cell surface that regulates macrophage phagocytosis (Oldenborg PA, et al. Role of CD47 as a marker of self on red blood cells. Science, 2000, 288 (5473): 2051-2054). CD47 can bind to SIRPα on the surface of macrophages by releasing the "don't eat me" signal, phosphorylate its immunoreceptor tyrosine-based inhibitory motif (ITIM), and then recruit SH2-containing protein tyrosine phosphatase SHP1 (SH2-containing protein tyrosine phosphatase 1) protein, resulting in a series of cascade reactions that inhibit the phagocytosis of macrophages. Young red blood cells express higher levels of CD47, sending a "don't eat me, we're one" signal to macrophages, whereas aged red blood cells have downregulated CD47 expression and are eventually cleared by macrophages.

[0007] Since CD47 is widely expressed on the surface of various cancer cells, this target can be used to treat various cancers. Moreover, the mouse xenograft tumor model has demonstrated the effectiveness of CD47 blockade. Therefore, CD47 has become a new target for cancer immune checkpoint therapy (Vonderheide RH.CD47 blockade as another immune checkpoint therapy for cancer.Nature Medicine,2015,21(10):1122-1123). Anti-CD47 antibodies, SIRPα-Fc fusion proteins and other drugs under development block the CD47-SIRPα signaling pathway, relieve the inhibitory effect of CD47 on immune cells, and show certain anti-tumor activity. However, because red blood cells also express a large amount of CD47 protein, anti-CD47 antibody therapy that binds to CD47 protein with high affinity will produce toxic side effects such as red blood cell agglutination and anemia (Mccracken MN, et al. Molecular Pathways: Activating T Cells after Cancer Cell Phagocytosis from Blockade of CD47 "Don't Eat Me" Signals. Clinical Cancer Research, 2015, 21 (16): 3597-3601). This makes the clinical development of anti-CD47 antibody drugs extremely difficult. So far, no anti-CD47 antibody drug has entered Phase III clinical trials. The wild-type SIRPα-Fc fusion protein has low affinity for CD47, so its efficacy is not significant. Some researchers have also mutated wild-type SIRPα with high affinity and obtained SIRPα mutants with affinity enhanced by thousands of times, which showed good anti-tumor efficacy (Weiskopf K, et al. Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies. Science, 2013, 341(6141):88-91), but whether the multi-point mutations have an impact on the safety, immunogenicity, stability and even target specificity of the fusion protein in the human body remains to be verified by clinical observation.

[0008] On the other hand, studies have shown that anti-CD47 antibodies can significantly improve the anti-tumor efficacy by combining them with other tumor-targeted therapeutic antibodies, such as CD47+CD20 and CD47+Her2 combined therapy (Chao MP, et al. Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell, 2010, 142(5): 699-713; Zhao XW, et al. CD47–signal regulatory protein-α(SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction. Proceedings of the National Academy of Sciences of the United States of America. 2011, 108(45): 18342-18347). However, there is great uncertainty as to whether and when anti-CD47 antibodies can be marketed; after being marketed, the combined use of tumor-targeted antibody drugs also makes it difficult for patients to afford the very expensive combined treatment costs.

[0009] Therefore, it is necessary to develop new tumor targeted drugs that can specifically target and treat tumors while activating and enhancing the patient's immune function, significantly improving the effect of tumor treatment while ensuring safety, while reducing patients' drug costs.

[0010] In addition, given the potential safety risks of monovalent or multivalent antibodies or recombinant proteins targeting CD47 (such as anemia, erythrocyte agglutination, killing of CD47-positive non-tumor target cells, etc.), and the species-specific binding of human SIRPα to human CD47, and the use of human blood is subject to ethical and genetic resource restrictions, there is an urgent need to develop an early in vivo / in vitro immune safety evaluation method to evaluate the immune safety of antibodies or recombinant proteins targeting CD47. Summary of the invention

[0011] The present invention provides a bispecific recombinant protein and application thereof, which significantly improves the tumor-targeted saturation binding abundance of a recombinant protein capable of regulating macrophage function and can significantly reduce non-tumor-targeted side effects.

[0012] In one aspect, the present invention provides a bispecific recombinant protein, wherein the bispecific recombinant protein comprises a high-affinity tumor-targeting arm and a fusion protein with a low affinity that blocks the interaction between CD47 and SIRPα.

[0013] In one embodiment, the high-affinity tumor-targeting arm does not bind to CD47, and the binding affinity of the antibody corresponding to the high-affinity tumor-targeting arm to the target antigen on tumor cells is at least 6 times, optionally 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, or higher times, or any value therebetween, of the monomeric fusion protein homodimer corresponding to the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity for binding to CD47 on tumor cells;

[0014] The binding affinity of the fusion protein for low affinity blocking the interaction between CD47 and SIRPα to CD47 is no higher than the binding affinity of the monomeric fusion protein homodimer containing the extracellular truncated body of SIRPα to CD47. The fusion protein for low affinity blocking the interaction between CD47 and SIRPα contains the extracellular truncated body of SIRPα.

[0015] In one embodiment, the extracellular truncation of SIRPα comprises part or all of the extracellular amino acid sequence of human SIRPα (wild type or CD47 non-high affinity mutant).

[0016] In another embodiment, the SIRPα extracellular truncation is a human SIRPα extracellular truncation. In a specific embodiment, the SIRPα extracellular truncation comprises an amino acid sequence selected from any one of the following a1)-a4): a1) SEQ ID No: 30; a2) SEQ ID No: 31; a3) SEQ ID No: 32; a4) an amino acid sequence obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, such as 1-5 amino acid residues, in any of the above amino acid sequences, and its monomer has a binding affinity for CD47 protein and the binding affinity is not higher than the binding affinity of a1), a2) or a3) monomer to CD47 protein.

[0017] In one embodiment, the bispecific recombinant protein has a structure of two arms arranged relatively to each other, wherein the arm for targeting tumors with high affinity is located at the position of the left arm, and the fusion protein for blocking the interaction between CD47 and SIRPα with low affinity is located at the position of the right arm; preferably, the left arm is in the form of Fab or Fab' of an immunoglobulin, and the right arm is an extracellular truncation of SIRPα. Among them, the "structure of two arms arranged relatively to each other" described in the present invention is a description of the conventional "Y"-shaped structure of an immunoglobulin. In this case, the left and right sides of the bispecific recombinant protein of the present invention have specific functional proteins with different targets; this structural description is mainly distinguished from the fact that the two specific functional proteins can be connected to form an upper and lower structure by connecting the C-terminus and the N-terminus. Therefore, the spatial position of the left and right arms described in the present invention is not a specific limitation on the structure of the bispecific recombinant protein, but only to distinguish the two arm structures of the bispecific recombinant protein from the upper and lower structures formed by connecting the C-terminus and the N-terminus. When one is located on one side, the other is correspondingly located on the other side of the opposite side; obviously, the left and right spatial positions of the two can be interchanged.

[0018] In another embodiment, the length of the right arm is adapted to the distance of the antigenic spatial epitope to be bound by the left arm from the surface of the target cell membrane. Preferably, when the high-affinity tumor-targeting arm needs to bind to an epitope near the membrane end of the target cell, the SIRPα extracellular truncation is selected to contain the shorter amino acid sequence of a1) to a4).

[0019] In one embodiment, the high affinity tumor targeting arm targets a target selected from the group consisting of 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5 , CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GCC, GD2, GPC -3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, integrin-4, Notch 2. Notch 1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138.

[0020] Preferably, when the target is CD20, EGFR or PD-L1, the SIRPα extracellular truncation selects a1); when the target is HER2, the SIRPα extracellular truncation selects a1) or a2).

[0021] In one embodiment, the high-affinity tumor-targeting arm and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα are bound by intermolecular forces, or by covalent bonds such as interchain disulfide bonds, or by salt bonds, or by a combination of two or three of the above binding modes.

[0022] In another embodiment, the fusion protein of the high affinity tumor targeting arm and the low affinity blocking CD47 and SIRPα interaction further comprises an Fc region. Generally, the Fc region of the present invention comprises the native sequence of the Fc region. However, the Fc region of the present invention may have one or more existing amino acid sequence changes or modifications in the native sequence of the Fc region, for example, an amino acid sequence change or modification that changes the C1q binding activity of the Fc region.

[0023] In another embodiment, the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity is a fusion protein comprising an extracellular truncation of SIRPα and a binding sequence for binding to the arm, wherein the extracellular truncation of SIRPα and the binding sequence for binding to the arm are optionally connected via a linker sequence, and the binding sequence for binding to the arm optionally comprises an Fc region.

[0024] In another embodiment, the Fc region of the present invention is conceptual, that is, although it may not actually exist, antibody engineering can be carried out according to the amino acid sequence of the desired Fc region variant to produce a polypeptide or fusion protein containing this sequence or a DNA encoding the amino acid sequence of the desired Fc region variant.

[0025] In another embodiment, the Fc region may be an Fc region variant. As used herein, "Fc region variant" refers to an Fc region obtained by modifying one or more amino acid residues in the native amino acid sequence of Fc. The method of modification is well known to those skilled in the art, including but not limited to site-directed mutagenesis of the DNA sequence encoding the Fc region, such as using PCR mutagenesis and cassette mutagenesis to prepare Fc region variants. For example, in order to improve binding to FcR, one or more amino acid residues in the Fc region may be deleted. For example, in one embodiment, in order to change the effector function of the Fc region, an amino acid insertion type Fc region variant may be prepared.

[0026] In one embodiment, for example, at least one amino acid residue (e.g., 1-2 amino acid residues, usually no more than 10 amino acid residues) can be introduced near one or more Fc region sites identified as affecting FcR binding. "Nearby" refers to within 1-2 amino acid residues of the identified Fc region sites that affect FcR binding. Such Fc region variants can exhibit enhanced or reduced FcR binding and / or ADCC activity. In order to prepare such insertion variants, the co-crystallization structure of a polypeptide containing an FcR binding region (e.g., the extracellular domain of a target FcR) and the Fc region into which the amino acid residue is to be inserted can be evaluated to relate to Fc region variants having, for example, enhanced FcR binding ability. Such insertions are usually placed in the loops of the Fc region.

[0027] In one embodiment, by introducing appropriate amino acid sequence modifications in the native Fc region, it is possible to prepare an Fc region variant that more effectively mediates antibody-dependent cell-mediated cytotoxicity (ADCC) and / or binds to Fcγ receptors (FcγR) with stronger affinity than a recombinant protein containing a native Fc region in the presence of human effector cells. The Fc region variants of the present invention generally include at least one amino acid modification in the Fc region. Preferably, multiple amino acid modifications are combined. For example, the Fc region variant may include substitutions of 2, 3, 4, 5 or more amino acid residues, such as in the identified specific FcR binding site.

[0028] The native Fc region is preferably a human Fc region, for example, a native sequence of the Fc region of human IgG1 (A or non-A isotype), IgG2, IgG3 or IgG4.

[0029] In another embodiment, the high-affinity tumor-targeting arm is combined with the low-affinity blocking CD47 and SIRPα interaction fusion protein by knobs-into-holes. For example, the knobs-into-holes are formed by the T366W mutation to form a protruding "knobs" type, and one amino acid mutation (Y407V) to form a recessed "holes" type or three amino acid mutations (T366S, L368A and Y407V) to form a recessed "holes" type. Mutations are numbered according to the Kabat numbering scheme [Eunumbering scheme of Kabat et al. (1991)], which are shown from left to right as the original amino acid residue, mutation site and substituted amino acid residue, for example, in T366W, T is the original amino acid residue, 366 is the mutation site and W is the amino acid residue that replaces T.

[0030] In one embodiment, the high affinity tumor targeting arm is a half antibody targeting a target selected from the following targets: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACA M5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GCC, GD2, GP C-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, integrin-4, Notch 2, Notch 1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138; preferably a half antibody of IgG1 antibody, optionally a human-mouse chimeric half antibody, a humanized half antibody, or a fully human half antibody; more preferably a half antibody of a humanized or fully human IgG1 antibody.

[0031] In one embodiment, the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity comprises an amino acid sequence selected from any one of the following b1)-b4): b1) SEQ ID No: 26; b2) SEQ ID No: 27; b3) SEQ ID No: 28; b4) an amino acid sequence obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, for example, 1-5 amino acid residues, in any of the above amino acid sequences, whose homodimer has a binding affinity to the CD47 protein and the binding affinity is not higher than the binding affinity of the homodimer to the CD47 protein in b1), b2) or b3). The signal peptide region sequence, linker sequence, hinge region, Fc region and / or binding sequence contained in the above sequence can be arbitrarily replaced according to methods well known to those skilled in the art or commonly used signal peptide sequences, linker sequences, hinge regions, Fc regions and / or binding sequences.

[0032] In another embodiment, the bispecific recombinant protein comprises the following sequence:

[0033] When the high-affinity tumor-targeting arm targets CD20, the high-affinity tumor-targeting arm comprises SEQ ID No: 16 and SEQ ID No: 17, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα comprises SEQ ID No: 26 or SEQ ID No: 27 or SEQ ID No: 28;

[0034] When the high-affinity tumor-targeting arm targets EGFR, the high-affinity tumor-targeting arm comprises SEQ ID No: 19 and SEQ ID No: 8, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα comprises SEQ ID No: 26 or SEQ ID No: 27 or SEQ ID No: 28;

[0035] When the high-affinity tumor-targeting arm targets Her2, the high-affinity tumor-targeting arm comprises SEQ ID No: 20 and SEQ ID No: 21, or SEQ ID No: 22 and SEQ ID No: 23, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα comprises SEQ ID No: 26 or SEQ ID No: 27 or SEQ ID No: 28; or,

[0036] When the high-affinity tumor-targeting arm targets PD-L1, the high-affinity tumor-targeting arm comprises SEQ ID No: 24 and SEQ ID No: 13, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα comprises SEQ ID No: 26 or SEQ ID No: 27 or SEQ ID No: 28.

[0037] In one aspect, the present invention provides a nucleic acid molecule encoding a bispecific recombinant protein. Preferably, the nucleic acid molecule encoding the high-affinity tumor-targeting arm and the nucleic acid encoding the low-affinity blocking CD47 and SIRPα fusion protein are in the same DNA chain, or the nucleic acid molecule encoding the high-affinity tumor-targeting arm and the nucleic acid encoding the low-affinity blocking CD47 and SIRPα fusion protein are in different DNA chains.

[0038] In another aspect, the present invention provides an expression vector comprising a nucleic acid molecule.

[0039] In one aspect, the invention provides a cell comprising an expression vector.

[0040] On the other hand, the present invention also provides a method for preparing a bispecific recombinant protein, which comprises: 1) providing an arm with high affinity for tumor targeting; 2) providing a fusion protein with low affinity for blocking the interaction between CD47 and SIRPα; 3) contacting the arm with high affinity for tumor targeting with the fusion protein with low affinity for blocking the interaction between CD47 and SIRPα to form the recombinant protein.

[0041] In one embodiment, the method of preparing a recombinant protein comprises expressing the recombinant protein in a host cell comprising an expression vector comprising a nucleic acid molecule encoding the recombinant protein.

[0042] In another aspect, the present invention also provides a method for tumor targeted therapy.

[0043] In one embodiment, the tumor is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, cutaneous fibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome and other blood tumors and solid tumors.

[0044] In yet another aspect, the present invention also provides a pharmaceutical composition comprising a recombinant protein or fusion protein of the present invention and an optional adjuvant, excipient or pharmaceutically acceptable carrier. The composition may contain a pharmaceutically acceptable carrier. The composition may exist in any form of pharmaceutical preparation, including but not limited to injection, powder, freeze-dried powder, etc. The pharmaceutical composition in the form of the pharmaceutical preparation may be prepared according to conventional techniques of pharmaceutics, including fusing the active pharmaceutical ingredient, the recombinant protein or fusion protein of the present invention with a pharmaceutical carrier, and preparing the required dosage form according to conventional techniques of pharmaceutics.

[0045] In one embodiment, the present invention also provides a pharmaceutical composition comprising an expression vector of a nucleic acid molecule encoding the recombinant protein or fusion protein of the present invention and an optional pharmaceutically acceptable carrier.

[0046] In another aspect, the present invention also provides a method for treating a tumor, comprising administering to a patient or subject a therapeutically effective amount of the pharmaceutical composition of the present invention. The tumor expresses additional target molecules, including but not limited to 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CL DN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GCC, GD2, GPC-3 , GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, integrin-4, Notch 2. Notch 1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138.

[0047] In yet another aspect, the present invention also provides an in vivo gene therapy method, comprising introducing into a patient or subject a therapeutically effective amount of a nucleic acid molecule encoding the recombinant protein or fusion protein of the present invention or a derivative thereof.

[0048] In another aspect, the present invention also provides a method for evaluating the early immune safety of a recombinant protein / antibody targeting CD47 and having ADCC activity in vitro, the method comprising: a) providing a recombinant protein / antibody (including monovalent or multivalent) targeting and having ADCC activity; b) detecting the ADCC activity of the recombinant protein / antibody; c) evaluating the early immune safety of the recombinant protein / antibody (including monovalent or multivalent).

[0049] In one embodiment, the present invention provides a method for evaluating the early immune safety of a recombinant protein / antibody targeting CD47 and having ADCC activity in vitro, comprising: a) preparing effector cells, such as but not limited to human NK92MI-CD16a effector cells; b) contacting the effector cells with the recombinant protein / antibody; c) detecting the ADCC activity of the recombinant protein / antibody; d) evaluating the early immune safety of the recombinant protein / antibody based on the ADCC activity results.

[0050] In another embodiment, the present invention provides a method for evaluating the early immune safety of a recombinant protein / antibody targeting CD47 and having ADCC activity in vitro, comprising: a) harvesting well-grown human NK92MI-CD16a effector cells, and resuspending the harvested effector cells to a cell density of 1×10 5 cells / ml to 5×10 6 cells / ml; b) incubating the gradiently diluted recombinant protein / antibody with the effector cells prepared in a) for 0.5-5h; c) after the incubation is completed, measuring LDH activity and calculating the cell lysis rate; d) evaluating the early immune safety of the recombinant protein / antibody according to the cell lysis rate, wherein the recombinant protein / antibody that results in a lower cell lysis rate has a higher immune safety.

[0051] In another aspect, the present invention also provides a method for evaluating the early immune safety of a recombinant protein / antibody (including monovalent or multivalent) targeting CD47 in vivo, the method comprising: a) providing a recombinant protein / antibody (including monovalent or multivalent) targeting CD47; b) providing Hu-NSG mice; c) contacting the recombinant protein / antibody with Hu-NSG mice; d) evaluating the early immune safety of the recombinant protein / antibody in the Hu-NSG mice.

[0052] In one embodiment, the present invention provides a method for evaluating the early immune safety of a recombinant protein / antibody (including monovalent or multivalent) targeting CD47 in vivo, the method comprising: a) providing Hu-NSG mice; b) administering a recombinant protein / antibody (including monovalent or multivalent); c) 24-96 hours after administration, taking venous blood from the mice, lysing red blood cells, and incubating the remaining cells with fluorescently labeled anti-human CD45, anti-human CD19 or anti-human CD3 antibodies for 15-60 minutes, and performing flow cytometry detection; d) evaluating the early immune safety of the recombinant protein / antibody based on the cell clearance rate, wherein the recombinant protein / antibody that results in a lower clearance rate of immune cells (excluding target cells) has a high immune safety.

[0053] Beneficial effects of the present invention:

[0054] The present invention reduces the binding of the recombinant protein of the present invention to the CD47 target on non-tumor cells (such as red blood cells, NK cells, T cells, etc.) by blocking the interaction between CD47 and SIRPα with low affinity, thereby improving the safety of the recombinant protein of the present invention (such as avoiding anemia, red blood cell agglutination, and CD47-positive non-tumor target cell killing caused by anti-CD47 antibodies / recombinant proteins such as Hu5F9-G4, and reducing the risks brought about by potential safety, immunogenicity, stability and other uncertainties caused by high-affinity mutated SIRPα-Fc fusion proteins, etc.).

