Recombinant fusion proteins targeting CD40 and CD47

Abstract

Provided is a recombinant fusion protein comprising (a) an anti-CD40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, and (b) a CD47 binding domain. Nucleic acid molecules encoding the recombinant fusion proteins, vectors comprising the nucleic acid molecules, and host cells transformed or transfected with the vectors or nucleic acid molecules are provided. In addition, the invention also provides a production method of the recombinant fusion protein, medical application of the recombinant fusion protein and a kit containing the recombinant fusion protein.

Description

Merge by reference

[0001] This application claims priority to PCT application number PCT / CN2022 / 091058 filed on May 6, 2022.

[0002] All documents or references cited herein (including but not limited to all documents, patents, published patent applications cited herein) ("references cited herein"), and all documents cited or referenced in references cited herein, and manufacturer's instructions, descriptions, product specifications, and product pages for any products mentioned herein or in any document cited herein, are incorporated herein by reference and may be used in the practice of the present invention. Specifically, all cited documents are incorporated by reference to the same extent as each individual document is specifically and individually indicated to be incorporated by reference. Any Genbank sequence mentioned in this disclosure is incorporated by reference, and the Genbank sequence is the sequence corresponding to the earliest effective filing date of this disclosure. Technical Field

[0003] The present disclosure relates to a recombinant fusion protein that binds CD40 and CD47 with high affinity and functionality. The present disclosure also provides a nucleic acid molecule encoding the recombinant fusion protein, an expression vector, a host cell, and a method for expressing the recombinant fusion protein. In addition, the present disclosure also provides a pharmaceutical composition comprising the recombinant fusion protein and a treatment method using the recombinant fusion protein. Background Art CD40 and CD40L

[0004] CD8 + Cytotoxic T lymphocyte (CTL) responses play an important role in immune defense against cancer and infection, and effective CTL responses are triggered by the binding of antigen-specific T cell receptors to peptide-loaded major histocompatibility complex (MHC) on antigen-presenting cells, followed by the binding of co-stimulatory molecules (Zhu Y et al., (2011) Immunity. 34(4):466-478).

[0005] CD40 and its main ligand CD40L are a pair of co-stimulatory molecules. CD40 is a type I transmembrane protein that was originally identified as a cell surface marker on B lymphocytes and bladder tumor cells. It was later found to be expressed on antigen presenting cells such as macrophages, dendritic cells and monocytes, while CD40L is mainly expressed by activated CD4 +T cell expression (Paulie S et al., (1989) J Immunol 142: 590-595; Bereznaya NM et al., (2007) Exp Oncol. 29 (1): 2-12). When CD40 binds to CD40L, TRAF1 to TRAF6 are recruited to its cytoplasmic domain to drive signal transduction (Ma DY et al., (2009) Semin Immunol 21 (5): 265-272). CD40 signaling in dendritic cells may lead to upregulation of MHC molecules and co-stimulatory molecules, as well as increased levels of T cell stimulating cytokines (such as interleukin 12), thereby activating dendritic cells and alternatively driving CD8 + CD4 required for T cell response + T cell help" (Ara A et al. (2018) Immunotargets Ther. 7:55-61). Immune activation mediated by CD40 may be independent of innate immune receptors, such as Toll-like receptors (Byrne KT & Vonderheide RH (2016) Cell Rep. 15:2719-2732) and may convert cold tumors into hot tumors, making them sensitive to checkpoint inhibitor therapy (Vonderheide RH (2020) Annu Rev Med. 71:47-58).

[0006] CD40 expression has also been found in tumor cells, including B cell malignancies, melanoma, lung cancer, bladder cancer, gastric cancer, breast cancer and ovarian cancer. Studies have shown that CD40-CD40L interaction may inhibit tumor growth, such as melanoma and breast cancer (Von Leoprechting A et al., (1999) Cancer Res 59:1287-1294; Hirano A et al., (1999) Blood 93:2999-3007). It is reported that in some cases, CD40 signaling in tumor cells promotes tumor growth, but at least enhances antigen presentation of tumor cells.

[0007] Agonistic anti-CD40 antibodies have been developed for disease treatment. In a first-in-human single-dose study, selicrelumab (Pfizer and VLST) has shown clinical efficacy in patients with advanced melanoma (Vonderheide RH et al., (2007) J. Clin. Oncol. 25:876-883; Bajor DL ​​et al., (2014) Cancer Immunol. Res. 2:1051-1058).

[0008] Biological agents that activate CD40 signaling have also shown efficacy in the treatment of infectious diseases, including HIV / AIDS, tuberculosis, and malaria (Elizabeth A Thompson et al. (2015) J Immunol. 195(3):1015–1024). CD47 and SIRPα

[0009] CD47 is a transmembrane protein expressed on the surface of a variety of cells, playing a role in cell proliferation, migration and apoptosis (Ratnikova NM et al., (2017) Mol Biol. 51(2): 251-261). Its ligands include signal regulatory protein α (SIRPα), also known as CD172a or phosphatase substrate containing Src homology 2 domain-1, which is present in myeloid cells such as macrophages and dendritic cells. After CD47 binds to SIRPα, it sends a "don't eat me" signal to prevent macrophages from phagocytosing cells, so cancer cells use this immune tolerance mechanism to escape immune surveillance.

[0010] CD47 is overexpressed in a variety of tumor cells, including myeloma, leiomyosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma, melanoma, hepatocellular carcinoma, liver cancer, bile duct cancer, ovarian cancer, bladder cancer, pancreatic ductal adenocarcinoma and gastric cancer (including Epstein-Barr virus-associated gastric cancer), and is associated with cancer metastasis and poor prognosis (Zhang W et al., (2020) Front Immunol. 11:18).

[0011] Studies have shown that inhibiting CD47-SIRPα signaling may promote the secretion of chemical factors and cytokines, recruit more immune cells to tumor cells, induce more macrophage-mediated phagocytosis of tumor cells, and promote antigen-specific CD8 +T cell proliferation, reduction of regulatory T cell number, increase of antibody-dependent cytotoxicity (e.g., NK cell-mediated cytotoxicity), initiation of tumor cell apoptosis through caspase-independent mechanisms, thereby inhibiting tumor growth (Weiskopf K et al., (2016) J Clin Investig. 126: 2610-2620; Tseng D et al., (2013) Proc Natl Acad Sci USA. 110: 11103-11108; Kim MJ et al., (2008) Tumour Biol. 29: 28-34). Specifically, disruption of CD47-SIRPα interaction may promote phagocytosis of cancer cells by macrophages, and antigenic peptides produced by cancer cells during phagocytosis may subsequently initiate adaptive immune responses (Chen, J et al. (2017) 544: 493-497).

[0012] Therapeutic approaches targeting CD47, including anti-CD47 antibodies and SIRPα-Fc fusion proteins, are being tested in clinical trials for the treatment of solid and hematological tumors (Zhang W et al., (2020) supra). SIRPα-Fc fusion proteins can contain the full length of SIRPα, or one or more of its extracellular immunoglobulin superfamily domains.

[0013] CD47 upregulation and immunosuppression by CD47 have also been found in viral and bacterial infections, and blocking CD47 by anti-CD47 antibodies can promote the activation and effector function of macrophages, dendritic cells, and T cells (Cham LB et al., (2020) Antibodies (Basel) 9(3):44). Bispecific or multispecific fusion proteins

[0014] Since CD47 is ubiquitously expressed in human cells and highly expressed in hematopoietic cells, monospecific anti-CD47 antibodies or proteins may bind to normal cells, especially red blood cells that occupy half of the blood volume, causing adverse reactions, including severe anemia, and some clinical trials have been terminated as a result.

[0015] Bispecific or multispecific fusion proteins can be designed to bind to CD47 and another antigen (e.g., CD40) to more precisely target immune cells in tumors and / or microenvironments, thereby reducing or eliminating off-target effects.

[0016] Citation or identification of any document in this application does not constitute an admission that the document is available as prior art to the present invention. SUMMARY OF THE INVENTION

[0017] The inventors of the present application designed and prepared a recombinant fusion protein that can bind to CD40 and CD47, which exhibits a low aggregation level and has comparable or higher binding affinity to human or monkey CD40 protein, comparable or higher blocking activity against CD40-CD40L binding, comparable or higher blocking activity against CD47-SIRPα binding, comparable or higher agonistic activity against CD40 signaling, and comparable or higher induction activity against CD47 compared to monospecific counterparts and prior art antibodies (e.g., Selicrelumab). + The ability of cells to engulf.

[0018] The recombinant fusion proteins disclosed herein can be used in in vitro and in vivo experiments, and in the treatment of diseases associated with CD40 and / or CD47 signaling, such as tumors.

[0019] In one aspect, the present disclosure provides a recombinant fusion protein that can bind to CD40 and CD47, comprising an anti-CD40 antibody or an antigen-binding fragment thereof, and a CD47 binding domain.

[0020] The anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises two identical heavy chain variable regions and two identical light chain variable regions. In some embodiments, the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and optionally, the light chain constant region is connected to the C-terminus of the light chain variable region.

[0021] The anti-CD40 antibody or antigen-binding fragment thereof can be a full-length antibody, a Fab fragment, a F(ab') 2 fragment, Fd fragment or Fv fragment.

[0022] The anti-CD40 antibody or antigen-binding fragment thereof has agonistic activity on CD40 signaling.

[0023] The heavy chain variable region of the anti-CD40 antibody or antigen-binding fragment thereof comprises VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 comprise the amino acid sequences shown in SEQ ID NO:1, 2 and 3, respectively. The heavy chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:7. The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7. The heavy chain constant region may be a heavy chain constant region of IgG1, IgG2 or IgG4, or a functional fragment thereof, such as an Fc fragment. The heavy chain constant region may be naturally occurring or may be engineered to have certain desired properties. In some embodiments, the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2 or IgG4 or a functional fragment thereof, having an amino acid sequence as shown in SEQ ID NO:9.