[0055] It was unexpectedly discovered in the present invention that the binding affinity of the recombinant protein of the present invention to tumor cells can be significantly improved through the high-affinity tumor cell targeting arm and the low-affinity blocking of the interaction between CD47 and SIRPα, thereby mediating the effect of highly efficient tumor cell killing (including but not limited to the effector function exerted after the antibody specifically binds to the tumor surface target antigen, and the targeted phagocytosis of macrophages).

[0056] The recombinant protein of the present invention achieves dual-targeting effect through a high-affinity tumor-targeting arm (for example, not limited to a half antibody) and a fusion protein with low affinity that blocks the interaction between CD47 and SIRPα. The results of the present invention show that the recombinant protein of the present invention exerts anti-tumor effects through multiple mechanisms with higher efficacy by virtue of the multiple interactions between the high-affinity tumor-targeting arm and the fusion protein with low affinity that blocks the interaction between CD47 and SIRPα.

[0057] Compared with the combined use of two separate targeting antibodies, the recombinant protein of the present invention has the advantages of lower cost and more convenient use, and can thus solve the problems of low patient compliance and excessively high treatment costs in the case of combined use of antibodies.

[0058] Compared with the classical bispecific antibody (composed of two half antibodies), the recombinant protein of the present invention has better tissue permeability and higher efficacy than the classical bispecific antibody, because the molecular weight of the fusion protein that blocks the interaction between CD47 and SIRPα is smaller than that of a single arm (half antibody) of the classical bispecific antibody.

[0059] The low-affinity fusion protein for blocking the interaction between CD47 and SIRPα of the present invention comprises extracellular truncations of SIRPα of different lengths, which can be quickly optimized and paired with a high-affinity tumor-targeting arm (for example, not limited to a half antibody), thereby avoiding the situation in which conventional bifunctional antibodies may be affected by the spatial structure of the two target antigens when simultaneously binding to the two target antigens (especially when the antibody recognition sites of the two target antigens are of a large difference in length from the cell membrane, the problem of difficulty in simultaneously binding the tumor-targeting antigen and the CD47 antigen), so that the obtained recombinant protein has a better binding with tumor cells, thereby exerting a better tumor killing effect.

[0060] The present invention provides a new in vitro safety evaluation method for immunotherapy drugs targeting CD47. The early safety evaluation of conventional anti-CD47 antibodies is generally evaluated by detecting the effect of the drug on red blood cell agglutination. However, due to the species-specific binding of human SIRPα to CD47, human blood must be used when evaluating whether the recombinant protein / antibody targeting CD47 has safety issues of red blood cell agglutination in the human body, and the use of human blood is restricted by ethics, genetic resources, etc. In addition, red blood cell agglutination experiments cannot evaluate the immune safety of drugs at an early stage. The present invention uses an optimized ADCC activity detection method (early in vitro immune safety evaluation experiment) to replace the traditional human red blood cell agglutination experiment, which is used to evaluate the early immune safety of recombinant proteins / antibodies (including monovalent or multivalent) that target CD47 and have ADCC activity. The evaluation method is simple and fast and is not limited by blood sources.

[0061] The present invention provides a new in vivo safety evaluation method for immunotherapy drugs targeting CD47. Preclinical in vivo immune safety evaluation of drugs generally uses non-human primates, and the number of samples required is large, which increases the difficulty and cost of early sample preparation. There are no mature early in vivo immune safety evaluation methods for other species, which leads to huge safety hazards of drugs in future clinical research and wastes research and development investment. The present invention provides Hu-NSG mice, wherein the early safety evaluation experiments conducted on Hu-NSG mice can simulate the safety of drugs in the human immune system. Compared with preclinical or clinical studies, the difficulty and cost of preparation are reduced, and it has the advantages of low detection cost and high detection efficiency, thereby reducing the risk of tumor immunotherapy drug research and development.

[0062] The present invention provides a recombinant protein, achieves high-affinity and specific targeting of tumors through a high-affinity tumor-targeting arm, and achieves blocking of CD47-SIRPα interaction through a fusion protein that blocks the interaction between CD47 and SIRPα with low affinity.

[0063] The recombinant protein of the present invention overcomes the technical problems of low yield and difficult purification of the target product of conventional bifunctional antibodies. In one embodiment of the present invention, the right arm (Fc knob mutation) of the recombinant protein is a single-chain protein. When the left arm light chain, the left arm heavy chain (Fc hole mutation) and the right arm (Fc knob mutation) are co-expressed in the cell, more than 80% of the expression products are the target protein after protein A purification, which effectively improves the expression yield. Although there are a small amount of left arm dimers, right arm dimers or left and right arm monomers, due to the obvious differences in the molecular weight, charge distribution, etc. of these products, they are easily removed by conventional purification means such as ion exchange chromatography, hydrophobic chromatography, molecular exclusion chromatography, affinity chromatography, and salting-out method (such as ammonium sulfate precipitation method), which is suitable for industrial scale-up production.

[0064] The low-affinity fusion protein that blocks the interaction between CD47 and SIRPα of the present invention can be combined with a high-affinity tumor-targeting arm (such as but not limited to a half antibody) to form a recombinant protein that can antagonize the immunosuppressive effect of CD47.

[0065] The present invention overcomes the defect of the existing SIRPα-Fc fusion protein in the prior art that the drug efficacy is not significant due to its low affinity with CD47, and avoids many adverse effects that may be caused by SIRPα high affinity mutants, such as high immunogenicity and high non-tumor targeting specificity. At the same time, because the recombinant protein of the present invention has a weak binding affinity to CD47 expressed on the surface of normal cells (such as red blood cells), it reduces or avoids the side effects of conventional anti-CD47 antibody treatment, such as red blood cell agglutination and anemia.

[0066] Those skilled in the art should understand that the scope of protection of the present invention will not be inappropriately limited because a certain technical solution of the present invention can achieve a certain beneficial effect explicitly or implicitly provided by the present invention but cannot achieve or fully achieve another beneficial effect. Those skilled in the art should understand that within the scope of protection of the present invention, any product, method or use, as long as it obtains any beneficial effect explicitly or implicitly provided by the present invention or optionally a combination of beneficial effects explicitly or implicitly provided by the present invention, means that it solves the technical problem that the present invention is intended to solve and achieves the corresponding technical effect.

[0067] Specifically, the present invention relates to the following embodiments:

[0068] Embodiment 1. A bispecific recombinant protein comprising a tumor-targeting arm and a fusion protein that blocks the interaction between CD47 and SIRPα.

[0069] Embodiment 2. The bispecific recombinant protein of Embodiment 1, wherein the fusion protein that blocks the interaction between CD47 and SIRPα comprises an extracellular truncation of SIRPα (including a wild-type extracellular truncation of human SIRPα or a CD47 non-high-affinity mutation of a human SIRPα extracellular truncation).

[0070] Embodiment 3. The bispecific recombinant protein of embodiment 2, wherein the extracellular truncation of SIRPα comprises part or all of the extracellular amino acid sequence of SIRPα.

[0071] Embodiment 4. A bispecific recombinant protein according to any one of Embodiments 1-3, wherein the high-affinity tumor-targeting arm does not bind to CD47, and the binding affinity of the antibody corresponding to the high-affinity tumor-targeting arm to the target antigen on tumor cells is at least 6 times the binding affinity of the monomeric fusion protein homodimer corresponding to the fusion protein with low affinity that blocks the interaction between CD47 and SIRPα to CD47 on tumor cells.

[0072] Embodiment 5. The bispecific recombinant protein of any one of embodiments 1-4, wherein the SIRPα extracellular truncation comprises an amino acid sequence selected from any one of the following a1)-a4):

[0073] a1) SEQ ID No: 30;

[0074] a2) SEQ ID No:31;

[0075] a3) SEQ ID No: 32;

[0076] a4) An amino acid sequence obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, for example, 1-5 amino acid residues, in any of the above amino acid sequences, wherein the monomer thereof has a binding affinity to the CD47 protein and the binding affinity is no higher than the binding affinity of a1), a2) or a3) monomer to the CD47 protein.

[0077] Embodiment 6. The bispecific recombinant protein of any one of Embodiments 1-5, wherein the bispecific recombinant protein has a left and right arm structure that are relatively arranged, the arm with high affinity for tumor targeting is located at the position of the left arm, and the fusion protein with low affinity for blocking the interaction between CD47 and SIRPα is located at the position of the right arm.

[0078] Embodiment 7. The bispecific recombinant protein of Embodiment 6, wherein the left arm is an Fab or Fab' form of an immunoglobulin, and the right arm is an extracellular truncation of SIRPα.

[0079] Embodiment 8. The bispecific recombinant protein of Embodiment 7, wherein the length of the right arm is adapted to the distance between the antigenic spatial epitope to which the left arm needs to bind and the target cell membrane surface.

[0080] Embodiment 9. In the bispecific recombinant protein of Embodiment 8, when the high-affinity tumor-targeting arm needs to bind to an epitope near the membrane end of the target cell, the SIRPα extracellular truncation is selected to contain a shorter amino acid sequence among a1) to a4).

[0081] Embodiment 10. The bispecific recombinant protein of any one of embodiments 1-9, wherein the tumor-targeting arm targets a target selected from the group consisting of 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GCC, GD2, GPC-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, integrin-4, Notch 2. Notch 1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138.

[0082] Embodiment 11. The bispecific recombinant protein of Embodiment 10, wherein when the target is CD20, EGFR or PD-L1, the SIRPα extracellular truncation selects a1); when the target is HER2, the SIRPα extracellular truncation selects a1 or a2).

[0083] Embodiment 12. The bispecific recombinant protein of any one of Embodiments 1-11, wherein the tumor-targeting arm is bound to the fusion protein that blocks the interaction between CD47 and SIRPα through intermolecular forces, or through covalent bonds such as interchain disulfide bonds, or through salt bonds, or through a combination of two or three of the above-mentioned binding modes.

[0084] Embodiment 13. The bispecific recombinant protein of any one of Embodiments 1-12, wherein the tumor-targeting arm and / or the fusion protein that blocks the interaction between CD47 and SIRPα further comprises an Fc region.

[0085] Embodiment 14. The bispecific recombinant protein of Embodiment 13, wherein the Fc region comprises a native Fc region sequence or a non-native Fc sequence.

[0086] Embodiment 15. The bispecific recombinant protein of Embodiment 14, wherein the Fc region is a human Fc region.

[0087] Embodiment 16. The bispecific recombinant protein of Embodiment 15, wherein the high-affinity tumor-targeting arm and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα are combined through knobs-into-holes.

[0088] Embodiment 17. The bispecific recombinant protein of any one of embodiments 1-16, wherein the tumor-targeting arm is a half antibody targeting a target selected from the group consisting of 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, C EA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GCC, GD 2. GPC-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, integrin-4, Notch 2, Notch 1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138; preferably a half antibody of IgG1 antibody, optionally a human-mouse chimeric half antibody, a humanized half antibody, or a fully human half antibody; more preferably a half antibody of a humanized or fully human IgG1 antibody.

[0089] Embodiment 18. The bispecific recombinant protein of any one of Embodiments 1-17, wherein the fusion protein that blocks the interaction between CD47 and SIRPα is a fusion protein comprising an extracellular truncation of SIRPα and a binding sequence for binding to the arm, the extracellular truncation of SIRPα and the binding sequence for binding to the arm are optionally connected by a linker sequence, and the binding sequence for binding to the arm is optionally an Fc region.

[0090] Embodiment 19. The bispecific recombinant protein of any one of embodiments 1-18, wherein the fusion protein that blocks the interaction between CD47 and SIRPα comprises an amino acid sequence selected from any one of the following b1)-b4):

[0091] b1) SEQ ID No: 26;

[0092] b2) SEQ ID No: 27;

[0093] b3) SEQ ID No: 28;

[0094] b4) An amino acid sequence obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, for example, 1-5 amino acid residues, in any of the above amino acid sequences, whose homodimer has binding affinity to the CD47 protein and the binding affinity is no higher than the binding affinity of the homodimer of b1), b2) or b3) to the CD47 protein.

[0095] Embodiment 20. The bispecific recombinant protein of Embodiment 19, wherein when the high-affinity tumor-targeting arm targets CD20, the high-affinity tumor-targeting arm comprises SEQ ID No: 16 and SEQ ID No: 17, and the low-affinity blocking fusion protein for interaction between CD47 and SIRPα comprises SEQ ID No: 26; wherein when the high-affinity tumor-targeting arm targets EGFR, the high-affinity tumor-targeting arm comprises SEQ ID No: 19 and SEQ ID No: 8, and the low-affinity blocking fusion protein for interaction between CD47 and SIRPα comprises SEQ ID No: 26; wherein when the high-affinity tumor-targeting arm targets Her2, the high-affinity tumor-targeting arm comprises SEQ ID No: 20 and SEQ ID No: 21, or SEQ ID No: 22 and SEQ ID No: 23, and the low-affinity blocking fusion protein for interaction between CD47 and SIRPα comprises SEQ ID No: 26 or SEQ ID No: 27; or, wherein when the high-affinity tumor-targeting arm targets PD-L1, the high-affinity tumor-targeting arm comprises SEQ ID No: 19 and SEQ ID No: 8, and the low-affinity blocking fusion protein for interaction between CD47 and SIRPα comprises SEQ ID No: 26. No:24 and SEQ ID No:13, and the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity comprises SEQ ID No:26.

[0096] Embodiment 21. A nucleic acid molecule encoding the bispecific recombinant protein of any one of embodiments 1-20.

[0097] Embodiment 22. The nucleic acid molecule of embodiment 21, wherein the nucleic acid molecule encoding the high-affinity tumor-targeting arm and the nucleic acid encoding the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity are in the same DNA chain, or in different DNA chains.

[0098] Embodiment 23. An expression vector comprising the nucleic acid molecule of embodiment 21 or 22.

[0099] Embodiment 24. A cell comprising the expression vector of embodiment 23.

[0100] Embodiment 25. A method for preparing the bispecific recombinant protein of any one of embodiments 1-20, comprising:

[0101] 1) Provide a tumor-targeting arm;

[0102] 2) providing a fusion protein that blocks the interaction between CD47 and SIRPα;

[0103] 3) contacting the tumor-targeting arm with a fusion protein that blocks the interaction between CD47 and SIRPα to form the recombinant protein.

[0104] Embodiment 26. The preparation method of embodiment 25, wherein the cell of embodiment 24 is allowed to express the recombinant protein.

[0105] Embodiment 27. The preparation method of embodiment 25, wherein the contact includes binding through intermolecular forces, or binding through covalent bonds such as interchain disulfide bonds, or binding through salt bonds, or binding through a combination of two or three of the above binding modes.

[0106] Embodiment 28. The method of any one of Embodiments 25-27, wherein the contacting comprises bonding via a knobs-into-holes technique.

[0107] Embodiment 29. A fusion protein, characterized in that the fusion protein comprises an extracellular truncation of SIRPα and a binding sequence for binding to another polypeptide.

[0108] Embodiment 30. The fusion protein of embodiment 29, wherein the binding sequence for binding to another polypeptide is an Fc region; optionally, wherein the Fc region comprises holes mutations and / or knobs mutations.

[0109] Embodiment 31. The fusion protein of embodiment 29 or 30, wherein the extracellular truncation of SIRPα comprises part or all of the extracellular amino acid sequence of wild-type human SIRPα and its non-high-affinity mutant.

[0110] Embodiment 32. The fusion protein of embodiment 31, wherein the SIRPα extracellular truncation comprises an amino acid sequence selected from any one of the following a1)-a4):

[0111] a1) SEQ ID No: 30;

[0112] a2) SEQ ID No:31;

[0113] a3) SEQ ID No: 32;

[0114] a4) An amino acid sequence obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, for example, 1-5 amino acid residues, in any of the above amino acid sequences, wherein the monomer thereof has a binding affinity to the CD47 protein and the binding affinity is no higher than the binding affinity of a1), a2) or a3) monomer to the CD47 protein.

[0115] Embodiment 33. The fusion protein of any one of embodiments 29-32, comprising an amino acid sequence selected from any one of the following b1)-b4):

[0116] b1) SEQ ID No: 26;

[0117] b2) SEQ ID No: 27;

[0118] b3) SEQ ID No: 28;

[0119] b4) An amino acid sequence obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, for example, 1-5 amino acid residues, in any of the above amino acid sequences, whose homodimer has binding affinity to the CD47 protein and the binding affinity is no higher than the binding affinity of the homodimer of b1), b2) or b3) to the CD47 protein.

[0120] Embodiment 34. A nucleic acid molecule encoding the fusion protein of any one of embodiments 29-33.

[0121] Embodiment 35. An expression vector comprising the nucleic acid molecule of embodiment 34.

[0122] Embodiment 36. A cell comprising the expression vector of embodiment 35.

[0123] Embodiment 37. A method for preparing the fusion protein of any one of embodiments 29-33, comprising:

[0124] 1) Provide extracellular truncations of SIRPα;

[0125] 2) providing a binding sequence for binding to another polypeptide;

[0126] 3) contacting the SIRPα extracellular truncation with a binding sequence for binding to another polypeptide to form the fusion protein.

[0127] Embodiment 38. A method for preparing the fusion protein of any one of embodiments 29-33, wherein the cell of embodiment 32 is caused to express the fusion protein.

[0128] Embodiment 39. A pharmaceutical composition comprising the recombinant protein of any one of embodiments 1-20 or the fusion protein of any one of embodiments 29-33, and optionally an adjuvant, an excipient or a pharmaceutically acceptable carrier.

[0129] Embodiment 40. The pharmaceutical composition of Embodiment 39, which is in the form of an injection or a lyophilized powder.

[0130] Embodiment 41. A pharmaceutical composition comprising a nucleic acid molecule encoding the recombinant protein of any one of embodiments 1-20 or the fusion protein of any one of embodiments 29-33, and optionally a pharmaceutically acceptable carrier.

[0131] Embodiment 42. Use of the fusion protein of any one of embodiments 29-33 for preparing the recombinant protein of any one of embodiments 1-20.

[0132] Embodiment 43. Use of the recombinant protein of any one of Embodiments 1-20 or the fusion protein of any one of Embodiments 29-33 in the preparation of a medicament for treating tumors.

[0133] Embodiment 44. The use of embodiment 43, wherein the tumor is selected from blood tumors and solid tumors such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, cutaneous fibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome.

[0134] Embodiment 45. Use of a nucleic acid molecule encoding a recombinant protein according to any one of embodiments 1 to 20 or a fusion protein according to any one of embodiments 29 to 33 or a derivative thereof in the preparation of a medicament for in vivo gene therapy.

[0135] Embodiment 46. A method for evaluating the early immune safety of a recombinant protein / antibody targeting CD47 and having ADCC activity in vitro, the method comprising:

[0136] a) Providing targeted recombinant proteins / antibodies (including monovalent or multivalent) with ADCC activity;

[0137] b) detecting the ADCC activity of the recombinant protein / antibody;

[0138] c) Evaluate the early immune safety of the recombinant protein / antibody.

[0139] Embodiment 47. A method for evaluating the early immune safety of a recombinant protein / antibody targeting CD47 and having ADCC activity in vitro, the method comprising:

[0140] a) preparing effector cells, such as but not limited to human NK92MI-CD16a effector cells;

[0141] b) contacting the effector cells with the recombinant protein / antibody (including monovalent or multivalent);

[0142] c) detecting the ADCC activity of the recombinant protein / antibody;

[0143] d) Evaluate the early immune safety of the recombinant protein / antibody based on the ADCC activity results.

[0144] Embodiment 48. The evaluation method of embodiment 47, comprising:

[0145] a) Harvest well-grown human NK92MI-CD16a effector cells and resuspend the harvested effector cells to a cell density of 1×10 5 cells / ml to 5×10 6 cells / ml;

[0146] b) incubating the gradiently diluted recombinant protein / antibody with the effector cells prepared in a) for 0.5-5 h;

[0147] c) After the incubation is completed, the LDH activity is measured and the cell lysis rate is calculated;

[0148] d) Evaluate the early immune safety of recombinant proteins / antibodies based on the cell lysis rate, where the recombinant proteins / antibodies that result in lower cell lysis rates have higher immune safety.

[0149] Embodiment 49. A method for in vivo evaluation of the early immune safety of a recombinant protein / antibody targeting CD47 (including monovalent or multivalent), the method comprising:

[0150] a) Providing recombinant proteins / antibodies targeting CD47 (including monovalent or multivalent);

[0151] b) providing Hu-NSG mice;

[0152] c) contacting the recombinant protein / antibody with Hu-NSG mice;

[0153] d) Evaluate the early immune safety of the recombinant protein / antibody in the Hu-NSG mice.

[0154] Embodiment 50. A method for in vivo evaluation of the early immune safety of a recombinant protein / antibody (including monovalent or multivalent) targeting CD47, the method comprising:

[0155] a) providing Hu-NSG mice;

[0156] b) administering recombinant proteins / antibodies (including monovalent or multivalent);

[0157] c) 24-96 hours after administration, venous blood was collected from the mice, red blood cells were lysed, and the remaining cells were incubated with fluorescently labeled anti-human CD45, anti-human CD19 or anti-human CD3 antibodies for 15-60 minutes, and flow cytometry was performed;

[0158] d) Evaluate the early immune safety of recombinant proteins / antibodies based on the cell clearance rate, where recombinant proteins / antibodies that result in lower immune cell clearance rates (excluding target cells) have higher immune safety.

[0159] The present invention also relates to but is not limited to the following items:

[0160] Item 1. A bispecific recombinant protein, wherein the bispecific recombinant protein comprises a high-affinity tumor-targeting arm and a fusion protein with low affinity that blocks the interaction between CD47 and SIRPα,

[0161] The high-affinity tumor-targeting arm does not bind to CD47, and the binding affinity of the antibody corresponding to the high-affinity tumor-targeting arm to the target antigen on tumor cells is at least 6 times the binding affinity of the monomeric fusion protein homodimer corresponding to the fusion protein with low affinity blocking the interaction between CD47 and SIRPα to CD47 on tumor cells;

[0162] The binding affinity of the fusion protein for CD47 that blocks the interaction between CD47 and SIRPα with low affinity is no higher than the binding affinity of the homodimer of the monomeric fusion protein containing the SIRPα extracellular truncation to CD47; the SIRPα extracellular truncation comprises a wild-type extracellular truncation of human SIRPα or a human SIRPα extracellular truncation with a non-high affinity mutation of CD47.

[0163] 2. The bispecific recombinant protein as described in Item 1, wherein the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity comprises an extracellular truncation of SIRPα, and the extracellular truncation of SIRPα comprises an amino acid sequence selected from any one of the following a1)-a4): a1) SEQ ID No: 30; a2) SEQ ID No: 31; a3) SEQ ID No: 32; a4) an amino acid sequence obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, for example, 1-5 amino acid residues, of any one of the above amino acid sequences, and its monomer has a binding affinity for CD47 protein and the binding affinity is not higher than the binding affinity of a1), a2) or a3) monomer for CD47 protein.

[0164] 3. The bispecific recombinant protein as described in item 1 or 2, wherein the bispecific recombinant protein has a left and right arm structure that are relatively arranged, wherein the high-affinity tumor-targeting arm is located at the position of the left arm, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα is located at the position of the right arm; preferably, the left arm is in the Fab or Fab' form of an immunoglobulin, and the right arm is an extracellular truncation of SIRPα.

[0165] 4. In the bispecific recombinant protein as described in Item 3, the length of the right arm is adapted to the distance between the spatial epitope of the antigen to which the left arm needs to bind and the surface of the target cell membrane; preferably, when the high-affinity tumor-targeting arm needs to bind to an epitope near the membrane end of the target cell, the SIRPα extracellular truncation selects a shorter amino acid sequence among a1) to a4).

[0166] 5. The bispecific recombinant protein according to any one of items 1 to 4, wherein the high affinity tumor-targeting arm targets a target selected from the following: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GCC, GD2, GPC-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, integrin-4, Notch 2. Notch 1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138;

[0167] Preferably, when the target is CD20, EGFR or PD-L1, the SIRPα extracellular truncation selects a1); when the target is HER2, the SIRPα extracellular truncation selects a1) or a2).

[0168] 6. A bispecific recombinant protein as described in any one of items 1 to 5, wherein the high-affinity tumor-targeting arm and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα are bound by intermolecular forces, or by covalent bonds such as interchain disulfide bonds, or by salt bonds, or by a combination of two or three of the above-mentioned binding modes.

[0169] 7. The bispecific recombinant protein as described in item 6, further comprising an Fc region; preferably, the Fc region comprises an Fc region native sequence or an Fc non-native sequence; more preferably, the Fc region is a human Fc region; further more preferably, the combination of the high-affinity tumor-targeting arm and the low-affinity blocking CD47 and SIRPα interaction fusion protein is through knobs-into-holes.

[0170] 8. The bispecific recombinant protein as described in item 7, wherein the high affinity tumor-targeting arm is a half antibody targeting a target selected from the following targets: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, C EACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GCC, GD2, GPC-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, integrin-4, Notch 2, Notch 1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138; preferably a half antibody of IgG1 antibody, optionally a human-mouse chimeric half antibody, a humanized half antibody, or a fully human half antibody; more preferably a half antibody of a humanized or fully human IgG1 antibody.

[0171] 9. The bispecific recombinant protein described in item 7 or 8, wherein the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity comprises an amino acid sequence selected from any one of the following b1)-b4): b1) SEQ ID No: 26; b2) SEQ ID No: 27; b3) SEQ ID No: 28; b4) an amino acid sequence obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, for example, 1-5 amino acid residues, to any one of the above amino acid sequences, whose homodimer has a binding affinity for the CD47 protein and the binding affinity is not higher than the binding affinity of the homodimer of b1), b2) or b3) to the CD47 protein.

[0172] 10. The bispecific recombinant protein as described in item 9,

[0173] When the high-affinity tumor-targeting arm targets CD20, the high-affinity tumor-targeting arm comprises SEQ ID No: 16 and SEQ ID No: 17, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα comprises SEQ ID No: 26;

[0174] When the high-affinity tumor-targeting arm targets EGFR, the high-affinity tumor-targeting arm comprises SEQ ID No: 19 and SEQ ID No: 8, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα comprises SEQ ID No: 26;

[0175] When the high-affinity tumor-targeting arm targets Her2, the high-affinity tumor-targeting arm comprises SEQ ID No: 20 and SEQ ID No: 21, or SEQ ID No: 22 and SEQ ID No: 23, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα comprises SEQ ID No: 26 or SEQ ID No: 27; or,

[0176] When the high-affinity tumor-targeting arm targets PD-L1, the high-affinity tumor-targeting arm comprises SEQ ID No: 24 and SEQ ID No: 13, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα comprises SEQ ID No: 26.

[0177] 11. A nucleic acid molecule encoding a bispecific recombinant protein of any one of items 1 to 10; preferably, the nucleic acid molecule encoding the high-affinity tumor-targeting arm and the nucleic acid encoding the fusion protein with low affinity for blocking the interaction between CD47 and SIRPα are in the same DNA chain, or the nucleic acid molecule encoding the high-affinity tumor-targeting arm and the nucleic acid encoding the fusion protein with low affinity for blocking the interaction between CD47 and SIRPα are in different DNA chains.

[0178] 12. An expression vector comprising the nucleic acid molecule of item 11.

[0179] 13. A host cell comprising the expression vector of item 12.

[0180] 14. A method for preparing a bispecific recombinant protein, comprising using the cell according to item 13 to express the recombinant protein.

[0181] 15. Use of the bispecific recombinant protein described in any one of items 1 to 10 in the preparation of a drug for treating tumors; preferably, the tumor is selected from blood tumors and solid tumors such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, cutaneous fibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0182] Figure 1 It is a schematic diagram of the structure of the insert to be inserted into the expression vector of the present invention.

[0183] Figure 2 Figure 1 is a plasmid map of an exemplary expression vector pCHO-TE2 used in the present invention.

[0184] Figure 3 Schematic diagram of the structure of an exemplary embodiment of the recombinant protein of the present invention.

[0185] Figure 4 A-4B is the SDS-PAGE electrophoresis diagram of the recombinant protein after purification by protein A. Figure 4 Lanes 1-6 of A are reducing samples, including: 1: Marker, 2: Anti-CD20 mAb (Ofatumumab), 3: Anti-CD47 mAb (Hu5F9-G4), 4: SIRPαD1-Fc, 5: Ofa-Fc1-D1-Fc2, 6: Ofa-Fc1-D1-D2-D3-Fc2; Figure 4 Lanes 7-12 of A are non-reduced samples, namely: 7: Marker, 8: Anti-CD20 mAb (Ofatumumab), 9: Anti-CD47 mAb (Hu5F9-G4), 10: SIRPαD1-Fc, 11: Ofa-Fc1-D1-Fc2, 12: Ofa-Fc1-D1-D2-D3-Fc2. Figure 4 Lane 1 of B is the Ofa-Fc1-D1-D2-Fc2 non-reduced sample, Lane 2 is the Marker, and Lane 3 is the Ofa-Fc1-D1-D2-Fc2 reduced sample.

[0186] Figure 5 The results (protein level) are obtained by ELISA to determine the binding affinity of the recombinant protein of the present invention to human CD47.

[0187] Figure 6 The results of flow cytometry determination of the binding affinity of the recombinant protein of the present invention to human CD47 (cell level).

[0188] Figure 7 The results of the binding force between the recombinant protein of the present invention and the corresponding left arm target were determined by flow cytometry. Hu5F9-G4, Ofa-Fc1-D1-Fc2, and SIRPαD1-Fc correspond to the average fluorescence intensity of samples Hu5F9-G4, Ofa-Fc1-D1-Fc2, and SIRPαD1-Fc after binding to Raji cells that have not been blocked by anti-CD47 antibody Hu5F9-G4 (Fab) 2, while Hu5F9-G4 (b), Ofa-Fc1-D1-Fc2 (b), and SIRPαD1-Fc (b) correspond to the average fluorescence intensity of samples Hu5F9-G4, Ofa-Fc1-D1-Fc2, and SIRPαD1-Fc after binding to Raji cells that have been blocked by anti-CD47 antibody Hu5F9-G4 (Fab) 2.

[0189] Figure 8 The results of flow cytometry determination of the binding affinity of the recombinant protein of the present invention to CD47 and left arm target double positive cells. Figure 8 A uses Raji cells (CD20+CD47), Figure 8 B uses SKBR-3 cells (Her2+CD47), Figure 8 C uses A431 cells (EGFR+CD47), Figure 8 D used NCI-H441 cells (PD-L1+CD47).

[0190] Fig. 9The competitive binding results of the recombinant protein of the present invention with SIRPαD1-Fc and anti-CD47 antibody to CD47 were determined by ELISA.

[0191] Fig.10 The results are flow cytometry results for determining the binding affinity of the recombinant protein of the present invention and its high-affinity CD47 mutant, anti-CD47 antibody and anti-CD20 antibody to human CD47.

[0192] Fig.11 These are the results of in vitro immune safety evaluation experiments of the recombinant protein of the present invention.

[0193] Fig.12 The results are the results of the pharmacodynamic experiments of the recombinant protein of the present invention in the subcutaneous transplanted NSG mouse model of Raji lymphoma.

[0194] Fig.13 The results of B cell content detection after 96 hours of treatment of Hu-NSG mice with different samples at the same dose. Fig.13 A uses 0.9% saline. Fig.13 B used Hu5F9-G4 (6.7 μg / mouse). Fig.13 In C, Ofa-Fc1-D1-Fc2 (5 μg / mouse) was used.

[0195] Fig.14 Ofa-Fc1-D1-Fc2 and Ofa-Fc1-D1 m -Results of early safety evaluation experiment of Fc2 in Hu-NSG mice (FACS immune cell typing test). Fig.14 Ofa-Fc1-D1-Fc2 (1 μg / mouse) was used in both AD and Fig.14 A and Fig.14 B is the test result before medication. Fig.14 C and Fig.14 D is the test result graph 72 hours after medication. Fig.14 EH used OFA-Fc1-D1 m -Fc2 (1 μg / mouse), Fig.14 E and Fig.14 F is the test result before medication. Fig.14 G and Fig.14 H is the test result graph after 72 hours of medication.

[0196] Fig.15 FACS immune cell typing detection 96 hours after Hu-NSG mice were treated with different samples at high doses. Fig.15 AB are the results 96 hours after drug administration, in which Hu5F9-G4 (200 μg / mouse) was used in Figure A. Fig.15B: A high dose of Ofa-Fc1-D1-Fc2 (150 μg / mouse). Fig.15 CD is the result 14 days after medication, Fig.15 C used Hu5F9-G4 (200 μg / mouse), Fig.15 D: A high dose of Ofa-Fc1-D1-Fc2 (150 μg / mouse).

[0197] Fig.16 The figure shows the binding of the recombinant protein of the present invention to dual targets (Her2 and CD47) in SKBR-3. Fig.16 A is the test results of Anti-Her2(T)-Fc1-D1-Fc2 and Anti-Her2(T)-Fc1-D1-D2-Fc2 samples. Fig.16 B is the test results of Anti-Her2(P)-Fc1-D1-Fc2 and Anti-Her2(P)-Fc1-D1-D2-Fc2 samples.

[0198] Fig.17 AE are the corresponding amino acid sequences and DNA sequences of the exemplary proteins of the present invention.

[0199] Fig.18 A shows the effects of two doses of Ofa-Fc1-D1-Fc2 on the number of red blood cells in cynomolgus monkeys. Fig.18 B shows the effects of two doses of Ofa-Fc1-D1-Fc2 on hemoglobin in cynomolgus monkeys.

[0200] Fig.19 The results of the test on B cell content in cynomolgus monkeys with two doses of Ofa-Fc1-D1-Fc2 are shown.

[0201] Fig. 20 Ofa-Fc1-D1-Fc2, Effects on the growth of subcutaneous xenografts of human B-cell lymphoma Daudi.

[0202] Fig.21 Ofa-Fc1-D1-Fc2, Effects on body weight of tumor-bearing mice.

[0203] Fig. 22 Ofa-Fc1-D1-Fc2, Tumor photographs showing efficacy against subcutaneous xenografts of human B-cell lymphoma Daudi. DETAILED DESCRIPTION

[0204] In order to promote understanding of the present invention, some embodiments will be referred to below, and specific language will be used to describe the present invention. However, it should be understood that these specific embodiments are not intended to limit the scope of the present invention. Any changes and further modifications in the described embodiments, as well as any further applications of the present invention, are generally expected by those skilled in the art.

[0205] Recombinant protein

[0206] As used herein, the term "recombinant protein" refers to an artificially designed / constructed protein, rather than a naturally occurring protein. The "recombinant" in the "recombinant protein" of the present invention does not represent its production method, it is only used to indicate that the "recombinant protein" does not exist naturally. The recombinant protein of the present invention can be an expressed protein or an assembled protein.

[0207] Optionally, the recombinant protein of the present invention comprises a high-affinity tumor-targeting arm and a fusion protein of a low-affinity blocking CD47-SIRPα interaction.

[0208] As used herein, "high affinity tumor targeting" means that the binding affinity of the recombinant protein of the present invention to the tumor is higher than or roughly equivalent to the binding affinity of the antibody drugs binding to the tumor in the prior art, and the binding affinity of the antibody drugs binding to the tumor in the prior art is usually at the nM or pM level. Preferably, in the recombinant protein of the present invention, the binding affinity of the antibody corresponding to the arm of the high affinity tumor targeting to the target antigen on the tumor cell is at least 6 times the binding affinity of the monomeric fusion protein homodimer corresponding to the fusion protein with low affinity blocking the interaction between CD47 and SIRPα to CD47 on the tumor cell, optionally 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, or higher multiple values, or any value therebetween. Optionally, in the recombinant protein of the present invention, the binding affinity of the antibody corresponding to the high-affinity tumor-targeting arm to the target antigen on tumor cells is at least 6 times the binding affinity of the SIRPα-Fc homodimer corresponding to the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity to CD47 on tumor cells, optionally 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, or a higher multiple value, or any value therebetween.

[0209] As used herein, "low affinity blocking of CD47 and SIRPα interaction" means that the recombinant protein of the present invention can block the interaction between CD47 and SIRPα with a relatively low affinity. Preferably, the binding affinity of the fusion protein of the recombinant protein of the present invention that blocks the interaction between CD47 and SIRPα with a low affinity is not higher than the binding affinity of the homodimer of the monomeric fusion protein containing the extracellular truncation of SIRPα to CD47. More preferably, it is not higher than the binding affinity of the human SIRPα-Fc fusion protein to CD47.

[0210] The bispecific recombinant protein described in this article can significantly improve the tumor-targeted saturation binding abundance of the recombinant protein that regulates macrophage function and reduce the defects of non-tumor targeting side effects when the antibody corresponding to its high-affinity tumor-targeting arm has a binding affinity for the target antigen on tumor cells that is at least 6 times the binding affinity of the monomeric fusion protein homodimer corresponding to its fusion protein arm that blocks the interaction between CD47 and SIRPα to CD47 on tumor cells.

[0211] The binding affinity determination methods used herein are well known to those skilled in the art, including, for example but not limited to, ELISA and / or flow cytometry.

[0212] Optionally, the signal regulatory protein alpha extracellular truncation (including wild-type extracellular truncation and non-high affinity mutant extracellular truncation)-Fc fusion protein and half antibody of the present invention can form heterodimers by modifying the antibody heavy chain. Specifically, for example, the recombinant protein can be obtained by Knobs-into-holes connection and / or interchain disulfide bonds and / or salt bonds.

[0213] Optionally, when the "tumor targeting arm" or "half antibody" or "left arm" or "half antibody structure" or "Ig molecule monomer" is connected to the "CD47 targeting arm" or "right arm" or "signal regulatory protein α truncated-Fc fusion protein" or "SIRPα-Fc" through knobs-into-holes to obtain a recombinant protein, the structure of the resulting recombinant protein is as follows Figure 3As shown. Wherein, the left arm is a tumor-targeted half antibody, including but not limited to anti-5T4 half antibody, anti-AGS-16 half antibody, anti-ALK1 half antibody, anti-ANG-2 half antibody, anti-B7-H3 half antibody, anti-B7-H4 half antibody, anti-c-fms half antibody, anti-c-Met half antibody, anti-CA6 half antibody, anti-CD123 half antibody, anti-CD19 half antibody, anti-CD20 half antibody, anti-CD22 half antibody, anti-EpCAM half antibody, anti-CD30 half antibody, anti-CD32b half antibody, anti-CD37 half antibody, anti-CD38 half antibody, anti-CD40 half antibody, anti-CD52 half antibody, anti-CD70 half antibody, anti-CD74 half antibody, anti-CD79b half antibody, anti-CD98 half antibody, anti-CEA half antibody, anti-CEACAM5 half antibody, anti-CLDN18.2 half antibody, anti-CL DN6 half antibody, anti-CS1 half antibody, anti-CXCR4 half antibody, anti-DLL-4 half antibody, anti-EGFR half antibody, anti-EGP-1 half antibody, anti-ENPP3 half antibody, anti-EphA3 half antibody, anti-ETBR half antibody, anti-FGFR2 half antibody, anti-FN half antibody, anti-FR-α half antibody, anti-GCC half antibody, anti-GD2 half antibody, anti-GPC-3 half antibody, anti-GPNMB half antibody, anti-HER2 half antibody, anti-HER3 half antibody, anti-HLA-DR half antibody, anti-ICAM-1 half antibody, anti-IGF-1R half antibody, anti-IL-3R half antibody, anti-LIV-1 half antibody, anti-MSLN half antibody, anti-MUC16 half antibody, anti-MUC1 half antibody, anti-NaPi2b half antibody, anti-Integrin-4 half antibody, anti-Notch2 half antibody, anti-Notch 1 half antibody, anti-PD-L1 half antibody, anti-PD-L2 half antibody, anti-PDGFR-α half antibody, anti-PS half antibody, anti-PSMA half antibody, anti-SLTRK6 half antibody, anti-STEAP1 half antibody, anti-TEM1 half antibody, anti-VEGFR half antibody, anti-CD25 half antibody, anti-CD27L half antibody, anti-DKK-1 half antibody, anti-CSF-1R half antibody, anti-MSB0010718C half antibody, anti-BCMA half antibody, anti-CD138 half antibody. The right arm is a fusion protein formed by connecting the extracellular truncation of SIRPα (including human SIRPα wild type and its non-high affinity mutant) with the hinge region and Fc region of IgG antibody. When the Fc region of the right arm is a hole mutant, the corresponding Fc region of the left arm is a knob mutant; when the right arm is a knob mutant, the corresponding Fc region of the left arm is a hole mutant. As known to those skilled in the art, the Fc region can also make multiple holes and / or knobs mutations at the same time.