[0024] The light chain variable region of the anti-CD40 antibody or antigen-binding fragment thereof comprises VL CDR1, VL CDR2 and VLCDR3, and the VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NO:4, 5 and 6, respectively. The light chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:8. The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8. The light chain constant region can be the constant region of a kappa and lambda chain. In some embodiments, the light chain constant region has the amino acid sequence shown in SEQ ID NO:10.

[0025] The anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively. The heavy chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8. In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.

[0026] In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises two identical heavy chains and two identical light chains.

[0027] The heavy chain of the anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a heavy chain constant region, wherein the heavy chain variable region and the heavy chain constant region consist of the above amino acid sequence. The heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 19. The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 19.

[0028] The light chain of the anti-CD40 antibody or antigen-binding fragment thereof comprises a light chain variable region and an optional light chain constant region, wherein the light chain variable region and the light chain constant region consist of the above amino acid sequence. The light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 20. The light chain comprises the amino acid sequence shown in SEQ ID NO: 20.

[0029] The heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19, and the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 19, and the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 20.

[0030] The CD47 binding domain is human SIRPα or a fragment thereof. In some embodiments, the CD47 binding domain may be the first Ig-like extracellular domain of human SIRPα. In some embodiments, the CD47 binding domain is a wild-type or mutant SIRPα isoform 2 (SIRPαV2). In some embodiments, the CD47 binding domain is the first Ig-like extracellular domain of SIRPαV2 (SIRPαV2D1). In some embodiments, SIRPαV2D1 is wild-type and has an amino acid sequence as shown in SEQ ID NO: 11 (X1 = V, X2 = K, X3 = S, X4 = K, X5 = F). In some embodiments, SIRPαV2 is a variant with higher CD47 binding affinity / ability, having an amino acid sequence as shown in SEQ ID NO: 11 (X1 = I, X2 = R, X3 = T, X4 = K, X5 = F; or X1 = I, X2 = R, X3 = T, X4 = R, X5 = V).

[0031] The CD47 binding domain can be connected to the N-terminus or C-terminus of the anti-CD40 antibody or its antigen-binding fragment. In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region connected to the C-terminus of the heavy chain variable region, a light chain constant region connected to the C-terminus of the light chain variable region, and a CD47 binding domain connected to the N-terminus of the heavy chain variable region or the light chain variable region or to the C-terminus of the heavy chain constant region or the light chain constant region. In some embodiments, the CD47 binding domain is connected to the N-terminus of the heavy chain variable region, or to the C-terminus of the heavy chain constant region or the light chain constant region. In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the CD47 binding domain is connected to the N-terminus or C-terminus of the heavy chain, or to the N-terminus or C-terminus of the light chain. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises two identical heavy chains and two identical light chains, and the CD47 binding domain is linked to the N-terminus or C-terminus of each heavy chain or to the N-terminus or C-terminus of each light chain. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises two identical heavy chains and two identical light chains, and the CD47 binding domain is linked to the N-terminus or C-terminus of each heavy chain or to the C-terminus of each light chain.

[0032] In some embodiments, the CD47 binding domain can be connected to the anti-CD40 antibody or antigen-binding fragment thereof through a linker. In some embodiments, the CD47 binding domain is connected to the heavy chain variable region or the light chain variable region through a linker. In some embodiments, the CD47 binding domain is connected to the N-terminus of the heavy chain variable region or the light chain variable region through a linker. In some embodiments, the CD47 binding domain is connected to the heavy chain constant region or the light chain constant region through a linker. In some embodiments, the CD47 binding domain is connected to the C-terminus of the heavy chain constant region or the light chain constant region through a linker. In some embodiments, the CD47 binding domain is connected to the heavy chain or the light chain through a linker. In some embodiments, the CD47 binding domain is connected to the N-terminus or C-terminus of the heavy chain, or to the C-terminus of the light chain through a linker. The linker can be a short peptide chain consisting of 5-20 amino acid residues. In some embodiments, the linker can be a GS linker having an amino acid sequence as shown in SEQ ID NO: 12, 13, 14 or 15.

[0033] In some embodiments, the recombinant fusion protein of the present disclosure comprises: i) an anti-CD40 antibody heavy chain variable region-heavy chain constant region-linker-SIRPαV2D1 polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 16; and an anti-CD40 antibody light chain variable region-light chain constant region polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 20; ii) SIRPαV2D1-linker-anti-CD40 antibody heavy chain variable region-heavy chain constant region polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 17 (X1=V, X2=K, X3=S, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=R, X5=V); and an anti-CD40 antibody light chain variable region-light chain constant region polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 20; or iii) an anti-CD40 antibody heavy chain variable region-heavy chain constant region polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 19; and an anti-CD40 antibody light chain variable region-light chain constant region-linker-SIRPαV2D1 polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 18.

[0034] The recombinant fusion protein disclosed herein comprises: i) a first polypeptide chain and a second polypeptide chain comprising an anti-CD40 antibody heavy chain variable region, a heavy chain constant region, and SIRPαV2D1, and a third polypeptide chain and a fourth polypeptide chain comprising an anti-CD40 antibody light chain variable region and optionally a light chain constant region; or ii) a first polypeptide chain and a second polypeptide chain comprising an anti-CD40 antibody heavy chain variable region and a heavy chain constant region, and a third polypeptide chain and a fourth polypeptide chain comprising an anti-CD40 antibody light chain variable region, an optional light chain constant region, and SIRPαV2D1; The heavy chain variable region of the first polypeptide chain and the light chain variable region of the third polypeptide chain associate to form a CD40 binding domain, the heavy chain variable region of the second polypeptide chain and the light chain variable region of the fourth polypeptide chain associate to form a CD40 binding domain, and the heavy chain constant region of the first polypeptide chain associates with the heavy chain constant region of the second polypeptide chain.

[0035] In some embodiments, the recombinant fusion protein comprises: i) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus, an anti-CD40 antibody heavy chain variable region, a heavy chain constant region, and SIRPαV2D1, and a third polypeptide chain and a fourth polypeptide chain comprising an anti-CD40 antibody light chain variable region; ii) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus, SIRPαV2D1, an anti-CD40 antibody heavy chain variable region and a heavy chain constant region, and a third polypeptide chain and a fourth polypeptide chain comprising an anti-CD40 antibody light chain variable region; or iii) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus, a heavy chain variable region and a heavy chain constant region of an anti-CD40 antibody, and a third polypeptide chain and a fourth polypeptide chain comprising a light chain variable region of an anti-CD40 antibody and SIRPαV2D1; The heavy chain variable region of the first polypeptide chain and the light chain variable region of the third polypeptide chain associate to form a CD40 binding domain, the heavy chain variable region of the second polypeptide chain and the light chain variable region of the fourth polypeptide chain associate to form a CD40 binding domain, and the heavy chain constant region of the first polypeptide chain associates with the heavy chain constant region of the second polypeptide chain.

[0036] In some embodiments, the recombinant fusion protein of the present disclosure comprises: i) from N-terminus to C-terminus, the first and second polypeptide chains comprising the heavy chain variable region, the heavy chain constant region, and SIRPαV2D1 of an anti-CD40 antibody, and from N-terminus to C-terminus, the third and fourth polypeptide chains comprising the light chain variable region and the light chain constant region of an anti-CD40 antibody; ii) from N-terminus to C-terminus, the first and second polypeptide chains comprising the heavy chain variable region and the heavy chain constant region of an anti-CD40 antibody, and from N-terminus to C-terminus, the third and fourth polypeptide chains comprising the light chain variable region and the light chain constant region of an anti-CD40 antibody; iii) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus, a heavy chain variable region and a heavy chain constant region of an anti-CD40 antibody, and a third polypeptide chain and a fourth polypeptide chain comprising, from N-terminus to C-terminus, a light chain variable region, and SIRPαV2D1 of an anti-CD40 antibody; wherein the heavy chain variable region of the first polypeptide chain and the light chain variable region of the third polypeptide chain associate to form a CD40 binding domain, the heavy chain variable region of the second polypeptide chain and the light chain variable region of the fourth polypeptide chain associate to form a CD40 binding domain, and the heavy chain constant region of the first polypeptide chain associates with the heavy chain constant region of the second polypeptide chain.

[0037] In some embodiments, the recombinant fusion protein of the present disclosure comprises: i) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus, an anti-CD40 antibody heavy chain variable region, a heavy chain constant region, a linker, and SIRPαV2D1, and a third polypeptide chain and a fourth polypeptide chain comprising, from N-terminus to C-terminus, an anti-CD40 antibody light chain variable region and a light chain constant region; ii) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus, SIRPαV2D1, a linker, a heavy chain variable region and a heavy chain constant region of an anti-CD40 antibody, and a third polypeptide chain and a fourth polypeptide chain comprising, from N-terminus to C-terminus, a light chain variable region and a light chain constant region of an anti-CD40 antibody; iii) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus, a heavy chain variable region and a heavy chain constant region of an anti-CD40 antibody, and a third polypeptide chain and a fourth polypeptide chain comprising, from N-terminus to C-terminus, a light chain variable region, a light chain constant region, a linker, and SIRPαV2D1 of an anti-CD40 antibody; The heavy chain variable region of the first polypeptide chain and the light chain variable region of the third polypeptide chain associate to form a CD40 binding domain, the heavy chain variable region of the second polypeptide chain and the light chain variable region of the fourth polypeptide chain associate to form a CD40 binding domain, and the heavy chain constant region of the first polypeptide chain associates with the heavy chain constant region of the second polypeptide chain.