[0214] Table 1 below shows exemplary molecular structures of recombinant proteins.

[0215] Table 1 Examples of molecular structures of recombinant proteins

[0216] Serial number Left Arm Right Arm 1 Anti-CD20-Fc1 D1-Fc2 2 Anti-CD20-Fc1 D1-D2-Fc2 3 Anti-CD20-Fc1 D1-D2-D3-Fc2 4 Anti-CD20-Fc1 <![CDATA[D1 m -Fc2]]> 5 Anti-CD20-Fc1 <h2 style=";text-align:left;direction:ltr"><![CDATA[D1 <h2 style=";text-align:left;direction:ltr"> m <h2 style=";text-align:left;direction:ltr"> -D2-Fc2]]><h2 style=";text-align:left;direction:ltr"> 6 Anti-PD-L1-Fc1 D1-Fc2 7 Anti-PD-L1-Fc1 D1-D2-Fc2 8 Anti-PD-L1-Fc1 D1-D2-D3-Fc2 9 Anti-PD-L1-Fc1 <![CDATA[D1 m -Fc2]]> 10 Anti-PD-L1-Fc1 <h2 style=";text-align:left;direction:ltr"><![CDATA[D1 <h2 style=";text-align:left;direction:ltr"> m <h2 style=";text-align:left;direction:ltr"> -D2-Fc2]]><h2 style=";text-align:left;direction:ltr"> 11 Anti-EGFR-Fc1 D1-Fc2 12 Anti-EGFR-Fc1 D1-D2-Fc2 13 Anti-EGFR-Fc1 D1-D2-D3-Fc2 14 Anti-EGFR-Fc1 <![CDATA[D1 m -Fc2]]> 15 Anti-EGFR-Fc1 <h2 style=";text-align:left;direction:ltr"><![CDATA[D1 <h2 style=";text-align:left;direction:ltr"> m <h2 style=";text-align:left;direction:ltr"> -D2-Fc2]]><h2 style=";text-align:left;direction:ltr"> 16 Anti-Her2-Fc1 D1-Fc2 17 Anti-Her2-Fc1 D1-D2-Fc2 18 Anti-Her2-Fc1 D1-D2-D3-Fc2 19 Anti-Her2-Fc1 <![CDATA[D1 m -Fc2]]> 20 Anti-Her2-Fc1 <h2 style=";text-align:left;direction:ltr"><![CDATA[D1 <h2 style=";text-align:left;direction:ltr"> m <h2 style=";text-align:left;direction:ltr"> -D2-Fc2]]><h2 style=";text-align:left;direction:ltr">

[0217] D1 m Represents the high-affinity mutant of the extracellular truncated form of SIRPα D1; D1 represents the extracellular D1 domain of the wild-type human SIRPα and its non-high-affinity mutant; Fc represents the wild-type Fc region, Fc1 represents the Fc region with hole or holes mutation, and Fc2 represents the Fc region with knob or knobs mutation

[0218] The corresponding amino acid sequence and DNA sequence of the recombinant protein of the present invention are shown in Fig.17 AE and see the sequence listing document of the present invention.

[0219] Antibody

[0220] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0221] Tumor-targeted arm

[0222] As used herein, the terms "tumor-targeting arm" or "half antibody" or "left arm" or "half antibody structure" or "Ig molecule monomer" refer to a heterodimeric glycoprotein composed of a light chain (L) and a heavy chain (H) of an antibody, which is the basic structure of an immunoglobulin molecule and can be used interchangeably in the present invention; its molecular weight is half of the molecular weight of the corresponding antibody, about 75,000 Daltons, wherein the light chain is connected to the heavy chain by a covalent disulfide bond. The heavy and light chains also have regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0223] As used herein, the term "tumor-targeted arm", "half antibody" or "left arm" or "half antibody structure" or "Ig molecule monomer" can be various tumor-targeted IgG proteins. Tumor target molecules include but are not limited to 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR 2. FN, FR-α, GCC, GD2, GPC-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, integrin-4, Notch2 , Notch1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138.

[0224] The Fc sequence of the "tumor targeting arm", "half antibody" or "left arm" or "half antibody structure" or "Ig molecule monomer" may adopt a hole or holes mutant and / or a knob or knobs mutant.

[0225] CD47-targeting arm

[0226] As used herein, the terms "arm targeting CD47" or "right arm" or "signal regulatory protein α truncation-Fc fusion protein" or "SIRPα-Fc" or "fusion protein that blocks the interaction between CD47 and SIRPα" can be used interchangeably in the present invention. As known to those skilled in the art, the "arm targeting CD47" or "right arm" or "signal regulatory protein α truncation-Fc fusion protein" or "SIRPα-Fc" or "fusion protein that blocks the interaction between CD47 and SIRPα" molecule length is variable. Optionally, the extracellular truncation of SIRPα (including human SIRPα wild type and its non-high affinity mutants) can be connected to the hinge region and Fc region of the IgG1 antibody to form a plurality of "arm targeting CD47" or "right arm" or "signal regulatory protein α truncation-Fc fusion protein" or "SIRPα-Fc" or "fusion protein that blocks the interaction between CD47 and SIRPα" of different molecular lengths. IgG1 can be human IgG1.

[0227] The Fc of the “CD47 targeting arm” or “right arm” or “signal regulatory protein α truncated-Fc fusion protein” or “SIRPα-Fc” or “fusion protein that blocks the interaction between CD47 and SIRPα” can adopt a knob or knobs mutant and / or a hole or holes mutant.

[0228] The “tumor-targeting arm” or “half-antibody” or “left arm” or “half-antibody structure” or “Ig molecule monomer” and the “CD47-targeting arm” or “right arm” or “signal regulatory protein α truncation-Fc fusion protein” or “SIRPα-Fc” or “fusion protein that blocks the interaction between CD47 and SIRPα” can be transformed into a heterodimeric recombinant protein as known to those skilled in the art by modifying the Fc fragment (region). Specifically, the recombinant protein of the present invention can be obtained by intermolecular forces, by covalent bonds such as interchain disulfide bonds, by salt bond mediation, or by any combination of two or three of the above three technologies. Optionally, the recombinant protein of the present invention is combined by knobs-into-holes technology.

[0229] knobs-into-holes technology

[0230] As used herein, the term "knobs-into-holes technology" or "knobs-into-holes" technology or "knobs-into-holes" technology or "buttons" technology refers to the use of genetic engineering technology to introduce different mutations into the two CH3 domains of the heavy chain to promote heterodimerization of the heavy chain, a knob is made on one heavy chain, a hole is made on the other heavy chain, and then the two preferentially bite together to form an asymmetric antibody (Ridgway JB, et al. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, 1996, 9(7): 617-621). As known to those skilled in the art, multiple knobs and / or holes can be made on one heavy chain, and correspondingly, multiple holes and / or knobs can be made on the other heavy chain.

[0231] SIRPα

[0232] As used herein, the term "SIRPα" is signal regulatory protein α, also known as CD172a. Signal regulatory protein (SIRP) is a transmembrane glycoprotein, including three family members, SIRPα (CD172a), SIRPβ (CD172b), and SIRPγ (CD172g). These three members have similar extramembrane ends, but different intramembrane regions. The extramembrane ends all contain three immunoglobulin (Ig)-like regions, of which the first region belongs to the IgV region, and the second and third regions belong to the IgC region. The intramembrane region of SIRPα (CD172a) contains two inhibitory signal domains, which can transmit inhibitory signals and inhibit the corresponding functions of cells. The intramembrane regions of SIRPβ (CD172b) and SIRPγ (CD172g) are very short and have no signal transmission region, but SIRPβ (CD172b) can transmit activation signals through adaptor proteins (Adaptor protein, such as DAP12). SIRP proteins are mainly expressed in macrophages (Mφ), dendritic cells (DC), and neurons. This article specifically refers to human SIRPα wild type and its CD47 non-high affinity mutant.

[0233] Extracellular truncations of SIRPα

[0234] "Extracellular truncation" refers to proteins with transmembrane function. "SIRPα extracellular truncation", as used herein, refers to wild-type human SIRPα and its CD47 non-high affinity mutant, which selectively truncates all or part of its amino acid sequence located in the space outside the cell membrane.

[0235] As used herein, the terms "D1, D2, D3" refer to the three extracellular Ig-like domains of SIRPα, starting from the amino terminus of the protein, namely the D1 domain (Ig variable region-like domain, IgV region), the D2 domain (Ig constant region-like domain, IgC region) and the D3 domain (Ig constant region-like domain, IgC region) (Lee WY, et al. The Role of cis Dimerization of Signal Regulatory Proteinα (SIRPα) in Binding to CD47. J Biol Chem, 2010, 285(49): 37953-37963).

[0236] SIRPα-Fc fusion protein

[0237] As used herein, the term "SIRPα-Fc fusion protein" refers to a fusion protein comprising an extracellular truncated body of SIRPα, a linker sequence and an Fc region. The linker sequence and / or Fc region contained in the above sequence can be arbitrarily replaced according to methods well known to those skilled in the art or commonly used linker sequences and / or Fc regions.

[0238] In order to avoid the influence of glycosylation, the present invention mutated D1 from asparagine to alanine (reference: Lee WY, et al. Novel Structural Determinants on SIRPα that Mediate Binding to CD47. Journal of Immunology, 2007, 179(11): 7741-7750).

[0239] D1, D2 and D3 of the present invention also include corresponding linker sequences.

[0240] Adapter sequence

[0241] As used herein, the term "linker sequence" refers to an amino acid sequence that connects the extracellular truncation of SIRPα and the binding sequence, optionally, the hinge region of an IgG antibody, optionally, comprising a hinge region and an IgG heavy chain CH1 domain. The linker sequence or hinge region sequence contained in the above sequence can be arbitrarily replaced according to methods well known to those skilled in the art or commonly used linker sequences or hinge region sequences.

[0242] Binding sequence

[0243] As used herein, the term "binding sequence" refers to a sequence that binds a high-affinity tumor-targeting arm to a fusion protein that blocks the interaction of CD47 and SIRPα with low affinity, and optionally, the binding sequence comprises a hinge region and an Fc region, and more optionally, the Fc region comprises a knob or knobs and / or a hole or holes mutation. The binding sequence or hinge region or Fc region sequence contained in the above sequence can be arbitrarily replaced according to methods well known to those skilled in the art or commonly used binding sequences or hinge region or Fc region sequences.

[0244] CD47

[0245] CD47 is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily and is expressed on the surface of almost all cells, including red blood cells. The ligands of CD47 include adhesion factors (integrin), thrombospondin-1, and signal regulatory proteins (SIRP). CD47 has a variety of biological functions, including cell migration, T cells, dendritic cell activation, axon development, etc. In addition, CD47 can inhibit the phagocytosis of macrophages by interacting with SIRPα. In this way, CD47 transmits a so-called "Don't eat me" signal, which can protect normal cells such as red blood cells, B cells, and T cells from being phagocytosed by macrophages.

[0246] Ofa

[0247] As used herein, the terms “Ofa”, “Ofatumumab”, and “Anti-CD20 (Ofatumumab)” are used interchangeably in the present invention to refer to the anti-CD20 antibody Ofatumumab.

[0248] Obi

[0249] As used herein, the terms “Obi”, “Obinutuzumab”, and “Anti-CD20 (Obinutuzumab)” are used interchangeably in the present invention to refer to the anti-CD20 antibody Obinutuzumab.

[0250] Hu5F9-G4

[0251] As used herein, the terms "Anti-CD47 mAb", "anti-CD47 antibody", and "Hu5F9-G4" are used interchangeably in the present invention to refer to the anti-CD47 antibody Hu5F9-G4.

[0252] Anti-EGFR mAb

[0253] As used herein, the terms "Anti-EGFR mAb" and "JMT101" can be used interchangeably in the present invention to refer to the anti-EGFR antibody JMT101. JMT101 is a humanized anti-EGFR monoclonal antibody, see ZL201210406288.3 patent BA03.

[0254] Trastuzumab

[0255] As used herein, the terms “trastuzumab”, “Trastuzumab”, “Anti-Her2 (T) mAb”, and “Herceptin” are used interchangeably in the present invention to refer to the anti-Her2 antibody Trastuzumab.

[0256] Pertuzumab

[0257] As used herein, the terms “Pertuzumab”, “Anti-Her2 (P) mAb”, and “Perjeta” are used interchangeably in the present invention to refer to the anti-Her2 antibody Pertuzumab.

[0258] Atezolizumab

[0259] As used herein, the terms "Tecentriq" and "Atezolizumab" are used interchangeably in the present invention to refer to the anti-PD-L1 antibody Atezolizumab.

[0260] SIRPαD1-Fc

[0261] As used herein, the terms “SIRPαD1-Fc” and “D1-Fc” are used interchangeably in the present invention to refer to a dimer of the single-chain fusion protein SIRPαD1-Fc.

[0262] Ofa-Fc1

[0263] Represents Ofatumumab half-antibody with hole mutation in the Fc region.

[0264] Anti-Her2(T)-Fc1

[0265] Represents the Transtuzumab half-antibody with a hole-mutated Fc region.

[0266] Anti-Her2(P)-Fc1

[0267] Represents Pertuzumab half-antibody with hole mutation in the Fc region.

[0268] Anti-EGFR-Fc1

[0269] represents an anti-EGFR half antibody with a hole-mutated Fc region.

[0270] D1-Fc2

[0271] represents a fusion protein comprising an extracellular truncation of the SIRPαD1 domain and an Fc region with a knob mutation.

[0272] D1-D2-Fc2

[0273] represents a fusion protein comprising the extracellular truncations of SIRPα D1 and D2 domains and an Fc region with a knob mutation.

[0274] D1-D2-D3-Fc2

[0275] It represents a fusion protein comprising the extracellular truncations of SIRPα D1, D2 and D3 domains and an Fc region with a knob mutation.

[0276] treat

[0277] As used herein, the terms "treat", "therapy" and "treatment" can be used interchangeably. The term "treat" includes controlling the progression of a disease, disorder, condition and associated symptoms, preferably reducing the disease, disorder, condition or alleviating the effects of one or more symptoms of the disease, disorder, condition. This term includes curing the disease or completely eliminating symptoms. This term includes symptom relief. This term also includes, but is not limited to, non-curative palliative treatment. The term "treatment" includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the present invention to prevent or delay, alleviate or relieve the progression of a disease, disorder, condition or the effects of one or more symptoms of a disease, disorder, condition.

[0278] Application

[0279] As used herein, the term "administering" refers to delivering a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the present invention to a subject. Administration may be systemic or topical. Administration may be performed by an administration device, such as a syringe. Modes of administration include, but are not limited to, embedding, nasal inhalation, spraying, injection, and the like. Routes of administration include inhalation, intranasal, oral, intravenous, subcutaneous, or intramuscular administration, and the like.

[0280] Table 2-1 Correspondence between sequence names and sequence numbers

[0281]

[0282]

[0283] Table 2-2 Correspondence between recombinant proteins and sequences

[0284]

[0285]

[0286]

[0287] Example 1 Construction of expression vector

[0288] According to the designed molecular structure, the amino acid sequences of each component are spliced ​​together front and back, and the optimal DNA coding sequence is designed according to the codon preference of Chinese hamsters (Cricetulus griseus), excluding the restriction sites required for gene cloning operations, and then the cloning site, Kozak sequence and signal peptide coding sequence are added to the 5' end of the sequence in sequence, and the termination codon and cloning site are added to the 3' end of the sequence in sequence, such as Figure 1 shown.

[0289] Perform full gene synthesis, and use the 5' and 3' cloning sites to clone the full gene into the corresponding cloning sites of the expression vector pCHO-TE2 (purchased from Thermo Fisher). After sequencing and verification, the expression plasmid can be obtained. The cloning sites used at the 5' and 3' ends are EcoRV and PacI sites. Figure 2 This is the plasmid map of the expression vector pCHO-TE2.

[0290] Example 2 Preparation of expression plasmid, cell transfection, and expression and purification of target protein

[0291] Preparation of expression plasmid

[0292] Inoculate the glycerol bacteria containing the expression plasmid (add 0.5 mL of 60% sterile glycerol solution to 1 mL of E. coli containing the expression plasmid and mix thoroughly) into the liquid LB medium at a ratio of 1:1000. Cultivate the cells by centrifugation at 37°C, 220 rpm, and shake for 16 hours. Use the endotoxin-free plasmid extraction kit (DP117, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the expression plasmid according to the standard process provided in the kit instructions.

[0293] Cell transfection and protein expression

[0294] After filtering the obtained expression plasmid with a 0.22 μm filter membrane, 3 mg of plasmid (wherein the product is a typical antibody molecule, the ratio of its light chain and heavy chain expression plasmids is 1:1 (molar ratio); the product is a recombinant protein, and the ratio of its light chain, heavy chain, and right arm expression plasmids is 1:1:1 (molar ratio), see Table 3 for details) is added to 50 mL of Opti MEM I Reduced Serum Medium (purchased from GIBCO) and mixed. 6 mg of transfection reagent polyetherimide (Polyetherimide, PEI, purchased from Polysciences, dissolved in sterile ultrapure water at a concentration of 1 mg / mL) is added to 50 mL of Opti MEM I Reduced Serum Medium and mixed. Add the obtained PEI solution to the Opti MEM I Reduced Serum Medium solution containing the plasmid and mix well. After standing at room temperature for 15 minutes, slowly and evenly add the mixture of plasmid and PEI to a volume of 1 L and a cell density of 3×10 6 The cells were placed in a suspension of host cells CHO-S (purchased from Thermo Fisher) at a concentration of 1 cell / mL and cultured in a 37°C, 5% CO2 incubator. After 4 hours, a feed medium equivalent to 7% of the initial volume was added (the feed medium formula is 80gCD Efficient FeedC AGT (purchased from Gibco) and 75g 5×00483 (purchased from Kerry) dissolved in each liter of water). The culture temperature was lowered to 33°C and harvested after 6 days of culture. The cell suspension was centrifuged at 10,000g and 10°C for 30 minutes, and the supernatant obtained by centrifugation, i.e., the cell culture harvest, was used for the purification of the target protein.

[0295] Protein purification

[0296] The following method uses Ofa-Fc1-D1-Fc2 as an example and protein A affinity capture.

[0297] The cell culture harvest was centrifuged at 10,000 rpm for 30 min to remove cells and their debris, and then loaded onto a protein A affinity column (Cat. No.: 17-5438-02, GE Healthcare) to elute the target protein. Protein purity was determined by SDS-PAGE.

[0298] The protein A purification method is a conventional protein purification method well known to those skilled in the art. For specific experimental methods, please refer to the GE Healthcare Protein A product instructions and the GE Antibody Purification Manual.

[0299] The theoretical molecular weights of the four proteins SIRPαD1-Fc, Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, and Ofa-Fc1-D1-D2-D3-Fc2 are 37.8 kD, 110.7 kD, 121.7 kD, and 131.4 kD, respectively. The results of SDS-PAGE protein electrophoresis are shown in Figure 4 A and Figure 4 As shown in B.