[0038] In some embodiments, the recombinant fusion protein of the present disclosure comprises: i) a first polypeptide chain and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 16, and a third polypeptide chain and a fourth polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 20; ii) a first polypeptide chain and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17 (X1 = V, X2 = K, X3 = S, X4 = K, X5 = F; or X1 = I, X2 = R, X3 = T, X4 = K, X5 = F; or X1 = I, X2 = R, X3 = T, X4 = R, X5 = V), and a third polypeptide chain and a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 20; or iii) a first polypeptide chain and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 19, and a third polypeptide chain and a fourth polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 18.

[0039] The present application also provides a nucleic acid molecule encoding the recombinant fusion protein of the present disclosure, an expression vector comprising the nucleic acid molecule, and a host cell transformed or transfected with the expression vector or nucleic acid molecule. The present disclosure also provides a method for preparing the recombinant fusion protein of the present disclosure using a host cell, comprising the following steps: (i) expressing the recombinant fusion protein in a host cell; (ii) isolating the recombinant fusion protein from the host cell or its culture medium.

[0040] The present application also provides a pharmaceutical composition, which comprises the recombinant fusion protein, nucleic acid molecule, expression vector or host cell described in the present disclosure, and a pharmaceutically acceptable carrier. The pharmaceutical composition may also comprise an additive, such as an anti-tumor agent.

[0041] In a second aspect, the present application provides a method for treating a disease related to CD40 and / or CD47 signaling in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure to the subject.

[0042] The disease can be cancer. The cancer can be a solid tumor or a hematological tumor, including but not limited to leiomyosarcoma, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, colon cancer, renal cancer, prostate cancer, cervical cancer, nasopharyngeal cancer, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, lung cancer (including small cell lung cancer and non-small cell lung cancer), multiple myeloma, melanoma, hepatocellular carcinoma, liver cancer, bile duct cancer, ovarian cancer, bladder cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma) and gastric cancer. In some embodiments, the pharmaceutical composition described in the present disclosure can be used in combination with chemotherapy and checkpoint inhibitors, such as anti-PD-L1 antibodies, anti-PD-1 antibodies or anti-CTLA-4 antibodies.

[0043] The disease may be an infectious disease. The infectious disease may be caused by bacterial, viral or parasitic infection.

[0044] In some embodiments, the subject is a human.

[0045] In a third aspect, the present application provides a method for enhancing an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the present disclosure.

[0046] In some embodiments, the subject is a human.

[0047] The present application also provides a method for reversing or alleviating immunosuppression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described in the present disclosure.

[0048] In some embodiments, the subject is a human.

[0049] The present application also provides use of the pharmaceutical composition disclosed herein in treating diseases associated with CD40 and / or CD47 signaling, enhancing immune response, and / or reversing or reducing immunosuppression.

[0050] In a fourth aspect, the present application provides a kit comprising a recombinant fusion protein, pharmaceutical composition, nucleic acid molecule, expression vector or host cell described in the present disclosure. The term "kit" refers to two or more components packaged in a container, a receiver or other device, one of which corresponds to the recombinant fusion protein, pharmaceutical composition, nucleic acid molecule, expression vector or host cell described in the present disclosure. Therefore, a kit can be described as a set of products and / or tools that can achieve a specific goal and can be sold as a single unit.

[0051] The kit comprises one or more containers of any suitable shape, size, material (preferably waterproof, such as plastic or glass), such as vials, ampoules, containers, syringes, bottles, bags, containing the recombinant fusion protein or pharmaceutical composition described in the present disclosure. The kit may also include instructions for use (e.g., in the form of a leaflet or instruction sheet).

[0052] Other features and advantages of the present disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank records, patents, and published patent applications cited in this application are expressly incorporated herein by reference.

[0053] Therefore, it is not the intention of the present invention to include within the scope of the present invention any previously known product, process for making a product, or method for using a product, and the applicants therefore reserve the right and hereby disclaim any previously known product, process, or method. In addition, it should also be noted that the present invention does not intend to include within the scope of the present invention any product, process, process for making a product, or method for using a product that does not meet the written description and enforceability requirements of the United States Patent and Trademark Office (USPTO) (35 U.S.C. §112, first paragraph) or the European Patent Office (EPO) (Article 83 of the EPC), and the applicants therefore reserve the right and hereby disclaim any previously described product, process for making a product, or method for using a product. In the practice of the present invention, it may be advantageous to comply with Article 53(c) of the European Patent Convention and Articles 28(b) and (c) of the European Patent Convention. All rights to any embodiments of any granted patents involved that are expressly rejected in the present application family or any other family or any third party's prior application are expressly reserved. Nothing in this application shall be construed as a commitment.

[0054] It is worth noting that in the present disclosure, especially in the claims and / or paragraphs, terms such as "comprises, comprised, comprising" and the like have the meanings assigned by the U.S. Patent Law, for example, they may mean "includes, included, including", etc. Terms such as "mainly comprised", "mainly comprised" and the like have the meanings assigned by the U.S. Patent Law, for example, allow the existence of elements not explicitly described, but exclude elements found in the prior art or elements that affect the basic or novel nature of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The following detailed description is given by way of example and is not intended to limit the invention to the particular embodiments described and is best understood in conjunction with the accompanying drawings.

[0056] Figure 1 .Schematic diagram of the structure of the recombinant fusion protein.

[0057] Figure 2 . Flow cytometry was used to detect the binding ability of the recombinant fusion protein disclosed in the present invention to human CD40 expressed on the surface of 293T-CD40 cells.

[0058] Figure 3 . Flow cytometry was used to detect the binding ability of the recombinant fusion protein disclosed in the present invention to human CD47 expressed on the surface of 293F-CD47 cells.

[0059] Figure 4 .Competition ELISA was used to detect the ability of the recombinant fusion protein described in the present disclosure to block the binding between human CD40 and human CD40L.

[0060] Figure 5 .Competition ELISA was used to detect the ability of the recombinant fusion protein described in the present disclosure to block the binding of human CD47 to human CD172a.

[0061] Figure 6 . Flow cytometry was used to detect the ability of the recombinant fusion protein described in the present disclosure to block the binding of human SIRPα to human CD47 on the cell surface.

[0062] Figure 7 . Flow cytometry was used to detect the ability of the recombinant fusion protein described in the present disclosure to block the binding of human SIRPα to human CD47 on the cell surface.

[0063] Figure 8 . A cell-based reporter gene assay was used to detect the CD40 agonistic activity of the recombinant fusion protein described in the present disclosure.

[0064] Fig. 9. A cell-based assay was used to detect the ability of the recombinant fusion protein disclosed in the present invention to induce macrophages to phagocytose human Jurkat cancer cells.

[0065] Fig.10 . Double antibody sandwich ELISA was used to detect the binding ability of the recombinant fusion protein described in the present disclosure to human CD40 and CD47. DETAILED DESCRIPTION

[0066] In order to better understand the present disclosure, some terms are first defined. Other definitions are listed throughout the detailed description.

[0067] The term "CD40" refers to cluster of differentiation 40. The term "CD40" encompasses variants, isoforms, homologs, orthologs, and paralogs. For example, in certain cases, an antibody specific for human CD40 protein may cross-react with CD40 protein from species other than human (e.g., monkey). In other embodiments, an antibody specific for human CD40 protein may be completely specific for human CD40 protein and not cross-react with other species or other types of proteins, or may cross-react with CD40 from certain specific species but not all other species.

[0068] The term "human CD40" refers to a CD40 protein having an amino acid sequence from humans, such as the human CD40 amino acid sequence of NCBI No. NP_001241.1 (Sasaki K et al., (2021) J Exp Clin Cancer Res 40(1):212). The term "monkey CD40" or "cynomolgus CD40" refers to a CD40 protein having an amino acid sequence from monkeys.

[0069] The term "SIRPα" refers to wild-type signal regulatory protein α, or a recombinant or non-recombinant polypeptide having wild-type signal regulatory protein α, or a natural or naturally occurring allelic variant of signal regulatory protein α, or an artificial variant of signal regulatory protein α. In one embodiment, SIRPα refers to wild-type mammalian SIRPα. In a preferred embodiment, SIRPα refers to wild-type human SIRPα. The term "human SIRPα" refers to a SIRPα protein having an amino acid sequence from humans, such as an amino acid sequence with GenBank accession number AAH75849.1 (Strausberg RL et al., (2002) Proc. Natl. Acad. Sci. USA 99 (26): 16899-16903). In one embodiment, SIRPα comprises a signal sequence; in other embodiments, SIRPα refers to the mature form of the protein. Currently, 10 human SIRPα alleles have been discovered, and it is reported that human SIRPα isoform 2 (or V2) has a reduced or lowest binding affinity to red blood cells.

[0070] The term "bispecific" as used herein refers to a binding molecule that is a fusion protein comprising at least a first and a second binding domain, wherein the first binding domain is capable of binding to an antigen or target, and the second binding domain is capable of binding to another antigen or target. Therefore, the bispecific fusion protein described in the present application comprises binding specificities for two different antigens or targets and is at least bispecific. The "bispecific fusion protein" described in the present application also includes multispecific binding molecules, such as trispecific binding molecules, which comprise three binding domains. The bispecific fusion protein described in the present application is also envisioned to have more functions in addition to binding to target molecules CD40 and CD47.

[0071] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines and complement), which result in selective damage, destruction or elimination of pathogens that invade the human body, cells or tissues infected by pathogens, cancer cells, or normal human cells or tissues in autoimmune or pathological inflammatory conditions.

[0072] The term "immunosuppression" refers to a decrease in the activity or effectiveness of the immune system due to age, ongoing illness, malnutrition, cancer, chemotherapy or radiation therapy, etc. In certain circumstances, immunosuppression can be reversed, for example by modulating certain pathways.

[0073] The term "antibody" as used herein refers to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., CD40) through at least one antigen binding site, wherein the antigen binding site is generally located within the variable region of the immunoglobulin molecule. The antibodies described herein include intact polyclonal antibodies, intact monoclonal antibodies, single-chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs), light chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, fusion proteins comprising an antigen binding site of an antibody, and any other modified immunoglobulin molecule (e.g., dual variable domain immunoglobulin molecule) comprising an antigen binding site, as long as the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are called alpha, delta, epsilon, gamma, and mu, respectively, based on the characteristics of their heavy chain constant regions. Different types of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or bound to other molecules, including but not limited to toxins and radioisotopes. Unless otherwise expressly stated, the term "antibody" as used herein includes the "antigen-binding portion" of a complete antibody. Conventional IgG is a glycoprotein comprising two identical heavy chains (H) and two identical light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region CH. The heavy chain constant region comprises three domains, namely CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region CL. The light chain constant region contains one domain, the CL. The VH and VL regions can be further divided into hypervariable regions, also known as complementarity determining regions (CDRs), which are separated by more conserved framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. A "functional fragment" of the heavy chain constant region refers to a portion of the constant region that retains the desired properties, such as binding affinity to Fc receptors and / or complement system proteins.

[0074] As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD40 protein). The antigen-binding function of an antibody has been demonstrated to be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab') 2 Fragments include: (i) a bivalent fragment of two Fab fragments connected by a disulfide bond in the hinge region; (ii) a Fd fragment consisting of a VH and CH1 domain; (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity determining region (CDR); and (viii) a nanobody, i.e., a heavy chain variable region containing a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, recombinant methods can be used to connect the two domains through a synthetic linker to form a single protein chain, in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and these fragments are functionally screened in the same manner as intact antibodies.

[0075] "Agonistic" anti-CD40 antibodies or antigen-binding fragments thereof, or "agonistic activity" of CD40 signaling, means that the anti-CD40 antibodies or antigen-binding fragments thereof bind to CD40 and activate / induce CD40 signaling, such as promoting the activation and proliferation of immune cells and the production of cytokines and chemokines. Agonistic anti-CD40 antibodies or antigen-binding fragments thereof can enhance the antigen presentation ability of APCs, activate tumor-specific CD4 + and CD8 + T cells, lymphocytes and monocytes secrete cytokines and chemokines, enhance the killing of tumor cells by cytotoxic lymphocytes and NK cells, etc., and promote the innate and adaptive immune responses of tumor patients to tumors.

[0076] As used herein, a recombinant fusion protein or bispecific fusion protein that "specifically binds to CD40 (e.g., human CD40)" refers to a protein that binds to CD40 protein (human CD40 and possibly CD40 protein from one or more non-human species), but does not substantially bind to non-CD40 proteins. Similarly, a fusion protein that "specifically binds to CD47 (e.g., human CD47)" refers to a protein that binds to human or non-human species CD47 protein but does not substantially bind to non-CD47 proteins. Preferably, the fusion protein binds to CD40 and CD47 with "high affinity", i.e., K D 5.0×10 -8 M or lower, more preferably, K D 1.0×10 -8 M or lower.

[0077] As used herein, the term "substantially not binding" a protein or cell means not binding to the protein or cell, or not binding to the protein or cell with high affinity, i.e., the K of binding to the protein or cell is less than 1. D 1.0×10 -6 M or higher, preferably 1.0×10 -5 M or more, more preferably 1.0×10 -4 M or more, more preferably 1.0×10 -3 M or more, even more preferably 1.0×10 -2 M or higher.

[0078] The term "high affinity" refers to the K D 1.0×10 -6 M or less, preferably 1.0×10 -8 M or less, more preferably 6.0×10 -9 M or less, even more preferably 1.0×10 -9 M or lower.

[0079] The term "K assoc ” or “K a " refers to the binding rate of a specific protein-protein interaction, such as an antibody-antigen or receptor-ligand interaction. As used herein, the term "K dis ” or “K d " refers to the dissociation rate of a specific protein-protein interaction. As used herein, the term "K D ” refers to the dissociation constant, which is given by K d With K a The ratio of Kd to Ka is obtained and expressed as molar concentration (M). D The value can be determined using methods known in the art. A preferred method for determining antibody K DA preferred method is to use surface plasmon resonance, more preferably a biosensor system such as Biacore TM system.

[0080] The term “EC 50 ", also known as half-maximal effect concentration, refers to the concentration of the fusion protein of the present disclosure that induces a response that is half of the baseline level and the maximum value after a specific exposure time.

[0081] The term "IC 50 "," also known as half maximal inhibitory concentration, refers to the concentration of the fusion protein of the present disclosure that inhibits a specific biological or biochemical function by 50% compared to the absence of the fusion protein.

[0082] The term "subject" includes humans or non-human animals. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, reptiles, etc. Preferred mammals are mice, rats, non-human primates, sheep, dogs, cats, cattle and horses.

[0083] The term "therapeutically effective amount" refers to an amount of the fusion protein disclosed herein that is sufficient to prevent or improve symptoms associated with a disease or condition (e.g., cancer), and / or reduce the severity of a disease or condition. A therapeutically effective amount should be understood to be related to the condition being treated, and those skilled in the art can easily discern the actual effective amount.

[0084] The "identity" percentage of two or more nucleic acids or polypeptides used herein refers to two or more sequences or subsequences when comparing and aligning (if necessary, introducing gaps) to achieve maximum correspondence, the sequences or subsequences are exactly the same or have a specified percentage of identical nucleotides or amino acid residues, and conservative amino acid substitutions may be considered as part of sequence identity, or may not be considered as part of sequence identity. The identity percentage can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignment results are well known in the art. These include but are not limited to BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and variants thereof. In some embodiments, two nucleic acids or polypeptides of the present disclosure are substantially identical, which means that when using a sequence comparison algorithm or by visual inspection to compare and align to obtain maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90% nucleotide or amino acid residue identity, and in some embodiments have at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity.

[0085] It is noted that, unless the context clearly indicates otherwise, singular forms used herein encompass plural references. Thus, for example, reference to "an agent" includes one or more such different agents, and reference to "the method" includes equivalent steps and methods known to those of ordinary skill in the art, which steps and methods may be modified or substituted for the methods described herein.

[0086] Various aspects of the disclosure are described in further detail below.

[0087] The recombinant fusion protein of the present disclosure comprises: (a) the anti-CD40 antibody or antigen-binding fragment thereof, and (b) a CD47 binding domain. The CD47 binding domain may be linked to the anti-CD40 antibody or antigen-binding fragment thereof, optionally via a linker.

[0088] The recombinant fusion proteins disclosed herein show low aggregation levels and have comparable or higher binding affinity to human and monkey CD40 proteins, comparable or higher blocking activity against CD40-CD40L binding, comparable or higher blocking activity against CD47-SIRPα binding, comparable or higher agonistic activity against CD40 signaling, and comparable or higher induction activity against CD47 compared to monospecific counterparts and existing antibodies (e.g., Selicrelumab). + The ability of cells to engulf (e.g., cancer cells, including Jurkat cells or HL-60 cells).

[0089] The three components in the recombinant fusion protein described in the present application are a CD47 binding domain, a linker, and an anti-CD40 antibody or an antigen-binding fragment thereof. One of ordinary skill in the art will recognize that the above three components can have a variety of designs. The CD47 binding domain can be connected to, for example, the N-terminus or C-terminus of an anti-CD40 antibody or an antigen-binding fragment thereof. Preferably, human sequences are used in the treatment of human cancers because the strong immunogenicity of proteins or peptides from non-human animals may cause allergies and other adverse reactions. However, depending on the purpose of different applications, other animal proteins or peptides may also be used in this application and humanized where appropriate.

[0090] The CD47 binding domain can be any protein or peptide that binds to CD47, such as SIRPα, a SIRPα variant, or an affinity-optimized SIRPα variant. A "variant" of SIRPα is defined as one or more amino acids that change the amino acid sequence of SIRPα compared to wild-type SIRPα. Variants can have "conservative" changes, in which the substituted amino acids have similar structures or chemical properties, such as replacing leucine with isoleucine. More rarely, variants can have "non-conservative" changes, such as replacing glycine with tryptophan. Similar minor changes can also include amino acid deletions or insertions, or both. In one embodiment, the SIRPα variant comprises a polypeptide having at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with wild-type SIRPα.

[0091] In some embodiments, the CD47 binding domain may be the first Ig-like extracellular domain of SIRPαV2 (SIRPαV2D1). SIRPαV2D1 may be a wild-type SIRPαV2D1 or a SIRPαV2D1 variant engineered to have a higher CD47 binding affinity / ability. In some embodiments, SIRPαV2D1 may be a wild-type comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 11 (X1=V, X2=K, X3=S, X4=K, X5=F). In some embodiments, SIRPαV2D1 may be a wild-type comprising an amino acid sequence as shown in SEQ ID NO: 11 (X1=V, X2=K, X3=S, X4=K, X5=F). In some embodiments, SIRPαV2D1 may be a SIRPαV2D1 variant comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 11 (X1=I, X2=R, X3=T, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=R, X5=V). In some embodiments, SIRPαV2D1 may be a SIRPαV2D1 variant comprising an amino acid sequence as shown in SEQ ID NO: 11 (X1=I, X2=R, X3=T, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=R, X5=V). A "variant" of SIRPαV2D1 is defined as one in which the amino acid sequence of SIRPαV2D1 has one or more amino acid changes compared to wild-type SIRPαV2D1. A variant may have "conservative" changes, in which the substituted amino acids have similar structural or chemical properties, such as replacing leucine with isoleucine. More rarely, variants may have "non-conservative" changes, such as replacing glycine with tryptophan. Similar minor changes may also include amino acid deletions or insertions, or both. In one embodiment, the SIRPαV2D1 variant comprises a polypeptide having at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to wild-type SIRPαV2D1.