[0300] Protein electrophoresis (SDS-PAGE): The results showed ( Figure 4 A and Figure 4 B), the target proteins in each lane were effectively expressed and purified, among which Ofa-Fc1-D1-Fc2 ( Figure 4 Lane A: 11), Ofa-Fc1-D1-D2-Fc2 ( Figure 4 Lane B 1), Ofa-Fc1-D1-D2-D3-Fc2 ( Figure 4 Lane A (12) shows left arm dimers (Ofa-Fc1-Ofa-Fc1), right arm dimers (SIRPα-Fc2) and / or multimers to varying degrees.

[0301] Table 3 Expression plasmid ratio

[0302]

[0303]

[0304] Note: D1 m Represents the high-affinity mutant of SIRPα extracellular truncated D1; D1 represents the extracellular D1 domain of human SIRPα wild type and its non-high-affinity mutant; Fc represents the wild-type Fc region, Fc1 represents the Fc region with hole or holes mutation, and Fc2 represents the Fc region with knob or knobs mutation

[0305] Example 3 Target affinity and target competitive binding activity detection

[0306] 1. CD47, CD20, EGFR, Her2 target affinity detection method

[0307] Recombinant proteins Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1 m -Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1 m -D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1 m-Fc2,Obi-Fc1-D1-D2-Fc2,Obi-Fc1-D1 m The binding affinity of Obi-Fc1-D1-D2-D3-Fc2 to the targets CD47 and CD20 was determined by ELISA and / or flow cytometry. The following method uses Ofa-Fc1-D1-Fc2 as an example and is suitable for the detection of recombinant proteins with CD20 as the target on the left arm.

[0308] Recombinant proteins Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1 m -Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1 m -D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1 m -Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1 m The binding affinity of -D2-Fc2 and Anti-Her2(P)-Fc1-D1-D2-D3-Fc2 to the targets CD47 and Her2 was determined by ELISA and / or flow cytometry. The following method takes Anti-Her2(T)-Fc1-D1-Fc2 as an example and is suitable for the detection of recombinant proteins with Her2 as the target on the left arm.

[0309] Recombinant proteins Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1 m -Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1 m The binding affinity of -D2-Fc2 and Anti-EGFR-Fc1-D1-D2-D3-Fc2 to the targets CD47 and EGFR is determined by ELISA and / or flow cytometry. The following method takes Anti-EGFR-Fc1-D1-Fc as an example and is suitable for the detection of recombinant proteins with EGFR as the target on the left arm.

[0310] Recombinant proteins Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1 m-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1 m -D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1 m -Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1 m -D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1 m -Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1 m The binding affinity of Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 to the targets CD47 and PD-L1 was determined by ELISA and / or. The following method takes Anti-PD-L1(Ate)-Fc1-D1-Fc2 as an example and is suitable for the detection of recombinant proteins with PD-L1 as the target on the left arm.

[0311] ELISA to detect the affinity of Ofa-Fc1-D1-Fc2 and Anti-EGFR-Fc1-D1-Fc2 to target CD47 :

[0312] 100 μl 1 μg / ml CD47-His (12283-H08H-200, Sino Biological) was used to coat the ELISA plate (Cat. No. 9018, Corning) and placed at 4°C overnight; after washing with PBST solution (PBS containing 0.1% Tween20), the ELISA plate was blocked with PBS+1% BSA at room temperature for 2 hours; after washing, diluted Ofa-Fc1-D1-Fc2 and Anti-EGFR-Fc1-D1-Fc2 (starting at 1000 ng / ml, 2.5-fold dilution, 11 points in total) were added to the coated plate, 100 μl per well, and incubated at 25°C for 1 hour; the sample was discarded and washed three times with PBST solution; 100 μl diluted mouse anti-human IgG was added After Fc-HRP (1:10000) (Ab7499, abcam), incubate at 25°C for 1 hour; discard the solution and wash three times with PBST solution; add TMB (P0209, beyotime) and color for about 20 minutes in the dark, then terminate the reaction with H2SO4 and read the OD (450-650nm) value on a microplate reader.

[0313] The results showed that anti-CD47 antibodies Hu5F9-G4, SIRPαD1-Fc, Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1 m -Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1 m -D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1 m -Fc2,Obi-Fc1-D1-D2-Fc2,Obi-Fc1-D1 m -D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1 m -Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1 m-D2-Fc2, and Anti-EGFR-Fc1-D1-D2-D3-Fc2 can bind to CD47 respectively; the binding affinity of Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, and Anti-EGFR-Fc1-D1-D2-D3-Fc2 to CD47 is slightly weaker than the binding affinity of anti-CD47 antibody Hu5F9-G4 and / or SIRPαD1-Fc to CD47.

[0314] The above experimental data prove that the recombinant protein of the present invention can specifically target the tumor cell CD47 antigen at the protein level, and the binding affinity to CD47 is no higher than the binding affinity of SIRPαD1-Fc fusion protein to CD47 protein; the recombinant protein of the present invention can reduce or avoid the side effects such as erythrocyte agglutination and anemia caused by anti-CD47 antibody treatment and / or non-tumor target cell killing caused by SIRPα high-affinity mutant treatment (Petrova PS, et al. TTI-621 (SIRPαFc): ACD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Antitumor Activity and Minimal Erythrocyte Binding. Clin Cancer Res, 2017, 23 (4): 1068-1079).

[0315] For example, Figure 5 As shown in the figure, except for the anti-CD20 antibody Ofatumumab and the anti-EGFR antibody JMT101, the anti-CD47 antibodies Hu5F9-G4, SIRPαD1-Fc, Ofa-Fc1-D1-Fc2, and Anti-EGFR-Fc1-D1-Fc2 can bind to CD47. However, since only the right arm of Ofa-Fc1-D1-Fc2 and Anti-EGFR-Fc1-D1-Fc2 can bind to the CD47 antigen, their affinity (EC 50 =52.57ng / mL / EC 50 =93.86 ng / mL) is weaker than anti-CD47 antibody (EC 50 =5.439 ng / mL) and / SIRPαD1-Fc (EC 50 =6.118ng / mL).

[0316] Flow cytometry detection of affinity of Ofa-Fc1-D1-Fc2 to target CD47 :

[0317] A431 cells (human epidermal carcinoma cells), well-growing cells were collected and counted, centrifuged and resuspended in PBS + 2% FBS (purchased from Gibco) to 3 × 10 6 The cells were added to a 96-well U-shaped plate (Cat. No. 3799, Corning) at 100 μl / well and allowed to stand for at least 15 minutes; the supernatant was centrifuged and 9 dilutions of Ofa-Fc1-D1-Fc2 (starting from 15000 ng / ml, 5-fold gradient dilution, a total of 9 concentrations) were added respectively, and the 96-well plate was incubated in a refrigerator at 4°C for 1 hour; after washing with PBS+2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4°C for 1 hour; after washing and resuspending with PBS+2% FBS, the fluorescence value was detected by flow cytometry (Accuri C6, BD).

[0318] Since A431 cells do not express CD20 antigen and cannot bind to Ofatumumab and Obinutuzumab, A431 cells can be used to evaluate OFA-Fc1-D1-Fc2 and OFA-Fc1-D1 at the cellular level. m -Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1 m -D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1 m -Fc2,Obi-Fc1-D1-D2-Fc2,Obi-Fc1-D1 m -D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2 and CD47 binding affinity.

[0319] The results showed that, except for the anti-CD20 antibodies Ofatumumab and Obinutuzumab, which could not bind to A431, the anti-CD47 antibodies Hu5F9-G4, SIRPαD1-Fc, Ofa-Fc1-D1-Fc2, and Ofa-Fc1-D1 m -Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1 m -D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1 m -Fc2,Obi-Fc1-D1-D2-Fc2,Obi-Fc1-D1 m-D2-Fc2, and Obi-Fc1-D1-D2-D3-Fc2 can all bind to A431 cells; the binding affinity of Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, and Obi-Fc1-D1-D2-D3-Fc2 to CD47 is slightly weaker than that of anti-CD47 antibodies and / or SIRPαD1-Fc to CD47, which is consistent with the data trend of ELISA.

[0320] The above experimental data prove that the recombinant protein of the present invention can specifically target the CD47 antigen of tumor cells at the cellular level, and the binding affinity to CD47 is no higher than the binding affinity of SIRPαD1-Fc fusion protein to CD47; the recombinant protein of the present invention can reduce or avoid the side effects such as red blood cell agglutination and anemia caused by anti-CD47 antibody treatment and / or non-tumor target cell killing caused by SIRPα high-affinity mutant treatment.

[0321] For example, Figure 6 As shown, anti-CD47 antibody Hu5F9-G4, SIRPαD1-Fc and Ofa-Fc1-D1-Fc2 can all bind to A431 cells. Specifically, the affinity of Ofa-Fc1-D1-Fc2 is slightly weaker than that of anti-CD47 antibody and / or SIRPαD1-Fc, which is consistent with the data trend of ELISA.

[0322] Flow cytometry detection of affinity of Ofa-Fc1-D1-Fc2 for target CD20 :

[0323] Raji cells (human B-cell lymphoma) (purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai), well-growing cells were collected and counted, centrifuged and resuspended in PBS + 2% FBS to 3 × 10 6 The cells were added to a 96-well U-shaped plate (Cat. No. 3799, Corning) at 100 μl / well and allowed to stand for at least 15 minutes; the supernatant was aspirated by centrifugation and 100 μL PBS + 2% FBS (control group) or 1.5 μg / mL anti-CD47 antibody Hu5F9-G4 (Fab) 2 (experimental group) (Fc removed by pepsin, kit: Thermo Fisher, 44988) was added, and incubated at 4°C for 1 hour; after washing with PBS + 2% FBS, 7 dilutions of Ofa-Fc1-D1-Fc2, Hu5F9-G4, and SIRPαD1-Fc were added respectively (starting from 6250 ng / ml, 4-fold gradient dilution, a total of 7 concentrations, Figure 7The cells were incubated at 4°C for 1 hour after washing with PBS + 2% FBS, and goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4°C for 1 hour. The cells were washed and resuspended with PBS + 2% FBS, and the fluorescence value was detected by flow cytometry (AccuriC6, BD).

[0324] The experimental results showed that the anti-CD47 antibody Hu5F9-G4 (Fab) 2 can effectively block the binding of the anti-CD47 antibody Hu5F9-G4 and / or SIRPαD1-Fc to CD47 on Raji cells; however, the blocking effect of Hu5F9-G4 (Fab) 2 on the CD47 antigen did not significantly inhibit the binding of Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi Fc1-D1-D2-Fc2, and Obi-Fc1-D1-D2-D3-Fc2 to Raji cells.

[0325] The above experimental data prove that when the CD47 antigen on the surface of tumor cells is blocked and the mutual binding of SIRPα-CD47 is blocked, the recombinant protein of the present invention can still rely on its left arm to specifically bind to the corresponding antigen on the tumor cells, and the affinity of the left arm is not significantly affected by the blocking of the binding of the right arm.

[0326] For example, Figure 7 As shown, the anti-CD47 antibody Hu5F9-G4 (Fab) 2 can effectively block the binding of anti-CD47 antibody Hu5F9-G4 and / or SIRPαD1-Fc to CD47 on Raji cells, but the blocking effect of Hu5F9-G4 (Fab) 2 on CD47 antigen did not significantly inhibit the binding of Ofa-Fc1-D1-Fc2 to Raji cells, indicating that Ofa-Fc1-D1-Fc2 can still rely on its left arm (anti-CD20 half antibody) to specifically bind to the CD20 antigen on Raji cells after the mutual binding of SIRPα-CD47 is blocked, and the affinity is not significantly affected by the blocking of the right arm binding.

[0327] Flow cytometry detection of bispecific binding to targets CD20 and CD47 :

[0328] Raji cells (human B-cell lymphoma) (purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai), well-growing cells were collected and counted, centrifuged and resuspended in PBS + 2% FBS to 3 × 10 6The cells were added to a 96-well U-shaped plate (Cat. No. 3799, Corning) at 100 μl / well and allowed to stand for at least 15 minutes. The supernatant was aspirated by centrifugation and 12 dilutions of Ofa-Fc1-D1-Fc2, Ofatumumab, Hu5F9-G4, SIRPαD1-Fc (50000 ng / ml, 25000 ng / ml, 6250 ng / ml) were added respectively, followed by 4-fold gradient dilutions for a total of 12 concentrations. Figure 8 A is the molar concentration after conversion), incubated at 4°C for 1 hour; after washing with PBS + 2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4°C for 1 hour; after washing and resuspending with PBS + 2% FBS, the fluorescence value was detected by flow cytometer (Accuri C6, BD).

[0329] Since Raji cells express both CD20 and CD47 antigens on their surface, anti-CD20 antibodies Ofatumumab, Obinutuzumab, anti-CD47 antibodies Hu5F9-G4 and SIRPαD1-Fc can all bind specifically to Raji cells, but the maximum mean fluorescence intensities that can be achieved are different.

[0330] The test results showed that Ofa-Fc1-D1-Fc2 and Ofa-Fc1-D1 m -Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1 m -D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1 m -Fc2,Obi-Fc1-D1-D2-Fc2,Obi-Fc1-D1 m -D2-Fc2 and Obi-Fc1-D1-D2-D3-Fc2 can also bind to Raji cells and have higher maximum mean fluorescence intensity.

[0331] The above test data prove that under the same supersaturated protein sample concentration environment, the recombinant protein of the present invention can specifically bind to tumor cells compared with the anti-CD20 antibody Ofatumumab, Obinutuzumab and / or the anti-CD47 antibody Hu5F9-G4 and / or SIRPαD1-Fc, and shows a significant molecular quantity advantage. Preferably, under the same supersaturated protein sample concentration environment, the saturated binding abundance of the recombinant protein of the present invention to tumor cells is greater than the sum of the saturated binding abundance of the anti-CD20 antibody and SIRPαD1-Fc to tumor cells.

[0332] Table 4 Maximum mean fluorescence intensity and EC50 (nM) of antibody / recombinant protein binding to Raji cells

[0333]

[0334] For example, Figure 8 As shown in Figure A and Table 4, anti-CD20 antibody Ofatumumab, anti-CD47 antibody Hu5F9-G4 and SIRPαD1-Fc can all specifically bind to Raji cells, but the maximum mean fluorescence intensities that can be achieved are different; at the same time, Ofa-Fc1-D1-Fc2 can also bind to Raji cells, and has a higher maximum mean fluorescence intensity, indicating that under the same supersaturated protein sample concentration environment, the number of Ofa-Fc1-D1-Fc2 protein molecules that can specifically bind to Raji cells is significantly higher than the number of anti-CD20 antibody Ofatumumab or anti-CD47 antibody Hu5F9-G4 or SIRPαD1-Fc molecules, higher than the sum of the number of anti-CD20 antibody Ofatumumab and anti-CD47 antibody Hu5F9-G4 molecules, and higher than the sum of the number of anti-CD20 antibody Ofatumumab and SIRPαD1-Fc molecules.

[0335] As known to those skilled in the art, the above test results suggest that, compared with the combined use of anti-CD47 antibodies or SIRPαD1-Fc with other anti-tumor targeted therapeutic antibodies, the recombinant protein capable of simultaneously binding to tumor targeting antigens and CD47 antigens of the present invention can bind to more tumor cells, thereby obtaining a more significant anti-tumor effect.

[0336] Flow cytometry detection of bispecific binding to targets Her2 and CD47 :

[0337] SKBR-3 cells (human breast cancer cells) (purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai), well-growing cells were collected and counted, centrifuged and resuspended in PBS + 2% FBS to 2 × 10 6 The cells were added to a 96-well U-shaped plate (Cat. No. 3799, Corning) at 100 μl / well and allowed to stand for at least 15 minutes; the supernatant was centrifuged and 10 dilutions of Anti-Her2(T)-Fc1-D1-Fc2, Trastuzumab and Hu5F9-G4 (433.2 nM starting point, 4-fold gradient dilution, 10 points in total) were added respectively and incubated at 4°C for 1 hour; after washing with PBS+2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4°C for 1 hour; after washing and resuspending with PBS+2% FBS, the fluorescence value was detected by flow cytometry (Accuri C6, BD).

[0338] Since SKBR-3 cells express both Her2 and CD47 antigens on their surface, anti-Her2 antibodies Trastuzumab, Pertuzumab, anti-CD47 antibodies Hu5F9-G4 and SIRPαD1-Fc can all bind specifically to SKBR-3 cells, but the maximum mean fluorescence intensities that can be achieved are different.

[0339] The test results showed that Anti-Her2(P)-Fc1-D1-Fc2 and Anti-Her2(P)-Fc1-D1 m -Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1 m -D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1 m -Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1 m -D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1 m -Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1 m -D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1 m -Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1 m -D2-Fc2 and Anti-Her2(T)-Fc1-D1-D2-D3-Fc2 can also bind to SKBR-3 cells and have higher maximum mean fluorescence intensity.

[0340] The above test data prove that under the same supersaturated protein sample concentration environment, the recombinant protein of the present invention can specifically bind to tumor cells compared with anti-Her2 antibody Trastuzumab, Pertuzumab and / or anti-CD47 antibody Hu5F9-G4 and / or SIRPαD1-Fc, and shows a significant molecular quantity advantage. Preferably, under the same supersaturated protein sample concentration environment, the saturated binding abundance of the recombinant protein of the present invention with tumor cells is greater than the sum of the saturated binding abundances of the corresponding anti-Her2 antibody and SIRPαD1-Fc with tumor cells.

[0341] Table 5 Maximum mean fluorescence intensity and EC50 (nM) of antibody / recombinant protein binding to SKBR-3 cells

[0342]

[0343] For example, Figure 8 As shown in Table B and Table 5, both anti-Her2 antibody Trastuzumab and anti-CD47 antibody Hu5F9-G4 can specifically bind to SKBR-3 cells, but the maximum average fluorescence intensities that can be achieved are different; at the same time, Anti-Her2(T)-Fc1-D1-Fc2 can also bind to SKBR-3 cells, and has a higher maximum average fluorescence intensity, indicating that under the same supersaturated protein sample concentration environment, the number of Anti-Her2(T)-Fc1-D1-Fc2 protein molecules that can specifically bind to SKBR-3 cells is significantly higher than the number of anti-Her2 antibody Trastuzumab or anti-CD47 antibody Hu5F9-G4 molecules, and higher than the sum of the number of anti-Her2 antibody Trastuzumab and anti-CD47 antibody Hu5F9-G4 molecules.

[0344] As known to those skilled in the art, the above test results suggest that, compared with the combined use of anti-CD47 antibodies or SIRPαD1-Fc with other anti-tumor targeted therapeutic antibodies, the use of the recombinant protein of the present invention that is capable of simultaneously binding to tumor targeting antigens and CD47 antigens can bind to more tumor cells, thereby obtaining a more significant anti-tumor effect.

[0345] Flow cytometry detection of bispecific binding to targets EGFR and CD47 :

[0346] A431 cells (human epidermal carcinoma cells) (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were collected and counted, centrifuged and resuspended in PBS + 2% FBS to 2 × 10 6The cells were added to a 96-well U-shaped plate (Cat. No. 3799, Corning) at 100 μl / well and allowed to stand for at least 15 minutes; the supernatant was centrifuged and 11 dilutions of Anti-EGFR-Fc1-D1-Fc2, JMT101, SIRPαD1-Fc and Hu5F9-G4 (starting at 216.6 nM, 4-fold gradient dilution, a total of 11 concentrations) were added, and incubated at 4°C for 1 hour; after washing with PBS + 2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4°C for 1 hour; after washing and resuspending with PBS + 2% FBS, the fluorescence value was detected by flow cytometry (AccuriC6, BD).

[0347] Since A431 cells express both EGFR and CD47 antigens on their surface, anti-EGFR antibody JMT101, anti-CD47 antibody Hu5F9-G4, and SIRPαD1-Fc can all bind specifically to A431 cells, but the maximum mean fluorescence intensities that can be achieved are different.