[0092] The linker mainly serves as a spacer between the CD47 binding domain and the anti-CD40 antibody or its antigen-binding fragment. The linker can be composed of amino acids connected by peptide bonds, preferably a linker composed of 5 to 30 amino acids connected by peptide bonds, or 10 to 30 amino acids connected by peptide bonds, or 10 to 20 amino acids connected by peptide bonds, or 15 amino acids connected by peptide bonds, wherein the amino acids are selected from 20 natural amino acids. It will be appreciated by those skilled in the art that one or more of these amino acids may be glycosylated. In one embodiment, 5 to 30 amino acids may be selected from glycine, alanine, proline, asparagine, glutamine, serine and lysine. In one embodiment, the linker is mainly composed of amino acids without steric hindrance, such as glycine and alanine. Exemplary linkers are polyglycine (particularly (Glys, poly (Gly-Ala)) and polyalanine, such as -GGGGS-(SEQ ID NO: 12), -GGGGSGGGGS-(SEQ ID NO: 13), -GGGGSGGGGSGGGGS-(SEQ ID NO: 14), and -GGGGSGGGGSGGGGSGGGGS-(SEQ ID NO: 15). The linker can also be a non-peptide linker. For example, alkyl linkers such as -NH-, -(CH 2 )sC(O)-, wherein s=2-20. These alkyl linkers can also be replaced by any non-sterically hindered groups such as lower alkyl groups (e.g., C 1-4 ), lower acyl, halogen (such as CI, Br), CN, NH 2 In other embodiments, the recombinant fusion protein of the present application can be assembled without a linker.

[0093] The anti-CD40 antibody may be an isolated monoclonal antibody, as disclosed in WO2021 / 197335. The anti-CD40 antibody or antigen-binding fragment thereof described in the present disclosure may be humanized.

[0094] The anti-CD40 antibodies or antigen-binding fragments thereof disclosed herein comprise a heavy chain variable region and a light chain variable region. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments thereof comprise two identical heavy chain variable regions and two identical light chain variable regions. In some embodiments, the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and optionally, the light chain constant region is connected to the C-terminus of the light chain variable region.

[0095] The anti-CD40 antibodies or antigen-binding fragments thereof described in the present disclosure comprise a heavy chain and a light chain. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments thereof described in the present disclosure comprise two identical heavy chains and two identical light chains. The heavy chain may comprise a heavy chain variable region and a heavy chain constant region, and the light chain may comprise a light chain variable region and an optional light chain constant region.

[0096] The heavy chain variable region of the present disclosure comprises VH CDR1, VH CDR2 and VH CDR3, wherein the VH CDR1, VH CDR2 and VH CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3. The heavy chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 7.

[0097] The heavy chain constant region can be a heavy chain constant region of human IgG1, IgG2 or IgG4, optionally modified to have altered functional properties, such as altered Fc receptor binding affinity. The heavy chain constant region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 9.

[0098] The light chain variable region of the present disclosure comprises VL CDR1, VL CDR2 and VL CDR3, wherein the VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively. The light chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 8. The light chain constant region can be the constant region of a human kappa or lambda light chain. The light chain constant region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 10.

[0099] The heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region comprises VL CDR1, VL CDR2 and VLCDR3, wherein VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively. The heavy chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. The heavy chain constant region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9, and the light chain constant region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:10. In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10.

[0100] The recombinant fusion protein disclosed herein comprises: i) a first polypeptide chain and a second polypeptide chain comprising an anti-CD40 antibody heavy chain variable region, a heavy chain constant region, and SIRPαV2D1, and a third polypeptide chain and a fourth polypeptide chain comprising an anti-CD40 antibody light chain variable region and optionally a light chain constant region; or ii) a first polypeptide chain and a second polypeptide chain comprising an anti-CD40 antibody heavy chain variable region and a heavy chain constant region, and a third polypeptide chain and a fourth polypeptide chain comprising an anti-CD40 antibody light chain variable region, an optional light chain constant region, and SIRPαV2D1; The heavy chain variable region of the first polypeptide chain and the light chain variable region of the third polypeptide chain associate to form a CD40 binding domain, the heavy chain variable region of the second polypeptide chain and the light chain variable region of the fourth polypeptide chain associate to form a CD40 binding domain, and the heavy chain constant region of the first polypeptide chain associates with the heavy chain constant region of the second polypeptide chain.

[0101] The CD47 binding domain can be connected to the N-terminus or C-terminus of the anti-CD40 antibody or its antigen-binding fragment. In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, the light chain constant region is connected to the C-terminus of the light chain variable region, and the CD47 binding domain is connected to the N-terminus of the heavy chain variable region or the light chain variable region or to the C-terminus of the heavy chain constant region or the light chain constant region. In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the CD47 binding domain is connected to the N-terminus or C-terminus of the heavy chain of the anti-CD40 antibody or its antigen-binding fragment, or to the N-terminus or C-terminus of the light chain of the anti-CD40 antibody or its antigen-binding fragment.

[0102] The recombinant fusion protein described in the present disclosure can be modified by modifying one or more residues in one or two variable regions (i.e., VH and / or VL) of an anti-CD40 antibody or an antigen-binding fragment thereof, such as one or more residues in one or more CDR regions and / or one or more framework regions. Additionally or alternatively, the recombinant fusion protein can be modified by modifying residues in the constant region of the anti-CD40 antibody or an antigen-binding fragment thereof, such as to change the effector function of the recombinant fusion protein. In addition, the recombinant fusion protein can be modified at residues in the CD47 binding domain to change the binding affinity or other functional properties to CD47.

[0103] In some embodiments, CDR grafting can be used to modify the variable region of an anti-CD40 antibody or its antigen-binding fragment. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). Therefore, the amino acid sequences within the CDRs of each antibody are more diverse than the sequences outside the CDRs. Since CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific natural antibody can be expressed by constructing an expression vector in which the CDR sequence of a specific natural antibody is transplanted to the framework region sequence of another antibody with different properties (see Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad. See also USA 86:10029-10033; US Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0104] Therefore, another embodiment of the present disclosure relates to a recombinant fusion protein comprising an anti-CD40 antibody heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3 sequences, which comprise the sequences described above in the present disclosure, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3 sequences, which comprise the sequences described above in the present disclosure. Although these fusion proteins contain the CDR sequences of the heavy chain variable region and the light chain variable region of the anti-CD40 antibody of the present disclosure, they may contain different framework sequences.

[0105] Such framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. For example, germline DNA sequences of human heavy chain variable region and light chain variable region genes can be obtained in the "VBase" human germline sequence database; for another example, germline DNA sequences of human heavy chain variable region and light chain variable region genes can be obtained in the Genbank database.

[0106] The antibody protein sequence is compared to compiled protein sequence databases using Gapped BLAST (Altschul et al., (1997), supra), a sequence similarity search method well known to those skilled in the art.

[0107] The framework sequences used for the anti-CD40 antibodies of the present disclosure are preferably those that are structurally similar to the framework sequences of the antibodies of the present disclosure. The VH CDR1, VH CDR2 and VH CDR3 sequences can be transplanted to a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequence can be transplanted to a framework region that contains one or more mutations compared to the germline sequence. For example, in certain cases, mutations of residues within the framework region have been found to be beneficial for maintaining or enhancing the antigen binding ability of the antibody (see US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0108] In addition, the anti-CD40 antibody or antigen-binding fragment thereof of the recombinant fusion protein of the present disclosure can be engineered to include modifications within the Fc region, or as an alternative to modifications made within the framework or CDR region, generally to alter one or more functional properties of the recombinant fusion protein, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular cytotoxicity. In addition, the recombinant fusion protein of the present disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the fusion protein) or modified to alter its glycosylation, thereby again altering one or more functional properties.

[0109] In one embodiment, the CH1-hinge region is modified so that the number of cysteine ​​residues in the hinge region is changed, for example, increased or decreased. This method is described in more detail in patent US5,677,425. Changing the number of cysteine ​​residues in the CH1-hinge region can, for example, promote the assembly of light chains and heavy chains, or increase or decrease the stability of the antibody.

[0110] In another embodiment, the Fc-hinge region of the anti-CD40 antibody or antigen-binding fragment thereof in the recombinant fusion protein disclosed herein is mutated to change the biological half-life of the recombinant fusion protein. Specifically, in another embodiment, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge region so that the binding ability of the fusion protein to Staphylococcus aureus protein A (SpA) is weakened relative to the binding ability of the native Fc hinge region to SpA. This method is described in more detail in U.S. Patent No. 6,165,745.

[0111] The amino acid changes near the junction of the Fc region and the non-Fc region can significantly increase the serum half-life of the Fc fusion protein. Therefore, the connection region of the recombinant fusion protein of the present disclosure can change relative to the naturally occurring immunoglobulin heavy chain sequence, preferably within about 10 amino acids at the junction. These amino acid changes can lead to increased hydrophobicity. In one embodiment, the constant region C-terminal lysine residue derived from the IgG sequence is replaced. Preferably, the C-terminal lysine of the IgG sequence is replaced with a non-lysine amino acid (e.g., alanine or leucine) to further increase the serum half-life.

[0112] In another embodiment, the glycosylation of the anti-CD40 antibody or antigen-binding fragment thereof in the recombinant fusion protein of the present disclosure can be altered. For example, the affinity of the anti-CD40 antibody or antigen-binding fragment thereof for the antigen can be increased by altering the glycosylation. Such sugar modification can be achieved, for example, by altering one or more glycosylation sites within the sequence of the anti-CD40 antibody or antigen-binding fragment thereof. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site. Such deglycosylation can increase the affinity of the antibody for the antigen. See US Pat. Nos. 5,714,350 and 6,350,861.

[0113] Another modification of the anti-CD40 antibody or antigen-binding fragment thereof in the recombinant fusion protein of the present disclosure is pegylation. For example, the antibody can be pegylated to increase the biological (e.g., serum) half-life of the antibody. The term "polyethylene glycol" as used herein is intended to encompass any form of PEG used to produce other proteins, such as mono(C 1 -C 10 ) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. Methods for pegylation of proteins are known in the art and can be applied to the antibodies described in the present disclosure. See EP 0,154,316 and EP 0,401,384.

[0114] The CD47 binding domain in the recombinant fusion protein disclosed herein can be modified to have a higher CD47 binding affinity. In some embodiments, the SIRPαV2D1 variant comprises one or more mutations within the SIRPαV2D1 region compared to wild-type SIRPαV2D1. Surface plasmon resonance (SPR) can be used to determine the binding affinity of the SIRPαV2D1 variant to CD47. Alternative cell binding assays can also be used to determine the binding affinity of the SIRPαV2D1 variant to CD47. When mutations are introduced into SIRPαV2D1 or the recombinant fusion protein disclosed herein, the resulting variant or fusion protein has sufficient SIRPα biological activity and can be used as a therapeutic protein. In some embodiments, the biological activity of the SIRPαV2D1 variant is at least 0.01 times, 0.03 times, 0.06 times, 0.1 times, 0.3 times, 1 times, 3 times, 5 times, 6 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times or 100 times that of wild-type SIRPαV2D1 or a fusion protein comprising wild-type SIRPαV2D1. The biological activity of the SIRPαV2D1 variant can be detected by in vitro or in vivo experiments. In vitro detection methods for determining the biological activity of SIRPαV2D1 on cells expressing CD47 have been established in the art. For example, as described by Liu et al. (J.Mol.Bio., 365:680, 2007), the biological activity can be determined by a leukocyte migration assay.

[0115] On the other hand, the present disclosure provides a nucleic acid molecule encoding the recombinant fusion protein of the present disclosure. In some embodiments, the nucleic acid molecule provided by the present disclosure encodes: i) an anti-CD40 antibody heavy chain variable region-heavy chain constant region-linker-SIRPαV2D1 polypeptide chain, which comprises the amino acid sequence shown in SEQ ID NO: 16, and an anti-CD40 antibody light chain variable region-light chain constant region polypeptide chain, which comprises the amino acid sequence shown in SEQ ID NO: 20; ii) SIRPαV2D1-linker-anti-CD40 antibody heavy chain variable region-heavy chain constant region polypeptide chain, which comprises the amino acid sequence shown in SEQ ID NO: 17 (X1=V, X2=K, X3=S, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=R, X5=V), and an anti-CD40 antibody light chain variable region-light chain constant region polypeptide chain, which comprises the amino acid sequence shown in SEQ ID NO: 21. NO:20; or iii) an anti-CD40 antibody heavy chain variable region-heavy chain constant region polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:19, and an anti-CD40 antibody light chain variable region-light chain constant region-linker-SIRPαV2D1 polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:18. The nucleic acid may be present in whole cells, in cell lysates, or in a partially purified or substantially pure form. When the nucleic acid is purified from other cellular components or other contaminants (such as other cellular nucleic acids or proteins) by standard techniques, the nucleic acid is "isolated" or "substantially pure". The nucleic acid described in the present disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a DNA molecule.

[0116] The nucleic acids described in the present disclosure can be obtained using standard molecular biology techniques. For example, the nucleic acid molecules described in the present disclosure can be chemically synthesized.

[0117] The present disclosure also provides an expression vector comprising the nucleic acid molecules described in the present disclosure. Vectors include but are not limited to plasmids, viral vectors, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), transforming artificial chromosomes (TAC), mammalian artificial chromosomes (MAC) and human artificial additional chromosomes (HAEC). The present disclosure also provides host cells transformed or transfected with the expression vectors or nucleic acids described in the present disclosure. Suitable host cells include Escherichia coli, yeast and other eukaryotic organisms. In one embodiment, the DNA encoding the polypeptide chain of the recombinant fusion protein described in the present disclosure is inserted into one or more expression vectors so that these genes are operably connected to transcription and translation regulatory sequences. In this article, the term "operably connected" means that the encoding nucleotides are connected to the vector so that the transcription and translation control sequences in the vector play their established functions of regulating and controlling the transcription and translation of the recombinant fusion protein genes.

[0118] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control nucleotide transcription or translation. Such regulatory sequences have been described in an article by Goeddel et al. (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus, such as adenovirus major late promoter (AdMLP) and polyoma enhancer. Alternatively, non-viral regulatory sequences such as ubiquitin promoters or β-globin promoters may be used. In addition, regulatory elements composed of sequences from different sources can also be used, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472). The choice of expression vector and expression regulatory sequence should be compatible with the expression host cell used.

[0119] In addition to the nucleotides and regulatory sequences encoding the recombinant fusion protein, the expression vector of the present invention can also carry other sequences, such as sequences (e.g., replication origins) and selective marker genes that regulate the replication of the vector in the host cell. The selective marker gene helps to select the host cell into which the vector has been introduced (see US Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, the selective marker gene usually confers resistance to drugs (e.g., G418, hygromycin or methotrexate) to the host cell into which the vector is introduced. Preferred selective marker genes include dihydrofolate reductase (DHFR) genes (for selection / amplification of methotrexate in DHFR-host cells) and neo genes (for G418 selection).

[0120] In order to express the peptide chains constituting the recombinant fusion protein, the expression vector encoding the peptide chains is transfected into the host cell by standard techniques. The term "transfection" covers a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although the recombinant fusion protein described in the present disclosure can be expressed in prokaryotic or eukaryotic host cells in theory, it is most preferred to express it in eukaryotic cells, especially in mammalian host cells. Because eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete correctly folded and immunologically active recombinant fusion proteins than prokaryotic cells.

[0121] Preferred mammalian host cells for expressing the recombinant fusion proteins of the present disclosure include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, see Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker, see, for example, RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells and SP2 cells. Particularly for use with NSO myeloma cells, another preferred expression system is the GS gene expression system, described in WO 87 / 04462, WO 89 / 01036 and EP 338,841. When the recombinant expression vector is introduced into a mammalian host cell, the recombinant fusion protein is prepared by culturing the host cell for a sufficient period of time to allow the recombinant fusion protein to be expressed in the host cell, or more preferably, to allow the recombinant fusion protein to be secreted into the culture medium in which the host cell is grown. The recombinant fusion protein can be recovered from the culture medium using standard protein purification methods.

[0122] On the other hand, the present disclosure provides a pharmaceutical composition comprising the recombinant fusion protein, nucleic acid molecule, expression vector, and / or host cell of the present disclosure formulated with a pharmaceutically acceptable carrier. When the pharmaceutical composition contains more than one recombinant fusion protein, nucleic acid molecule, expression vector or host cell, the recombinant fusion protein, nucleic acid molecule, expression vector and / or host cell can be administered separately. The pharmaceutical composition may optionally contain one or more additional active pharmaceutical ingredients, such as another antibody or drug, such as an anti-tumor drug.

[0123] The pharmaceutical composition may contain any number of excipients. Available excipients include carriers, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, and combinations thereof. The selection and use of suitable excipients is taught in Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), edited by Gennaro, the disclosure of which is incorporated herein by reference.

[0124] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient can be wrapped in a certain material to protect it from the influence of acid and other natural conditions that may inactivate it. The phrase "parenteral administration" used herein refers to, except for enteral and topical administration, a mode of administration usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. In addition, the pharmaceutical composition described in the present disclosure can be used by non-parenteral route, such as local, epidermal or mucosal route, such as intranasal, oral, vaginal, rectal, sublingual or topical.

[0125] The pharmaceutical composition can be in the form of a sterile aqueous solution or dispersion and can also be formulated as a microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0126] The "therapeutically effective dose" of the recombinant fusion protein, nucleic acid molecule, expression vector or host cell disclosed herein is preferably an amount that causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom periods, or the prevention of damage or disability caused by the disease. For example, for the treatment of subjects with tumors, a "therapeutically effective dose" is preferably an amount that inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and more preferably at least about 80%, relative to untreated subjects. The therapeutically effective amount of the therapeutic recombinant fusion protein, nucleic acid molecule, expression vector or host cell disclosed herein can reduce tumor size, or otherwise improve symptoms in a subject, who is typically a human or other mammal.

[0127] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. For example, see "Sustained and Controlled Release Drug Delivery Systems", edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0128] The therapeutic composition can be administered by medical devices, such as (1) needle-free subcutaneous injection devices (U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 and 4,596,556); (2) microinfusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion devices (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0129] The pharmaceutical compositions comprising the recombinant fusion proteins, nucleic acid molecules, expression vectors or host cells disclosed herein have various in vitro and in vivo uses, for example, involving the treatment of diseases associated with CD40 and / or CD47 signaling, such as tumors or infectious diseases.

[0130] The present disclosure provides a method for treating a disease associated with CD40 signaling and / or CD47 signaling, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition described in the present disclosure.

[0131] The disease can be a tumor or cancer. The tumor includes but is not limited to leiomyosarcoma, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, colon cancer, renal cancer, prostate cancer, cervical cancer, nasopharyngeal carcinoma, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, lung cancer (including small cell lung cancer and non-small cell lung cancer), multiple myeloma, melanoma, hepatocellular carcinoma, liver cancer, bile duct cancer, ovarian cancer, bladder cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma) and gastric cancer. In some embodiments, the subject is a person.

[0132] The disease can be an infectious disease. The infectious disease can be caused by bacterial, viral or parasitic infection. In some embodiments, the subject is a human.

[0133] In another aspect, the present disclosure provides a method for regulating or enhancing an immune response in a subject in need thereof, comprising administering the pharmaceutical composition of the present disclosure to the subject, such that the immune response of the subject is regulated / enhanced.