[0348] The test results showed that Anti-EGFR-Fc1-D1-Fc2 and Anti-EGFR-Fc1-D1 m -Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1 m -D2-Fc2 and Anti-EGFR-Fc1-D1-D2-D3-Fc2 can also bind to A431 cells and have higher maximum mean fluorescence intensity.

[0349] The above test data prove that under the same supersaturated protein sample concentration environment, the recombinant protein of the present invention can specifically bind to tumor cells compared with the anti-EGFR antibody JMT101 and / or the anti-CD47 antibody Hu5F9-G4 and / or SIRPαD1-Fc, and shows a significant molecular quantity advantage. Preferably, under the same supersaturated protein sample concentration environment, the saturated binding abundance of the recombinant protein of the present invention with tumor cells is greater than the sum of the saturated binding abundances of the corresponding anti-EGFR antibodies and SIRPαD1-Fc with tumor cells.

[0350] Table 6 Maximum mean fluorescence intensity and EC50 (nM) of antibody / recombinant protein binding to A431 cells

[0351]

[0352] For example, Figure 8As shown in Table 6, anti-EGFR antibody JMT101, anti-CD47 antibody Hu5F9-G4, and SIRPαD1-Fc can all bind specifically to A431 cells, but the maximum mean fluorescence intensity they can achieve is different, and the EC 50 EC 50 (JMT101)=0.598nM、EC 50 (SIRPαD1-Fc)=3.677nM, EC 50 (Hu5F9-G4) = 0.865 nM, which shows that the binding affinity of JMT101 to A431 cells is more than 6 times that of SIRPαD1-Fc to A431 cells; at the same time, Anti-EGFR-Fc1-D1-Fc2 can also bind to A431 cells, and has a higher maximum average fluorescence intensity, indicating that under the same supersaturated protein sample concentration environment, the number of Anti-EGFR-Fc1-D1-Fc2 protein molecules that can specifically bind to A431 cells is significantly higher than the number of molecules of anti-EGFR antibody JMT101 or SIRPαD1-Fc or anti-CD47 antibody Hu5F9-G4, and higher than the sum of the number of molecules of anti-EGFR antibody JMT101 and SIRPαD1-Fc.

[0353] As known to those skilled in the art, the above test results suggest that, compared with the combined use of anti-CD47 antibodies or SIRPαD1-Fc with other anti-tumor targeted therapeutic antibodies, the use of the recombinant protein of the present invention that is capable of simultaneously binding to tumor targeting antigens and CD47 antigens can bind to more tumor cells, thereby obtaining a more significant anti-tumor effect.

[0354] Flow cytometry detection of bispecific binding to targets PD-L1 and CD47 :

[0355] NCI-H441 cells (human lung adenocarcinoma cells) (purchased from Beijing Beina Chuanglian Biotechnology Research Institute) were stimulated with 10 ng / ml hIFN-γ (BD, Cat. No. 554616) at 2 × 10 7 After 18 h, the cells were digested, collected and counted, centrifuged and resuspended in PBS + 2% FBS to 3 × 10 6The cells were added to a 96-well U-shaped plate (Cat. No. 3799, Corning) at 100 μl / well and allowed to stand for at least 15 minutes; the supernatant was centrifuged and 12 dilutions of Anti-PD-L1 (Ate) -Fc1-D1-Fc2 and Atezolizumab (433.2 nM, 216.6 nM, then 4-fold gradient dilution, a total of 12 concentrations) were added, and incubated at 4°C for 1 hour; after washing with PBS + 2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4°C for 1 hour; after washing and resuspending with PBS + 2% FBS, the fluorescence value was detected by flow cytometry (Accuri C6, BD).

[0356] Since NCI-H441 cells express both PD-L1 and CD47 antigens on their surface, anti-PD-L1 antibodies Atezolizumab, 13G4, 12A4, anti-CD47 antibodies Hu5F9-G4, and SIRPαD1-Fc can specifically bind to NCI-H441 cells.

[0357] The test results showed that Anti-PD-L1(Ate)-Fc1-D1-Fc2 and Anti-PD-L1(Ate)-Fc1-D1 m -Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1 m -D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1 m -Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1 m -D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1 m -Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1 m -D2-Fc2 and Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 can also bind to NCI-H441 cells and have higher maximum mean fluorescence intensity.

[0358] The above test data prove that under the same supersaturated protein sample concentration environment, the recombinant protein of the present invention can specifically bind to tumor cells compared with anti-PD-L1 antibodies Atezolizumab, 13G4, and 12A4, and shows a certain molecular quantity advantage. For example, under the same supersaturated protein sample concentration environment, the recombinant protein of the present invention can bind to tumor cells more than the anti-PD-L1 antibody Atezolizumab, showing a significant molecular quantity advantage.

[0359] Table 7 Maximum mean fluorescence intensity and EC50 (nM) of antibody / recombinant protein binding to NCI-H441 cells

[0360]

[0361] For example, Figure 8 D and Table 7 show that the anti-PD-L1 antibody Atezolizumab can specifically bind to NCI-H441 cells; at the same time, Anti-PD-L1(Ate)-Fc1-D1-Fc2 can also bind to NCI-H441 cells and has a higher maximum mean fluorescence intensity, indicating that the number of Anti-PD-L1(Ate)-Fc1-D1-Fc2 protein molecules that can specifically bind to NCI-H441 cells is higher than the number of anti-PD-L1 antibody Atezolizumab molecules under the same supersaturated protein sample concentration environment. EC 50 EC 50 (Atezolizumab)=0.2006nM, EC 50 (SIRPαD1-Fc)=1.643nM, EC 50 (Hu5F9-G4) = 0.3865 nM, indicating that the binding affinity of Atezolizumab to NCI-H441 cells is more than 6 times that of SIRPαD1-Fc to NCI-H441 cells.

[0362] As known to those skilled in the art, the above test results suggest that, compared with the combined use of anti-CD47 antibodies and other anti-tumor targeted therapeutic antibodies, the use of the recombinant protein of the present invention that is capable of simultaneously binding to tumor targeting antigens and CD47 antigens can bind to more tumor cells, thereby obtaining a more significant anti-tumor effect.

[0363] 2. Target competition binding activity detection

[0364] The following method uses Ofa-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-Fc2 as an example to measure the competitive binding activity of target CD47 and SIRPα by ELISA.

[0365] ELISA to detect the competitive binding activity of Ofa-Fc1-D1-Fc2 and Anti-EGFR-Fc1-D1-Fc2 :

[0366] 100 μl 1 μg / ml CD47-His (12283-H08H-200, Sino Biological) was used to coat an ELISA plate (9018, Corning) and placed at 4°C overnight; after washing with PBST, the ELISA plate was blocked with PBS + 1% BSA at room temperature for 2 h; after washing, Ofa-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-Fc2 with different concentrations (starting at 1000 ng / ml, 3-fold gradient dilution, a total of 11 concentrations) and biotin-labeled SIRPαD1-Fc (biotin labeling kit, 21925, Thermo, loading concentration was 2.34 μg / ml, 1.5 μg / ml, 2.5 ... 100ng / ml) mixture, 100μl per well, incubate at 25℃ for 1 hour; discard the sample and wash three times with PBST solution; add 100μl diluted streptavidin-HRP (1:10000) (ML-0437P-HRP, ZI501-1 Yanyu Chemical Reagents), incubate at 25℃ for 1 hour; discard the solution and wash three times with PBST solution; add TMB (P0209, beyotime) to develop color in the dark for about 20 minutes, terminate the reaction with H2SO4, and read the OD (450-650nm) value on the microplate reader.

[0367] The test results showed that anti-CD47 antibody Hu5F9-G4, SIRPαD1-Fc, Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti- EGFR-Fc1-D1-D2-D3-Fc2, Anti-Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2- D3-Fc2, Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGF R-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti- PD-L1(Ate)-Fc1-D1-D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1 (13G4)-Fc1-D1-D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, and Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 can all compete with biotin-labeled SIRPαD1-Fc for binding to CD47 antigen to varying degrees, exerting competitive binding activity.

[0368] For example, Fig. 9As shown, anti-CD47 antibodies Hu5F9-G4, SIRPαD1-Fc, Ofa-Fc1-D1-Fc2 and Anti-EGFR-Fc1-D1-Fc2 can all compete with biotin-labeled SIRPαD1-Fc for binding to CD47 antigen to varying degrees, exerting competitive binding activity; the competitive binding ability of Ofa-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-Fc2 with CD47 is weaker than that of anti-CD47 antibody Hu5F9-G4 or SIRPαD1-Fc, respectively, which is consistent with the affinity results described above in this example.

[0369] Example 4 Early in vitro immune safety evaluation experiment of recombinant protein

[0370] Recombinant protein Ofa-Fc1-D1 m -Fc2, Ofa-Fc1-D1 m -D2-Fc2, Anti-EGFR-Fc1-D1 m -Fc2、Anti-EGFR-Fc1-D1 m -D2-Fc2、Anti-Her2(T)-Fc1-D1 m -Fc2、Anti-Her2(T)-Fc1-D1 m -D2-Fc2、Anti-Her2(P)-Fc1-D1 m -Fc2、Anti-Her2(P)-Fc1-D1 m -D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1 m -Fc2、Anti-PD-L1(Ate)-Fc1-D1 m -D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1 m -Fc2, Anti-PD-L1(13G4)-Fc1-D1 m -D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1 m -Fc2, Anti-PD-L1(12A4)-Fc1-D1 m -D2-Fc2 early in vitro immune safety evaluation experiment, the following method is based on Ofa-Fc1-D1 m -Fc2 is used as an example, which is suitable for the detection of recombinant proteins containing high-affinity mutants of extracellular truncations of SIRPα in the right arm.

[0371] Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc 1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-He r2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1-D1- Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc 1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-F c2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2 , Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 early in vitro immune safety evaluation experiment. The following method takes Ofa-Fc1-D1-Fc2 as an example and is suitable for the detection of recombinant proteins containing SIRPα extracellular truncations in the right arm.

[0372] Flow cytometry was used to detect the specific binding of the bispecific antibody to CD47 before and after the specific right arm mutation :

[0373] NCI-H441 cells (human lung adenocarcinoma cells) (purchased from Beijing Beinachuanglian Biotechnology Research Institute) were digested, collected and counted, centrifuged and resuspended in PBS + 2% FBS to 3 × 10 6 The cells were added to a 96-well U-shaped plate (Cat. No. 3799, Corning) at a concentration of 100 μl / well and allowed to stand for at least 15 minutes. The supernatant was aspirated by centrifugation and 12 dilutions of Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1 were added respectively.m -Fc2, Hu5F9-G4, Ofatumumab (433.2nM, 216.6nM, then 4-fold gradient dilution, a total of 12 concentrations), incubated at 4°C for 1 hour; after washing with PBS + 2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4°C for 1 hour; after washing and resuspending with PBS + 2% FBS, the fluorescence value was detected by flow cytometer (Accuri C6, BD).

[0374] Table 8 Flow cytometry detection of specific binding of dual antibodies to CD47 before and after specific right arm mutation

[0375]

[0376] For example, Fig.10 As shown in Table 8, except for the anti-CD20 antibody Ofatumumab, which could not bind to CD47, the anti-CD47 antibodies Hu5F9-G4, Ofa-Fc1-D1-Fc2, and Ofa-Fc1-D1 m -Fc2 can bind to CD47, and Ofa-Fc1-D1 m -Fc2(EC 50 =0.529 nM) had a higher binding affinity to NCI-H441 cells than Ofa-Fc1-D1-Fc2 (EC 50 =8.68nM), where D1 m It is a high-affinity mutant of SIRPα in the D1 region (ie, Seq ID No: 10 of CN106519036A).

[0377] Early in vitro immune safety evaluation experiment of recombinant protein

[0378] (1) Preparation of human effector cell suspension:

[0379] Take well-grown human NK92MI-CD16a effector cells (purchased from Huabo Biotechnology) that stably and highly express CD16a, centrifuge (201g, 5min), discard the supernatant, resuspend in 5ml MEM (phenol red-free) basal medium (purchased from Gibco, 51200-038), count, and adjust the target cell density to 2.4×10 with MEM (phenol red-free) basal medium. 6 cells / ml, used as human effector cell suspension.

[0380] (2) Incubation of effector cells and antibodies:

[0381] Dispense MEM (phenol red-free) basal medium (50 μl / well) into the corresponding wells of a 96-well black bottom transparent culture plate, and add the diluted Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1m -Fc2 bispecific antibody dilution gradient 25μl / well, and set up duplicate wells. Add 25μl of the human effector cell suspension prepared in (1) (60,000 cells / well). After mixing, Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1 m The -Fc2 bispecific antibody reached a final concentration gradient (433.2 nM starting point, 4-fold gradient dilution, a total of ten concentrations), and then reacted at 37°C for 5.5 hours, and a lysis buffer (from a Promega kit, G7891) was added to the control group and incubated for 0.5 h.

[0382] (3) Detection of ADCC activity:

[0383] After incubation, place the culture plate in a safety cabinet, open the cover and cool naturally to room temperature for about 15 minutes. Add the LDH substrate reaction solution (derived from Promega's kit, G7891) that has been equilibrated at room temperature for 30 minutes to the culture plate, 100 μl per well, mix gently and incubate at room temperature for 15 minutes, then immediately add 50 μl / well of the stop solution (derived from Promega's kit, G7891), mix well, and read the fluorescence value on a microplate reader.

[0384] The results of the experiment showed that due to the expression of CD47 antigen on NK cells, recombinant proteins and / or antibodies targeting CD47 with ADCC activity can lead to mutual killing of NK cells. m -Fc2 or Ofa-Fc1-D1 m -D2-Fc2 or Anti-EGFR-Fc1-D1 m -Fc2 or Anti-EGFR-Fc1-D1 m -D2-Fc2 or Anti-Her2(T)-Fc1-D1 m -Fc2 or Anti-Her2(T)-Fc1-D1 m -D2-Fc2 or Anti-Her2(P)-Fc1-D1 m -Fc2 or Anti-Her2(P)-Fc1-D1 m -D2-Fc2 or Anti-PD-L1(Ate)-Fc1-D1 m -Fc2 or Anti-PD-L1(Ate)-Fc1-D1 m -D2-Fc2 or Anti-PD-L1(13G4)-Fc1-D1 m -Fc2 or Anti-PD-L1(13G4)-Fc1-D1 m-D2-Fc2 or Anti-PD-L1(12A4)-Fc1-D1 m -Fc2 or Anti-PD-L1(12A4)-Fc1-D1 m -D2-Fc2, Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1-D2-Fc2 or Ofa-Fc1-D1-D2-D3-Fc2 or Obi-Fc1-D1-Fc2 or Obi-Fc1-D1-D2-Fc2 or Obi-Fc1-D1-D2-D3-Fc2 or Anti-EGFR-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-D2-Fc2 or Anti-EGFR-Fc1-D1-D2-D3-Fc 2 or Anti-Her2(P)-Fc1-D1-Fc2 or Anti-Her2(P)-Fc1-D1-D2-Fc2 or Anti-Her2(P)-Fc1-D1-D2-D3-Fc2 or Anti-Her2(T)-Fc1-D1-Fc2 or Anti-Her2(T)-Fc1-D1-D2-Fc2 or Anti-Her2(T)-Fc1-D1-D2-D3-Fc2 or Anti-EGFR-Fc1-D1-Fc2 or A nti-EGFR-Fc1-D1-D2-Fc2 or Anti-EGFR-Fc1-D1-D2-D3-Fc2 or Anti-PD-L1(Ate)-Fc1-D1-Fc2 or Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2 or Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2 or Anti-PD-L1(13G4)-Fc1-D1-Fc2 or Anti-PD-L1(13G4)-Fc1-D1-Fc2 c1-D1-D2-Fc2 or Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2 or Anti-PD-L1(12A4)-Fc1-D1-Fc2 or Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2 or Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, due to their weak affinity to CD47 antigen, significantly reduce the toxic side effects caused by NK cells, and the toxic side effects are reduced by at least 1,000 times.

[0385] For example, Fig.11 As shown, when the antibody / recombinant protein concentration reaches 10 -1 Ofa-Fc1-D1 m -Fc2 has begun to lyse cells, and when the antibody / recombinant protein concentration reaches 10 3 Ofa-Fc1-D1 m-Fc2 has a cell lysis rate of 15.25%, while Ofa-Fc1-D1-Fc2 has a cell lysis rate of 10 3 No cell lysis was observed at nM.

[0386] As known to those skilled in the art, the above test results indicate that the recombinant protein having ADCC activity and targeting CD47 antigen with low affinity according to the present invention has higher immune safety.

[0387] As known to those skilled in the art, the above test results indicate that the optimized ADCC activity detection method described in the present invention (i.e., early in vitro immune safety evaluation experiment) can be used to evaluate the early immune safety of recombinant proteins (including monovalent or multivalent) or antibodies (including monovalent or multivalent) that target CD47 and have ADCC activity. This evaluation method is simple, fast and is not limited by blood source.

[0388] Example 5 Experiment on the inhibition of tumor cell growth by recombinant protein in vivo

[0389] Recombinant protein Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi -Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti -Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1- D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR -Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2 -Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-F c2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 in vivo inhibition of tumor cell growth experiment. The following method takes Ofa-Fc1-D1-Fc2 as an example and is suitable for the detection of recombinant proteins containing extracellular truncations of SIRPα in the right arm.

[0390] Male NSG mice were subcutaneously inoculated with human B-cell lymphoma Raji cells (purchased from Edmo). After tumor cell inoculation, the tumor tissue reached 80 mm. 3 -150mm 3, divided into the following 2 groups (6 mice in each group, each group was intraperitoneally injected according to the labeled amount): 1) vehicle control group (Tris-citrate, pH 6.5); 2) Ofa-Fc1-D1-Fc2 group (150μg / mouse); administration was done twice a week for a total of 2 weeks. Tumor growth was observed before administration (0 day), 3 days, 5 days, 7 days, 10 days, 12 days, and 14 days after administration, and tumor size was measured to evaluate the tumor inhibition effect of Ofa-Fc1-D1-Fc2.

[0391] The experimental results showed that in the NSG mouse model of Raji lymphoma subcutaneously transplanted, the recombinant proteins Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR- Fc1-D1-D2-D3-Fc2, Anti-Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1- Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)- Fc1-D1-D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1 -D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, and Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 activated the targeted phagocytosis of macrophages and / or macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) by blocking the CD47-SIRPα signaling pathway, thereby showing significant tumor inhibitory effects.

[0392] For example, Fig.12 As shown, the horizontal axis is the time (days) that NSG mice with subcutaneous transplantation of Raji lymphoma received drug treatment, and the vertical axis is the tumor volume (mm 3 ). Fig.12The results showed that after a period of treatment with the drug Ofa-Fc1-D1-Fc2, the Ofa-Fc1-D1-Fc2 group showed a significant tumor inhibition trend compared with the vehicle control group. On the 14th day, the tumor inhibition rate of the Ofa-Fc1-D1-Fc2 group reached 63.14%.

[0393] As known to those skilled in the art, the above test results indicate that the recombinant protein of the present invention that can simultaneously bind to the tumor targeting antigen and the CD47 antigen can achieve significant tumor inhibition effect in NSG mice subcutaneously transplanted with tumor cells.

[0394] Example 6 Early in vivo immune safety evaluation experiment of recombinant protein

[0395] Recombinant protein Ofa-Fc1-D1 m -Fc2, Ofa-Fc1-D1 m -D2-Fc2, Anti-EGFR-Fc1-D1 m -Fc2、Anti-EGFR-Fc1-D1 m -D2-Fc2、Anti-Her2(T)-Fc1-D1 m -Fc2、Anti-Her2(T)-Fc1-D1 m -D2-Fc2、Anti-Her2(P)-Fc1-D1 m -Fc2、Anti-Her2(P)-Fc1-D1 m -D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1 m -Fc2、Anti-PD-L1(Ate)-Fc1-D1 m -D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1 m -Fc2, Anti-PD-L1(13G4)-Fc1-D1 m -D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1 m -Fc2, Anti-PD-L1(12A4)-Fc1-D1 m -D2-Fc2 in vivo early immune safety evaluation, the following method is based on Ofa-Fc1-D1 m -Fc2 is used as an example, which is suitable for the detection of recombinant proteins containing high-affinity mutants of extracellular truncations of SIRPα in the right arm.