[0134] The present disclosure also provides a method of reversing or alleviating immunosuppression in a subject in need thereof, comprising administering the pharmaceutical composition of the present disclosure to the subject.

[0135] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

[0136] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting.All figures and all references, Genbank sequences, patents and published patent applications cited in this application are expressly incorporated herein by reference. Examples Example Example 1 Construction of an exemplary recombinant fusion protein

[0137] A recombinant fusion protein is constructed by connecting the first Ig-like extracellular domain of human SIRPα isoform 2 (SIRPαV2D1), optionally through a linker, to the N-terminus or C-terminus of the heavy chain, or the C-terminus of the light chain, of a humanized anti-CD40 antibody having a human IgG2 heavy chain constant region (as described in WO2021 / 197335, named HuCD40-C1H1-V2). The SIRPαV2D1 is wild-type, having an amino acid sequence as shown in SEQ ID NO: 11 (X1 = V, X2 = K, X3 = S, X4 = K, X5 = F); or a variant mutated to increase binding affinity to CD47, having an amino acid sequence as shown in SEQ ID NO: 11 (X1 = I, X2 = R, X3 = T, X4 = K, X5 = F; or X1 = I, X2 = R, X3 = T, X4 = R, X5 = V). The anti-CD40 antibody HuCD40-C1H1-V2 is an IgG antibody having two identical heavy chains and two identical light chains, wherein each heavy chain comprises a heavy chain variable region and a heavy chain constant region from N-terminus to C-terminus, and each light chain comprises a light chain variable region and a light chain constant region from N-terminus to C-terminus, wherein the heavy chain variable region, the light chain variable region, the heavy chain constant region and the light chain constant region comprise the amino acid sequences shown in SEQ ID NOs: 7, 8, 9 and 10, respectively, wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the VH CDR1, VH CDR2 and VH CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region comprises VLCDR1, VL CDR2 and VL CDR3, and the VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0138] The specific structure and sequence ID of the exemplary recombinant fusion protein are shown in Tables 1 and Figure 1 shown. Table 1. Molecular structures and amino acid sequence IDs of exemplary recombinant fusion proteins Example 2 Expression and identification of exemplary recombinant fusion proteins

[0139] In brief, nucleic acids encoding each polypeptide chain of the recombinant fusion protein were first synthesized and inserted into pTT5 vectors, respectively. The plasmid DNA of the vector was then transfected into mammalian cells (CHO cells), and the CHO cells expressed and secreted the exemplary recombinant fusion protein of the present disclosure. The recombinant fusion protein was then purified by protein A affinity chromatography column, and analyzed by SDS-PAGE and SEC-HPLC, and the results are shown in Tables 2-1 and 2-2. Table 2-1 SDS-PAGE results of exemplary recombinant fusion proteins sample purity(%) BSI038×S-001 >90％ BSI038×S-002 >90％ BSI038×S-003 >90％ BSI038×S-004 >90％ BSI038×S-005 >90％ Table 2-2 SEC-HPLC results of exemplary recombinant fusion proteins sample monomer(%) BSI038×S-001 98.76 BSI038×S-002 99.70 BSI038×S-003 99.53 BSI038×S-004 98.94 BSI038×S-005 95.89

[0140] It can be seen from the results that the recombinant fusion protein disclosed in the present invention has a high purity, and most of the fusion protein exists in monomeric form. Example 3 Using BIACORE surface plasmon resonance to detect the binding affinity of exemplary recombinant fusion proteins force

[0141] The binding affinity and binding kinetics of the purified recombinant fusion proteins were characterized using the Biacore T200 system (GE healthcare, Pittsburgh, PA, USA). Anti-CD40 antibody HuCD40-C1H1-V2 (having heavy and light chains as shown in SEQ ID NOs: 19 and 20), Selicrelumab (Roche's anti-CD40 antibody agonist, prepared in-house, having heavy and light chains as shown in SEQ ID NOs: 27 and 28), SIRPα-Fc fusion proteins (including SIRPαV2D1M1-Fc (IgG2) (SEQ ID NO: 21, X1 = I, X2 = R, X3 = T, X4 = K, X5 = F), SIRPαV2D1M2-Fc (IgG2) (SEQ ID NO: 21, X1 = I, X2 = R, X3 = T, X4 = R, X5 = V) and SIRPα-isoform2-Fc (SEQ ID NO: 25) were used as positive controls.

[0142] Protein A chip (Cat#: 29-1275-56, GE healthcare) was used for affinity determination. The recombinant fusion protein of the present disclosure and the positive control at a concentration of 2 μg / ml were flowed through the chip at a flow rate of 10 μL / min. Then, the starting concentration was 200 nM and HBS-EP was used. + Recombinant human CD40-his protein (Cat#:CD0-H5228, Acro biosystems), human CD47-his protein (Cat#:CD7-H5227, Acrobiosystems), cynomolgus CD40-his protein (Biosion homemade, SEQ ID NO:29) or cynomolgus CD47-his protein (Cat#:CD7-C52H1, Acro biosystems) diluted 2 times in buffer (provided by Biacore) flowed through the chip at a flow rate of 30 μl / min. The binding kinetics were tracked for 2 minutes and the dissociation kinetics were tracked for 10 minutes. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore evaluation software to determine KD , K a and K d The values ​​are summarized in Tables 3-1, 3-2, 3-3 and 3-4 below. Table 3-1 Binding affinity of recombinant fusion protein to human CD40 Table 3-2 Binding affinity of recombinant fusion protein to human CD47 BDL:Below Detection Limit Table 3-3 Binding affinity of recombinant fusion protein to cynomolgus monkey CD40 Table 3-4 Binding affinity of recombinant fusion protein to cynomolgus monkey CD47

[0143] All recombinant fusion proteins disclosed herein specifically bind to human CD40 and cynomolgus CD40 with high binding affinity.

[0144] Furthermore, the recombinant fusion proteins disclosed in the present invention also specifically bind to human CD47 and cynomolgus monkey CD47, wherein the CD47 binding affinity of BSI038×S-004 and BSI038×S-005 is comparable to that of SIRPαV2D1M1-Fc (IgG2) and SIRPαV2D1M2-Fc (IgG2), and is superior to that of BSI038×S-002. Example 4 Binding Activity of Exemplary Recombinant Fusion Proteins

[0145] Using 293T-CD40 cells stably expressing full-length human CD40 (uniprot#P25942-1) produced by Biosion, the binding activity of the recombinant fusion protein described in the present disclosure to human CD40 was further determined by flow cytometry (FACS). According to the instructions of lipofectamine 3000 transfection reagent (Thermo Fisher), the pCMV-TP plasmid with DNA encoding human CD40 (uniprot#P25942-1) inserted between the EcoRI and XbaI sites was transfected into 293T cells to prepare 293T-CD40 cells.

[0146] Briefly, 293T-CD40 cells were collected from cell culture flasks, washed twice, and resuspended in phosphate-buffered saline (PBS) containing 2% v / v fetal bovine serum (FACS buffer). Then, 1×10 5293T-CD40 cells were incubated on ice for 40 minutes with 100 μL of gradient dilutions of the recombinant fusion protein or control described in the present disclosure (starting concentration was 10 μg / mL, and 5-fold gradient dilutions were performed using FACS buffer). The cells were washed twice with FACS buffer, and 100 μL of goat anti-human IgG (Fab)-PE (diluted 1:1000 using FACS buffer, Cat#:109-116-097, Jackson ImmunoResearch) was added to each well. After incubation at 4°C in the dark for 40 minutes, the cells were washed three times with FACS buffer and resuspended with FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS device, and graphs were drawn based on the concentration of the recombinant fusion protein or control. The data were analyzed using Graphpad Prism and EC was obtained. 50 The result is Figure 2 shown.

[0147] Using 293F-CD47 cells stably expressing full-length human CD47 (NCBI# NP_942088.1) produced by Biosion, the binding activity of the recombinant fusion protein described in the present disclosure to human CD47 was further determined by flow cytometry (FACS). According to the instructions of lipofectamine 3000 transfection reagent (Thermo Fisher), pCMV-TP plasmid with DNA encoding human CD47 (NP_942088.1) inserted between EcoRI and XbaI sites was transfected into 293F cells to prepare 293F-CD47 cells (Thermo Fisher).

[0148] 293F-CD47 cells were collected from the cell culture flask, washed twice, and resuspended in phosphate-buffered saline (PBS) containing 2% v / v fetal bovine serum (FACS buffer). Then, 1×10 5293T-CD40 cells were incubated on ice for 40 minutes with 100 μL of gradient dilutions of the recombinant fusion protein or control described in the present disclosure (starting concentration was 10 μg / mL, and 5-fold gradient dilutions were performed using FACS buffer). The cells were washed twice with FACS buffer, and 100 μL of goat anti-human IgG (Fc)-PE (diluted 1:1000 using FACS buffer, Cat#:109-115-098, Jackson ImmunoResearch) was added to each well. After incubation at 4°C in the dark for 50 minutes, the cells were washed three times with FACS buffer and resuspended with FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS device, and a mean fluorescence intensity graph was prepared based on the concentration of the recombinant fusion protein or control. The data was analyzed using Graphpad Prism and EC was obtained. 50 The result is Figure 3 shown.

[0149] from Figure 2 It can be seen that the recombinant fusion protein described in the present disclosure specifically binds to human CD40.

[0150] from Figure 3 It can be seen that the recombinant fusion protein described in the present disclosure specifically binds to human CD47. The binding activity of BSI038×S-002 to human CD47 is comparable to that of the positive control SIRPa isoform 2-Fc.