[0396] Recombinant protein Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi -Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti -Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1- D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR -Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2 -Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-F c2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 early in vivo immune safety evaluation. The following method takes Ofa-Fc1-D1-Fc2 as an example and is suitable for the detection of recombinant proteins containing extracellular truncations of SIRPα in the right arm.

[0397] Since CD20 antigen is highly expressed on B cells, this experiment measures the B cell content to represent the killing of tumor cells. When evaluating the early immune safety of recombinant proteins targeting other tumor antigens and CD47 antigens in vivo, the corresponding tumor cells need to be subcutaneously transplanted into the experimental mice described in this example.

[0398] Tumor-specific targeting:

[0399] Select human CD34 +HSC-transplanted NSG (Hu-NSG) female mice (purchased from Edmore) were divided into the following 3 groups (3 mice in each group, each group was intravenously injected according to the indicated amount): 1) 0.9% saline control group; 2) Hu5F9-G4 (6.7 μg / mouse) group; 3) Ofa-Fc1-D1-Fc2 (5 μg / mouse) group. The mice were given the drug once. 96 h after the drug administration, 80 μl of blood was collected from the tail vein of the mice and placed in an anticoagulant tube containing sodium heparin. The red blood cells were lysed with freshly prepared lysate (the lysate was lysate (BD Pharm Lyse TM , Catalog No.: 555899): double distilled water (mixed in equal volumes), the remaining cells were washed and resuspended with PBS + 2% FBS, fluorescent antibodies (PE anti-human CD45 (catalog No.: 304039) / FITC anti-human CD19 (catalog No.: 302206) / APC anti-human CD3 (catalog No.: 300312, all purchased from BioLegend) were incubated with the cells for 30 min, washed and resuspended with PBS + 2% FBS, and analyzed by flow cytometry (Accurate TM C6, BD) detection.

[0400] The experimental results showed that when the same dose of Ofa-Fc1-D1-Fc2 and anti-CD47 antibody Hu5F9-G4 were used in the Hu-NSG mouse model, 96 hours after administration, Ofa-Fc1-D1-Fc2 preferentially eliminated B cells (i.e., target cells) expressing the CD20 antigen, while the anti-CD47 antibody Hu5F9-G4 preferentially eliminated non-target cells with a higher abundance of CD47 expression, such as T cells, due to its high affinity for the CD47 antigen.

[0401] For example, Fig.13 As shown, anti-CD47 antibody Hu5F9-G4 ( Fig.13 B) compared with 0.9% saline ( Fig.13 A), non-target cells with high CD47 expression abundance (such as T cells) were significantly eliminated (B cells, i.e. CD20 antigen target cells, accounted for 40.9% of all detected cells before treatment and increased to 73.5% after 96 hours of treatment); while the recombinant protein Ofa-Fc1-D1-Fc2 ( Fig.13 C) compared with 0.9% saline ( Fig.13 A), B cells (i.e., CD20 antigen target cells) were significantly eliminated, that is, 96 hours after administration, Ofa-Fc1-D1-Fc2 preferentially eliminated B cells (the proportion of B cells in all detected cells decreased from 40.9% before administration to 3.7% after 96 hours of administration).

[0402] As known to those skilled in the art, the above test results indicate that the recombinant protein of the present invention that can simultaneously bind to the tumor targeting antigen and the CD47 antigen preferentially eliminates the cells where the tumor targeting antigen is located and / or tumor cells under the same dose condition.

[0403] Immunological safety at low doses:

[0404] Select human CD34 + HSC-transplanted NSG (Hu-NSG) female mice (purchased from Edmore) were divided into the following 2 groups (3 mice in each group, each group was intravenously injected according to the indicated amount): 1) Ofa-Fc1-D1-Fc2 (1 μg / mouse) group; 2) Ofa-Fc1-D1 m -Fc2 (1 μg / mouse) group was given once. 72 hours after the administration, 80 μl of blood was collected from the tail vein of the mice and placed in an anticoagulant tube containing sodium heparin. The red blood cells were lysed with freshly prepared lysate (the lysate was lysate (BD Pharm Lyse TM , Catalog No.: 555899): double distilled water (mixed in equal volumes), the remaining cells were washed and resuspended with PBS + 2% FBS, fluorescent antibodies (PE anti-human CD45 (Catalog No.: 304039) / FITC anti-human CD19 (Catalog No.: 302206) / APC anti-human CD3 (Catalog No.: 300312, all purchased from BioLegend) were incubated with the cells for 30 min, washed and resuspended with PBS + 2% FBS, and the samples of the OFA-Fc1-D1-Fc2 (1 μg / cell) group were analyzed by flow cytometry (Accurate flow cytometry). TM C6, BD) detection, Ofa-Fc1-D1 m -Fc2 (1 μg / mouse) group samples were analyzed by flow cytometry (NovoCyte TM 3130, ACEA) detection.

[0405] The experimental results showed that in the Hu-NSG mouse model, when Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2- D3-Fc2, Anti-Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)- Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1 -D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2, Anti-P D-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2, Anti-PD-L1(13G4) After a single dose of -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, and Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 for 72 hours, target cells expressing the left arm antigen (such as tumor cells) were significantly eliminated, while cells that did not express the left arm antigen (such as T cells and other immune cells) were not significantly affected; while Ofa-Fc1-D1 containing the high-affinity mutant of SIRPα extracellular truncated m -D2-Fc2 or Anti-EGFR-Fc1-D1 m -Fc2 or Anti-EGFR-Fc1-D1 m -D2-Fc2 or Anti-Her2(T)-Fc1-D1 m -Fc2 or Anti-Her2(T)-Fc1-D1 m -D2-Fc2 or Anti-Her2(P)-Fc1-D1m -Fc2 or Anti-Her2(P)-Fc1-D1 m -D2-Fc2 or Anti-PD-L1(Ate)-Fc1-D1 m -Fc2 or Anti-PD-L1(Ate)-Fc1-D1 m -D2-Fc2 or Anti-PD-L1(13G4)-Fc1-D1 m -Fc2 or Anti-PD-L1(13G4)-Fc1-D1 m -D2-Fc2 or Anti-PD-L1(12A4)-Fc1-D1 m -Fc2 or Anti-PD-L1(12A4)-Fc1-D1 m -72 hours after a single dose of D2-Fc2, although target cells expressing the left arm antigen (such as tumor cells) were significantly eliminated, cells that did not express the left arm antigen (such as T cells and other immune cells) were also significantly eliminated.

[0406] For example, Fig.14 As shown, 72 hours after a single administration of Ofa-Fc1-D1-Fc2 ( Fig.14 C. Fig.14 D) compared with before administration ( Fig.14 A. Fig.14 B), B cells (CD20 antigen target cells) were significantly eliminated (almost completely eliminated), while cells that do not express CD20 antigen (such as T cells, other immune cells) were not significantly affected. m -72 hours after single administration of Fc2 ( Fig.14 G. Fig.14 H) compared with before administration ( Fig.14 E. Fig.14 F) Although B cells (CD20 antigen target cells) were significantly eliminated, cells that did not express the CD20 antigen (such as T cells and other immune cells) were also significantly eliminated.

[0407] As known to those skilled in the art, the above test results indicate that the recombinant protein containing the extracellular truncation of SIRPα in the right arm has a higher tumor-specific targeting effect and exhibits higher immune safety than the recombinant protein containing the high-affinity mutant of the extracellular truncation of SIRPα in the right arm under the same dose conditions.

[0408] Immuno-restorative effects at high doses:

[0409] Select human CD34 +HSC-transplanted NSG (Hu-NSG) female mice (purchased from Edmore) were divided into the following 2 groups (3 mice in each group, each group was intravenously injected according to the indicated amount): 1) Hu5F9-G4 (200 μg / mouse) group; 2) Ofa-Fc1-D1-Fc2 (150 μg / mouse) group, which were administered once. 4 days and 14 days after administration, 80 μl of blood was collected from the tail vein of the mice and placed in an anticoagulant tube containing sodium heparin, and freshly prepared lysate was used to lyse red blood cells (the lysate was lysate (BD Pharm Lyse TM , Catalog No.: 555899): double distilled water (mixed in equal volumes), the remaining cells were washed and resuspended with PBS + 2% FBS, fluorescent antibodies (PE anti-human CD45 (catalog No.: 304039) / FITC anti-human CD19 (catalog No.: 302206) / APC anti-human CD3 (catalog No.: 300312, all purchased from BioLegend) were incubated with the cells for 30 min, washed and resuspended with PBS + 2% FBS, and analyzed by flow cytometry (Accurate TM C6, BD) detection.

[0410] The results showed that in the Hu-NSG mouse model, high doses of Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1-D2-Fc2 or Ofa-Fc1-D1-D2-D3-Fc2 or Obi-Fc1-D1-Fc2 or Obi-Fc1-D1-D2-Fc2 or Obi-Fc1-D1-D2-D3-Fc2 or Anti-EGFR-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-D2-Fc2 or Anti-EGF R-Fc1-D1-D2-D3-Fc2 or Anti-Her2(P)-Fc1-D1-Fc2 or Anti-Her2(P)-Fc1-D1-D2-Fc2 or Anti-Her2(P)-Fc1-D1-D2-D3-Fc2 or Anti-Her2(T)-Fc1-D1-Fc2 or Anti-Her2(T)-Fc1-D1-D2-Fc2 or Anti-Her2(T)-Fc1-D1-D2-D3-Fc2 or Anti -EGFR-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-D2-Fc2 or Anti-EGFR-Fc1-D1-D2-D3-Fc2 or Anti-PD-L1(Ate)-Fc1-D1-Fc2 or Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2 or Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2 or Anti-PD-L1(13G4)-Fc1-D1-Fc2 or Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2 or Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2 or Anti-PD-L1(12A4)-Fc1-D1-Fc2 or Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2 or Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 and anti-CD47 antibody Hu5F9-G4, 96 hours after a single dose,Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc 1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-He r2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1-D1- Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc 1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-Fc 2. Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 and anti-CD47 antibody Hu5F9-G4 all showed a significant degree of clearance of B cells (CD20 antigen target cells) and non-target cells (such as T cells and other immune cells). After 14 days,Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1-D2-Fc2 or Ofa-Fc1-D1-D2-D3-Fc2 or Obi-Fc1-D1-Fc2 or Obi-Fc1-D1-D2-Fc2 or Obi-Fc1-D1-D2-D3-Fc2 or Anti-EGFR-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-D2-Fc2 or Anti-EGFR-Fc1-D1-D2-D3-Fc2 or Anti-Her2(P)-Fc1-D1-F c2 or Anti-Her2(P)-Fc1-D1-D2-Fc2 or Anti-Her2(P)-Fc1-D1-D2-D3-Fc2 or Anti-Her2(T)-Fc1-D1-Fc2 or Anti-Her2(T)-Fc1-D1-D2-Fc2 or Anti-Her2(T)-Fc1-D1-D2-D3-Fc2 or Anti-EGFR-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-D2-Fc2 or Anti-EGFR-Fc1-D 1-D2-D3-Fc2 or Anti-PD-L1(Ate)-Fc1-D1-Fc2 or Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2 or Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2 or Anti-PD-L1(13G4)-Fc1-D1-Fc2 or Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2 or Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2 or Anti-PD-L1 In the mice of the D-L1(12A4)-Fc1-D1-Fc2 or Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2 or Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 groups, the B cells (CD20 antigen target cells) were still in a state of being eliminated, while other non-target cells expressing CD47 (such as T cells) were significantly restored; while in the mice of the anti-CD47 antibody Hu5F9-G4 group, neither the B cells (CD20 antigen target cells) nor the non-target cells expressing CD47 were restored.

[0411] For example, Fig.15 As shown, high doses of anti-CD47 antibody Hu5F9-G4 ( Fig.15 A) and Ofa-Fc1-D1-Fc2( Fig.15 B) Although a large number of B cells (CD20 antigen target cells) and non-target cells expressing CD47 (such as T cells) were eliminated 96 hours after administration, 14 days later, Ofa-Fc1-D1-Fc2 ( Fig.15Although B cells (CD20 antigen target cells) of mice in group D were still in a state of being eliminated, non-target cells (such as T cells) expressing CD47 other than B cells (CD20 antigen target cells) were significantly restored, and the anti-CD47 antibody Hu5F9-G4 ( Fig.15 In group C), the B cells (CD20 antigen target cells) and non-target cells expressing CD47 (such as T cells) of mice showed no signs of recovery.

[0412] As known to those skilled in the art, the above test results indicate that the recombinant protein of the present invention that can simultaneously bind to the tumor targeting antigen and the CD47 antigen has recoverable non-target cells (such as immune cells such as T cells) expressing CD47 under high-dose administration conditions, and the recombinant protein has higher immune safety.

[0413] As known to those skilled in the art, the above test results indicate that the in vivo early immune safety evaluation method described in the present invention can be used to evaluate the early immune safety of recombinant proteins (including monovalent or multivalent) or antibodies (including monovalent or multivalent) targeting CD47.

[0414] Example 7 Effects of different truncations on target affinity binding activity

[0415] Recombinant protein Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D 2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-Her2(P)-Fc1-D1-Fc2 , Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1-D2 -Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-F c2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1( 13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D 1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2, taking Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-Fc2 and Anti-Her2(P)-Fc1-D1-D2-Fc2 as examples, are suitable for recombinant proteins with the same left arm and different lengths of SIRPα extracellular truncations in the right arm.

[0416] Flow cytometry detection of bispecific binding to targets Her2 and CD47 :

[0417] SKBR-3 cells (human breast cancer cells) (purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai), well-growing cells were collected and counted, centrifuged and resuspended in PBS + 2% FBS to 2 × 10 6The concentration of cells / ml. The cells were added to a 96-well U-shaped plate (Cat. No.: 3799, Corning) at 100 μl / well and allowed to stand for at least 15 minutes; the supernatant was aspirated by centrifugation and 11 dilutions of Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-Fc2 and Anti-Her2(P)-Fc1-D1-Fc2 (433.2 nM starting point, 4-fold serial dilutions, a total of 11 concentrations) were added respectively and incubated at 4°C for 1 hour; after washing with PBS+2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4°C for 1 hour; after washing and resuspending with PBS+2% FBS, the fluorescence value was detected by flow cytometer (Accuri C6, BD).

[0418] Since trastuzumab and pertuzumab act on different epitopes of Her2 antigen, respectively, and there is a large difference in the distance between the two antigen epitopes and the cell membrane, the recombinant proteins Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-Fc2, and Anti-Her2(P)-Fc1-D1-D2-Fc2 can all bind specifically to SKBR-3 cells, but the binding affinity and the maximum mean fluorescence intensity that can be achieved are different.

[0419] The test results showed that because Trastuzumab and the Her2 antigen epitope (Pedersen MW, et al. Targeting Three Distinct HER2 Domains with a Recombinant Antibody Mixture Overcomes Trastuzumab Resistance. Molecular Cancer Therapeutics, 2015, 14 (3): 669-680) are closer to the cell membrane surface, Anti-Her2 (T) -Fc1-D1-Fc2 has a better affinity for SKBR-3 cells than Anti-Her2 (T) -Fc1-D1-D2-Fc2. Pertuzumab and the Her2 antigen epitope (Her2 extracellular domain II) are farther away from the cell membrane surface, so Anti-Her2 (P) -Fc1-D1-Fc2 has a comparable affinity for SKBR-3 cells than Anti-Her2 (P) -Fc1-D1-D2-Fc2.

[0420] The above experimental data prove that the recombinant protein composed of different length truncations of the right arm will affect the affinity of the recombinant protein to the target cells. For the left arm that binds to the proximal membrane epitope, when the right arm selects a shorter SIRPα truncation, the ability of the recombinant protein to bind to dual targets can be greatly improved. However, for the left arm that binds to the distal membrane epitope, the right arm loses its advantage by selecting a shorter SIRPα truncation. The farther the antigen target of the left arm is from the cell membrane, the longer the SIRPα truncation of the right arm should be to achieve the best match.

[0421] For example, Fig.16 As shown, Anti-Her2(T)-Fc1-D1-Fc2(EC 50 =2.04 nM) was significantly better than Anti-Her2(T)-Fc1-D1-D2-Fc2(EC 50 =25.95nM)( Fig.16 A). The ability of Anti-Her2(P)-Fc1-D1-Fc2 to bind to SKBR-3 cells (EC 50 =15.22 nM) and Anti-Her2(P)-Fc1-D1-D2-Fc2(EC 50 =11.03nM) equivalent to ( Fig.16 B).

[0422] As known to those skilled in the art, the above test results suggest that, based on the distance between the spatial epitope of the target antigen and the surface of the target cell membrane, the right arm adaptively selects a human SIRPα extracellular truncation of appropriate length, which can effectively improve the binding ability of the recombinant protein to the target cells.

[0423] Example 8 Recombinant protein acute toxicity test

[0424] Recombinant proteins Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Ob i-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Ant i-Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc 1-D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EG FR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1 -D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D Acute toxicity test of Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2. The following method takes Ofa-Fc1-D1-Fc2 as an example and is suitable for the detection of recombinant proteins containing extracellular truncations of SIRPα in the right arm.

[0425] Take an appropriate amount of Ofa-Fc1-D1-Fc2 solution (4.02 mg / mL) and dilute it to 0.25 and 2.5 mg / mL respectively with Ofa-Fc1-D1-Fc2 buffer for injection (Tris-citrate, pH 6.5), and use it for administration to the first and second groups of experimental animals, respectively.

[0426] Four healthy female cynomolgus monkeys were purchased from Guangxi Guidong Primate Development Experiment Co., Ltd., aged 4 years. The production of the above experimental animals was permitted by the Guangxi Zhuang Autonomous Region Science and Technology Department, and the experimental animal production license number is SCXK Gui 2016-0001. A detailed clinical observation and weighing were performed before administration, and no abnormalities were found. The body weight range on the day of administration was 2.47-2.85 kg.

[0427] Table 9 Experimental design

[0428]

[0429] *Administered by intravenous injection

[0430] Four female crab-eating macaques were divided into two groups, two in each group, and administered by intravenous injection at a dose of 0.5 mg / kg and 5 mg / kg, with a dosing volume of 2 mL / kg. The experimental design is shown in Table 9 above. The drug was administered once on the day of administration, and the drug was observed continuously for 28 days after the end of administration. Crab-eating macaques were kept in stainless steel mobile cages, one in each cage. Approximately 12 hours of alternating light and dark lighting were provided every day. The experimental monkey compound feed was purchased from Beijing Keao Xieli Feed Co., Ltd., and the animals were free to eat during the experiment, except for specific fasting. This batch of feed was tested by Shanghai Pony Testing Technology Co., Ltd. (PONY) for specific microorganisms, heavy metals and pesticide residues. During the experiment, all animals drank water freely through water bottles. The drinking water used was purified water filtered and sterilized by a reverse osmosis system, and the pH, hardness, heavy metals and microorganisms of the drinking water were tested by the meter.

[0431] During the experiment, all experimental animals were observed twice a day (once in the morning and once in the afternoon) by the cage. The observation contents included but were not limited to the incidence, damage, death and food and water supply. All experimental animals were observed clinically once before the experiment. During the experiment, all experimental animals were observed clinically at least once a day after administration. The observation contents included but were not limited to the incidence, mortality, damage and food and water supply, skin, hair, eyes, ears, nose, mouth, chest, abdomen, external genitalia, limbs, respiratory and circulatory systems, autonomic effects (such as salivation), nervous system (such as tremor, convulsion, stress response and abnormal behavior). The weight of animals was measured on D-1 (before administration), D1, D4, D8, D11, D15, D18, D22, D25 and before dissection. The food consumption of animals within 24 hours (24h±1h) was measured on D2, D4, D8, D11, D15, D18, D22, and D25. Electrocardiograms were measured at D-1, D2, D14, and D28, using standard II leads (8 leads) for recording at a speed of 50 mm / sec.

[0432] Clinical pathology samples were collected and hematology, coagulation, blood biochemistry and lymphocyte typing tests were performed before administration (D-1) and on D2, D7, D14 and D28 after administration; urine samples were collected before administration and on the 28th day after administration and urine analysis was performed.

[0433] All animals were fasted overnight (at least 10 hours) before sample collection, but water was not allowed. Blood samples (4.5-6 mL) were collected from the femoral vein, of which approximately 1.8 mL of whole blood was placed in an anticoagulant tube containing sodium citrate for coagulation analysis; approximately 1 mL of whole blood was placed in an anticoagulant tube containing K3-EDTA for hematological analysis; approximately 2 mL of whole blood was placed in a separation gel collection tube (without anticoagulant) for blood biochemistry analysis, and serum was separated by centrifugation according to standard operating procedures. At the same time, the serum samples separated on D-1 before administration and D2 after administration were tested for T / B cell typing by flow cytometry.

[0434] The animals were euthanized on D29, and the heart, liver, spleen, lung and kidney tissues were collected and preserved. The liver, lung (including the main bronchi), kidney, spleen, heart, adrenal gland, pituitary gland, thyroid and parathyroid gland, thymus, ovary, uterus (including cervix), brain and other tissues were weighed.

[0435] The results showed that when Ofa-Fc1-D1-Fc2 was administered by single intravenous injection at doses of 0.5 and 5 mg / kg, respectively, the animals were observed continuously for 28 days after administration, and no obvious abnormalities related to drug effects were observed in the animals. During this period, the food consumption and body weight of the animals fluctuated within the normal range. There were no significant changes in the coagulation, urine and electrocardiogram related test data of the animals after administration compared with those before administration. After the animals were dissected, the gross observations of all organs were within the normal range, and the organ weights, organ system numbers, and organ-brain ratio of the animals were also within the normal range.

[0436] On D2 after administration, the lymphocyte counts and lymphocyte percentages of animals in the low- and high-dose groups decreased significantly, and returned to normal levels after D7. This change may be related to the drug effect.

[0437] Recombinant protein Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D 2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-Her 2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc 1-D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, An ti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(A te)-Fc1-D1-D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1 The results of (13G4)-Fc1-D1-D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, and Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 were similar to those of Ofa-Fc1-D1-Fc2 at the same dose.

[0438] Table 10 Effect of recombinant protein on the number of erythrocytes in cynomolgus monkeys (1 12 cells / L)

[0439]

[0440] like Fig.18 As shown in A and Table 10, Ofa-Fc1-D1-Fc2 at doses of 0.5 mg / kg and 5 mg / kg had no effect on the number of red blood cells in cynomolgus monkeys; Fig.18 B and Table 10, Ofa-Fc1-D1-Fc2 at doses of 0.5 mg / kg and 5 mg / kg had no effect on cynomolgus monkey hemoglobin.

[0441] Table 11 Effect of recombinant protein on cynomolgus monkey hemoglobin (g / L)

[0442]

[0443] Recombinant protein Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2- D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-Her2(P) -Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1-D1-Fc 2. Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-F c1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1- D2-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1 -D2-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, and Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 at the same doses had similar effects to those of Ofa-Fc1-D1-Fc2.

[0444] like Fig.19 As shown, according to the results of T / B cell typing test, Ofa-Fc1-D1-Fc2 at doses of 0.5 mg / kg and 5 mg / kg can lead to significant clearance of the animal's B cells (CD20 antigen target cells).

[0445] As known to those skilled in the art, the above test results indicate that after a single administration of the recombinant protein capable of simultaneously binding to the tumor targeting antigen and the CD47 antigen described in the present invention, no obvious drug-related abnormalities were observed in the animals, and during this period, the food consumption and body weight of the animals fluctuated within the normal range; after administration, there were no significant changes in the coagulation, urine and electrocardiogram related test data of the animals compared with those before administration; after the animals were dissected, gross observations of all organs were within the normal range, and the organ weights, organ system numbers, and organ-brain ratios of the animals were also within the normal range.

[0446] As known to those skilled in the art, the above test results indicate that the recombinant protein of the present invention that can simultaneously bind to the tumor targeting antigen and the CD47 antigen has no effect on the number of red blood cells and hemoglobin in animals after a single administration.

[0447] As known to those skilled in the art, the above test results indicate that the recombinant protein of the present invention that can simultaneously bind to the tumor targeting antigen and the CD47 antigen preferentially eliminates the cells where the tumor targeting antigen is located and / or tumor cells under the same dosage conditions.

[0448] Example 9 In vivo tumor growth inhibition experiment of recombinant protein

[0449] Recombinant proteins Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Ob i-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Ant i-Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1 -D1-Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGF R-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D 2-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2- In vivo tumor growth inhibition experiment of Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2. The following method takes Ofa-Fc1-D1-Fc2 as an example and is suitable for the detection of recombinant proteins containing extracellular truncations of SIRPα in the right arm.

[0450] Ofa-Fc1-D1-Fc2: colorless clear liquid, concentration 1.14-4.02 mg / ml, stored in aliquots at -80℃; (Rituximab injection): colorless clear liquid, specification 100mg / 10ml, batch number H0205, stored at 2-8℃ away from light. Preparation buffer (Tris-citrate, pH6.5): colorless clear liquid, stored at 2-8℃. Preparation method: Ofa-Fc1-D1-Fc2, All were diluted with formulation buffer; formulation buffer was used as a solvent for direct administration.

[0451] Cells: CD20-positive human B-cell lymphoma Daudi cells were purchased from the cell bank of the Chinese Academy of Sciences. The culture conditions were RPMI 1640 medium with 10% fetal bovine serum and penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2 air. The cells were passaged twice a week, and when the cells were in the exponential growth phase, the cells were collected, counted, and inoculated.

[0452] Experimental animals: Female NOD-SCID mice, 6-7 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd. Production license number: SCXK (Shanghai) 2013-0018; Animal qualification certificate number 2013001829463, 2013001827545. Housing environment: SPF grade. The use and welfare of experimental animals were carried out in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health status and death of animals were monitored daily, and routine examinations included observing the effects of test substances and drugs on the daily behavior of animals, such as behavioral activities, weight changes, and physical signs.

[0453] Each mouse was subcutaneously inoculated with 1.5 × 10 7 Daudi cells were inoculated on day 18 when the average tumor volume reached 100-150 mm 3 At 14:00, mice were divided into groups according to tumor volume and administrated (D0). The mice were intravenously injected (IV) with drugs; the solvent group was injected with the same volume of solvent, with an injection volume of 0.1 mL / 10 g body weight; the dosage and administration schedule are shown in Table 12.

[0454] Tumor diameter was measured with a vernier caliper twice a week, and tumor volume (V) was calculated as:

[0455] The tumor volume (V) was calculated as:

[0456] V=1 / 2×a×b2

[0457] Where a and b represent length and width respectively.

[0458] T / C(%)=(T-T0) / (C-C0)×100

[0459] Wherein, T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment; T is the drug-treated group, and C is the control group.

[0460] Tumor inhibition rate (TGI) (%) = 100-T / C ​​(%).

[0461] When the tumor regresses, tumor inhibition rate (TGI) (%) = 100-(T-T0) / T0×100

[0462] If the tumor shrinks compared to the initial volume, i.e., when T < T0 or C < C0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR).

[0463] After the experiment, or when the tumor volume of the animals reached 1500 mm 3 of the euthanasia endpoint, the animals were sacrificed by carbon dioxide anesthesia, and then the tumors were dissected, weighed, and photographed.

[0464] The comparison between the tumor volumes or tumor weights of the two groups was performed using a two-tailed Student's t-test, and P < 0.05 was defined as having a statistically significant difference.

[0465] Ofa-Fc1-D1-Fc2 (5 mg / kg, IV, twice a week for 5 times) significantly inhibited the growth of Daudi subcutaneous xenografts, with a tumor inhibition rate of 80.8%, and partial tumor regression occurred in 1 / 6 of the mice; (7 mg / kg, IV, twice a week for 5 times) had a tumor inhibition rate of 24.5% on Daudi subcutaneous xenografts. The tumor-bearing mice could generally tolerate the above drugs well (Table 12, Fig. 20 、 21 、22).

[0466] Table 12 Efficacy of Ofa-Fc1-D1-Fc2, on subcutaneous xenografts of human B-cell lymphoma Daudi cells

[0467]

[0468] Note: Random grouping, the first administration time was D0; IV: intravenous injection.

[0469] It can be seen that both Ofa-Fc1-D1-Fc2 and had varying degrees of inhibitory effects on the growth of subcutaneous xenografts of CD20-positive human B-cell lymphoma Daudi cells, and among them, Ofa-Fc1-D1-Fc2 was significantly superior to The tumor-bearing mice could generally tolerate the above drugs well.

[0470] Recombinant protein Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2-D3 -Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-Her2(P)-Fc1 -D1-Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1-D2-D3-Fc2, Anti-Her2(T)-Fc1-D1-Fc2, Anti -Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2 -Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate)-Fc1-D1-Fc2, Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2, Ant i-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4)-Fc1-D1-Fc2, Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2, Anti The tumor inhibitory effects of -PD-L1(13G4)-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4)-Fc1-D1-Fc2, Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2, and Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2 on transplanted tumors in NOD-SCID mice at the same dose and the tolerance of mice to recombinant proteins were similar to the above results of Ofa-Fc1-D1-Fc2.

[0471] As known to those skilled in the art, the above test results indicate that the recombinant protein of the present invention that can simultaneously bind to the tumor targeting antigen and the CD47 antigen has an unexpectedly significant tumor inhibition effect in mice compared with the tumor targeting antibody under the same dosage conditions.

[0472] The use and welfare of experimental animals were carried out in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health status and mortality of the animals were monitored daily, and routine examinations included observing the effects of the test substances and drugs on the daily behavior of the animals, such as behavioral activities, weight changes, and physical signs.

[0473] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise required. No language in the specification should be construed as indicating any non-claimed element is essential to the practice of the invention.

[0474] All publications and patent applications cited in this specification are incorporated herein by reference, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof or discovery described herein is intended to further enhance the understanding of the present invention, and is not intended to limit the present invention to such theory, mechanism, proof or discovery in any way. Although the present invention has been shown and described in detail in the accompanying drawings and the preceding description, the present invention should be considered illustrative rather than restrictive.

Claims

1. A bispecific recombinant protein, It is characterized in that The bispecific recombinant protein comprises a high-affinity tumor-targeting arm and a low-affinity fusion protein that blocks the interaction between CD47 and SIRPα. The bispecific recombinant protein has a left and right arm structure that are relatively arranged, wherein the high-affinity tumor-targeting arm is located at the position of the left arm, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα is located at the position of the right arm; the left arm is in the form of Fab or Fab' of an immunoglobulin, and the right arm is an extracellular truncated body of SIRPα; The left arm comprises a VH and a VL selected from antibodies targeting CD20, HER2, EGFR, and PD-L1, wherein the antibody targeting CD20 is Ofatumumab or Obinutuzumab, the antibody targeting HER2 is Trastuzumab or Pertuzumab, the antibody targeting EGFR is JMT101, and the antibody targeting PD-L1 is Atezolizumab, 13G4, or 12A4; The fusion protein for low-affinity blocking of the interaction between CD47 and SIRPα comprises an extracellular truncation of SIRPα, wherein the extracellular truncation of SIRPα comprises an amino acid sequence selected from any one of the following a1)-a3): a1) SEQ ID No: 30; a2) SEQ ID No: 31; and a3) an amino acid sequence obtained by replacing the 80th amino acid sequence of any one of the above amino acid sequences with alanine to asparagine; The bispecific recombinant protein further comprises an Fc region.

2. A bispecific recombinant protein, It is characterized in that The bispecific recombinant protein comprises a high-affinity tumor-targeting arm and a low-affinity fusion protein that blocks the interaction between CD47 and SIRPα. The bispecific recombinant protein has a left and right arm structure that are relatively arranged, wherein the high-affinity tumor-targeting arm is located at the position of the left arm, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα is located at the position of the right arm; the left arm is composed of the Fab or Fab' region and the Fc region of the immunoglobulin, and the right arm is composed of the SIRPα extracellular truncation and the Fc region; The left arm comprises Fab or Fab' selected from antibodies targeting CD20, HER2, EGFR, and PD-L1, wherein the antibody targeting CD20 is Ofatumumab or Obinutuzumab, the antibody targeting HER2 is Trastuzumab or Pertuzumab, the antibody targeting EGFR is JMT101, and the antibody targeting PD-L1 is Atezolizumab, 13G4, or 12A4; The fusion protein for low-affinity blocking of the interaction between CD47 and SIRPα comprises an extracellular truncation of SIRPα, and the extracellular truncation of SIRPα comprises an amino acid sequence selected from any one of the following a1)-a3): a1) SEQ ID No: 30; a2) SEQ ID No: 31; and a3) an amino acid sequence obtained by replacing the 80th position of any one of the above amino acid sequences by alanine to asparagine.

3. A bispecific recombinant protein, It is characterized in that The bispecific recombinant protein comprises a high-affinity tumor-targeting arm and a low-affinity fusion protein that blocks the interaction between CD47 and SIRPα. The bispecific recombinant protein has a left and right arm structure that are relatively arranged, wherein the high-affinity tumor-targeting arm is located at the position of the left arm, and the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα is located at the position of the right arm; the left arm is composed of the Fab or Fab' region and the Fc region of the immunoglobulin, and the right arm is composed of the SIRPα extracellular truncation and the Fc region; The high-affinity tumor-targeting arm targets a target selected from the following: CD20, EGFR, HER2, PD-L1; the high-affinity tumor-targeting arm comprises an amino acid sequence selected from any one of the following groups: 1) SEQ ID NO: 16 and SEQ ID NO: 17; 2) SEQ ID NO: 18 and SEQ ID NO: 4; 3) SEQ ID NO: 22 and SEQ ID NO: 23; 4) SEQ ID NO: 20 and SEQ ID NO: 21; 5) SEQ ID NO: 19 and SEQ ID NO: 8; 6) SEQ ID NO: 24 and SEQ ID NO: 13; 7) SEQ ID NO: 10 in CN104356236A and SEQ ID NO: 20 in CN104356236A; and 8) SEQ ID NO: 2 in CN104356236A and SEQ ID NO: 12 in CN104356236A; The fusion protein for low-affinity blocking of the interaction between CD47 and SIRPα comprises an extracellular truncation of SIRPα, and the extracellular truncation of SIRPα comprises an amino acid sequence selected from any one of the following a1)-a3): a1) SEQ ID No: 30; a2) SEQ ID No: 31; and a3) an amino acid sequence obtained by replacing the 80th position of any one of the above amino acid sequences by alanine to asparagine.

4. The bispecific recombinant protein according to any one of claims 1 to 3, It is characterized in that When the target is CD20, EGFR or PD-L1, the SIRPα extracellular truncation selects a1) or an amino acid sequence obtained by replacing the 80th position of the amino acid sequence with alanine to asparagine; when the target is HER2, the SIRPα extracellular truncation selects a1 or a2) or an amino acid sequence obtained by replacing the 80th position of the amino acid sequence with alanine to asparagine in any one of the above amino acid sequences.

5. The bispecific recombinant protein according to any one of claims 1 to 2, It is characterized in that The Fc region comprises a native Fc region sequence or a non-native Fc sequence; preferably, the Fc region is a human Fc region; more preferably, the Fc region is an Fc region of an IgG1, IgG2, IgG3 or IgG4 antibody.

6. The bispecific recombinant protein according to claim 5, It is characterized in that The Fc region contains a knobs-into-holes mutation; preferably, when the Fc region of the left arm contains a hole or holes mutation, the Fc region of the right arm contains a knob or knobs mutation; or when the Fc region of the left arm contains a knob or knobs mutation, the Fc region of the right arm contains a hole or holes mutation.

7. The bispecific recombinant protein according to claim 3, It is characterized in that The high-affinity tumor-targeting arm is coupled to the low-affinity fusion protein that blocks the interaction between CD47 and SIRPα via knobs-into-holes.

8. The bispecific recombinant protein according to any one of claims 1 to 5 and 7, It is characterized in that The fusion protein that blocks the interaction between CD47 and SIRPα with low affinity comprises an amino acid sequence selected from any one of the following b1)-b3): b1) SEQ ID No: 26; b2) SEQ ID No: 27; and b3) an amino acid sequence obtained by replacing the 99th amino acid sequence of any one of the above amino acid sequences by alanine to asparagine.

9. A nucleic acid molecule encoding the bispecific recombinant protein as described in any one of claims 1 to 8; preferably, the nucleic acid molecule encoding the high-affinity tumor-targeting arm and the nucleic acid encoding the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity are in the same DNA chain, or the nucleic acid molecule encoding the high-affinity tumor-targeting arm and the nucleic acid encoding the fusion protein that blocks the interaction between CD47 and SIRPα with low affinity are in different DNA chains.

10. An expression vector comprising the nucleic acid molecule of claim 9.

11. A host cell comprising the expression vector according to claim 10.

12. A method for preparing a bispecific recombinant protein, comprising using the cell according to claim 11 to express the recombinant protein; preferably, the cell is a eukaryotic cell; more preferably, the cell is a CHO cell.

13. Use of the bispecific recombinant protein according to any one of claims 1 to 8, or the nucleic acid molecule according to claim 9 in the preparation of a medicament for treating a blood tumor and a solid tumor; preferably, the blood tumor and the solid tumor are selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, cutaneous fibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome.

14. A method for in vitro evaluation of the early immune safety of a recombinant protein / antibody targeting CD47 and having ADCC activity, It is characterized in that The method comprises the following steps: a) Providing a recombinant protein / antibody (including monovalent or multivalent) targeting CD47 and having ADCC activity; b) detecting the ADCC activity of the recombinant protein / antibody; c) evaluating the early immune safety of the recombinant protein / antibody; Preferably, the method further comprises the following steps between steps a) and b): a1) preparing effector cells; a2) contacting effector cells with the recombinant protein / antibody; More preferably, the effector cells are NK cells; preferably, the effector cells are human NK92MI-CD16a effector cells; More preferably, in a1), the harvested effector cells are resuspended to a cell density of 1×10 5 cells / ml to 5×10 6 cells / ml; in a2), the gradiently diluted recombinant protein / antibody is incubated with the effector cells prepared in a1), and the incubation time is 0.5-5h; in b), the LDH activity is determined and the cell lysis rate is calculated; and / or, in c), the early immune safety of the recombinant protein / antibody is evaluated according to the cell lysis rate, wherein the recombinant protein / antibody that results in a lower cell lysis rate has a higher immune safety.

15. Method for in vivo evaluation of early immune safety of recombinant proteins / antibodies targeting CD47, It is characterized in that The method includes: a) providing a recombinant protein / antibody targeting CD47, wherein the recombinant protein / antibody is monovalent or multivalent; b) providing Hu-NSG mice; c) contacting the recombinant protein / antibody with Hu-NSG mice; d) evaluating the early immune safety of the recombinant protein / antibody in the Hu-NSG mice; Preferably, in c), 24-96 hours after administration, venous blood is collected from mice, red blood cells are lysed, and the remaining cells are incubated with fluorescently labeled anti-human CD45, anti-human CD19 or anti-human CD3 antibodies for 15-60 minutes, and flow cytometry is performed; in d), the early immune safety of the recombinant protein / antibody is evaluated based on the cell clearance rate, wherein the recombinant protein / antibody that results in a lower immune cell clearance rate has a higher immune safety.

16. A pharmaceutical composition comprising the bispecific recombinant protein according to any one of claims 1 to 8, or the nucleic acid molecule according to claim 9; Preferably, the pharmaceutical composition comprises a therapeutically effective amount of the bispecific recombinant protein or the nucleic acid molecule, and optionally a pharmaceutically acceptable carrier; More preferably, the pharmaceutical composition is in the form of an injection or a lyophilized powder; More preferably, the pharmaceutical composition is administered to the subject systemically or topically; preferably, the pharmaceutical composition is administered to the subject by inhalation, intranasal, oral, intravenous, subcutaneous or intramuscular administration.

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