[0151] Double antibody sandwich ELISA was used to detect the binding activity of the recombinant fusion protein to human CD40 and CD47. In brief, human CD40-Fc protein (Cat#: CD0-H5253, ACRO Biosystems) was diluted to 0.2 μg / mL using PBS solution, coated in 96-well plates with 100 μL per well, and incubated overnight at 2-8°C. The next day, the 96-well plates were washed 3 times with washing buffer (PBS + 0.05% v / v Tween-20, PBST), and then blocked at 37°C for 3 hours with blocking buffer (PBS + 1% w / v BSA). Finally, the 96-well plates were washed 3 times with washing buffer.

[0152] The recombinant fusion protein or control described in the present disclosure was diluted with blocking buffer (initial concentration of 100 nM, 6-fold gradient dilution) and added to a 96-well plate coated with CD40-Fc, 100 μL per well, and incubated at 37°C for 1 hour. The 96-well plate was washed 3 times with washing buffer, and then 100 μL of human CD47-his protein (Cat#: CD7-H5227, ACRO Biosystems) at a concentration of 3 μg / mL was added to each well and incubated at 37°C for 1 hour. The 96-well plate was washed again with washing buffer, and 100 μL of anti-His tag antibody (HRP) (diluted 1:2000 with blocking buffer, Cat#: 105327-MM02T-H, Sino Biological) was added to each well and incubated at 37°C for 1 hour. The 96-well plate was washed again with washing buffer. Finally, TMB was added to color the 96-well plate, and 1M H was added. 2 SO 4 Terminate the reaction. Read the absorbance at 450 nm using a microplate reader, and then plot the OD (450) value versus the recombinant fusion protein or control concentration. Analyze the data using Graphpad Prism and derive the EC 50 The result is Fig.10 shown.

[0153] from Fig.10 It can be seen that the recombinant fusion protein described in the present disclosure specifically binds to human CD40 and CD47, and has higher binding activity than SIRPα-Fc-CD40L. Example 5 Exemplary recombinant fusion protein blocking activity of CD40-CD40L or CD47-SIRPα binding 5.1 Ligand blocking ELISA to detect inhibition of CD40-CD40L binding

[0154] Competitive ELISA was used to detect the ability of the recombinant fusion protein described in the present disclosure to block CD40-CD40L binding. Briefly, 2 μg / mL human CD40-Fc protein (prepared in-house, amino acid sequence is SEQ ID NO: 22) prepared in coating buffer (carbonate / bicarbonate buffer) was coated on a 96-well plate at 100 μL per well and incubated overnight at 4°C. The next day, the plate was washed once with washing buffer (PBS+0.05% v / v Tween-20, PBST), then blocked with 5% w / v skim milk in PBST at 37°C for 2 hours, and then washed 4 times with washing buffer.

[0155] The recombinant fusion protein or control described in the present disclosure was diluted with PBST containing 2.5% w / v skim milk (starting concentration of 10 μg / mL, 5-fold gradient dilution), and 100 μL per well was added to a 96-well plate coated with CD40-Fc, and incubated at 37°C for 40 minutes. The plate was washed 4 times with wash buffer, and then 100 μL of 95 ng / mL biotin-labeled human CD40L-his protein (Cat#:10239-H08E, Sino Biological) was added to each well and incubated at 37°C for 40 minutes. The plate was washed again with wash buffer, and then 100 μL of HRP-coupled streptavidin (diluted 1:10000 with PBST buffer, Cat#:016-030-084, Jackson ImmunoResearch) was added to each well, incubated at 37°C for 40 minutes, and the plate was washed again with wash buffer. Finally, TMB was added to develop the color of the 96-well plate, and then 1M H 2 SO 4 The reaction was terminated. The absorbance was read in the dual wavelength mode of the microplate reader, with TMB at 450 nm and the reference wavelength at 630 nm. A curve was then drawn with OD (450-630) as the ordinate and the recombinant fusion protein or control concentration as the abscissa. The data were analyzed using Graphpad Prism and the IC 50 The result is Figure 4 shown. 5.2 Ligand blocking ELISA to detect inhibition of CD47-CD172a binding

[0156] Competitive ELISA was used to detect the ability of the recombinant fusion protein described in the present disclosure to block CD47-CD172a binding. In brief, 2 μg / mL human CD172a-Fc protein (prepared internally, amino acid sequence is SEQ ID NO: 24) configured with coating buffer (carbonate / bicarbonate buffer) was coated on a 96-well plate with 100 μL per well and incubated overnight at 4°C. The next day, the plate was washed once with washing buffer (PBS+0.05% v / vTween-20, PBST), then blocked at 37°C for 2 hours with 5% w / v skim milk configured with PBST, and then washed 4 times with washing buffer.

[0157] The recombinant fusion protein or control described in the present disclosure was diluted in a gradient (starting concentration of 100 nM, 5-fold gradient dilution) and mixed with 20 ng / mL biotin-labeled human CD47-Fc (Biosion homemade, SEQ ID NO: 23) in PBST containing 2.5% w / v skim milk. After incubation at 37°C for 40 minutes, 100 μL per well was added to a 96-well plate coated with CD172a-Fc. After incubation at 37°C for 40 minutes, the plate was washed 4 times with washing buffer, and then 100 μL HRP-coupled streptavidin (diluted 1:10000 with PBST buffer, Cat#: 016-030-084, Jackson ImmunoResearch) was added to each well. After incubation at 37°C for 40 minutes, the plate was washed again with washing buffer. Finally, TMB was added to color the 96-well plate, and then 1M H 2 SO 4 The reaction was terminated. The absorbance was read in the dual wavelength mode of the microplate reader, with TMB at 450 nm and the reference wavelength at 630 nm. A curve was then drawn with OD (450-630) as the ordinate and the recombinant fusion protein or control concentration as the abscissa. GraphpadPrism was used to analyze the data and derive the IC 50 Wild-type SIRPαV2D1-Fc (IgG2) (SEQ ID NO: 21, X1 = V, X2 = K, X3 = S, X4 = K, X5 = F) was used as a positive control. Figure 5 shown. 5.3 Ligand blocking ELISA to detect inhibition of CD47-SIRPα binding

[0158] The activity of the recombinant fusion protein described in the present disclosure in blocking the binding of SIRPα to cell surface CD47 was detected by flow cytometry using homemade 293F-CD47 cells.

[0159] Briefly, according to the instructions of lipofectamine 3000 transfection reagent (Thermo Fisher), pCMV-TP plasmid with DNA encoding human CD47 (NP_942088.1) inserted between EcoRI and XbaI sites was transfected into 293F cells to prepare 293F-CD47 cells.

[0160] The recombinant fusion protein or control described in the present disclosure was diluted in a gradient manner using PBS (FACS buffer) containing 2% v / v fetal bovine serum (initial concentration was 100 nM, 3-fold gradient dilution). At the same time, 293F-CD47 cells in the logarithmic growth phase were harvested, washed twice with FACS buffer, and then centrifuged and collected. 1×10 5The cells were seeded in a 96-well plate containing 100 μL / well diluted antibody or control solution and incubated at 4°C for 60 minutes. The plate was washed twice, and then 100 μL of 148 ng / mL biotin-labeled human CD172a-Fc protein (prepared in-house, amino acid sequence is SEQ ID NO: 24) prepared with FACS buffer was added to each well and incubated at 4°C for 60 minutes. The plate was washed twice with FACS buffer, and then 100 μL of R-phycoerythrin-streptavidin (diluted 1:500 with FACS buffer, Cat#: 016-110-084, JacksonImmunoResearch) was added to each well and incubated in the dark at 4°C for 40 minutes. The cells were washed twice and resuspended in FACS buffer. Fluorescence was detected using a Becton Dickinson FACS Canto II-HTS device, and a curve graph was drawn with MFI (mean fluorescence intensity) as the ordinate and recombinant fusion protein or control concentration as the abscissa. Data were analyzed using Graphpad Prism and IC was derived 50 The result is Figure 6 and Figure 7 shown.

[0161] from Figure 4 It can be seen that the recombinant fusion protein disclosed in the present disclosure is able to block the binding of human CD40 to human CD40L, and its activity is comparable to that of the positive control.

[0162] from Figure 5 It can be seen that BSI038×S-002 can block the binding of human CD47 to human CD172a, and its activity is higher than that of wild-type SIRPαV2D1-Fc (IgG2). In addition, BSI038×S-004 and BSI038×S-005 can block the binding of human CD47 to human CD172a, and their activity is higher than that of positive control SIRPαV2D1M1-Fc (IgG2) and SIRPαV2D1M2-Fc (IgG2).

[0163] also, Figure 6 and Figure 7 The results showed that the recombinant fusion proteins described in the present disclosure were able to block the binding of human SIRPα to human CD47 on the cell surface, and most of them had blocking activity comparable to that of the positive control. In addition, BSI038×S-004 and BSI038×S-005 exhibited higher CD47-SIRPα blocking activity than BSI038×S-002. Example 6 Exemplary cell-based functional detection of recombinant fusion proteins 6.1 Detection of CD40 agonist activity in a cell-based reporter gene assay

[0164] The CD40 agonistic activity of the recombinant fusion protein described in the present disclosure was detected using 293T-NF-κB-Luc-CD40 reporter cells stably expressing full-length human CD40 (uniprot No. P25942-1). According to the instructions of lipofectamine 3000 transfection reagent (Thermo Fisher), the pGL4.32[luc2P / NF-κB-RE / Hygro] vector and the pCMV-TP plasmid with DNA encoding human CD40 inserted between EcoRI and XbaI sites were transfected into 293F cells to prepare 293T-NF-κB-Luc-CD40 cells. When the CD40 agonist contacts these cells, the CD40 signal will be activated, and the expression of luciferase will be upregulated by the NF-κB transcription factor, which can be measured by fluorescence analysis. The recombinant fusion protein SIRPα-Fc-CD40L having the amino acid sequence shown in SEQ ID NO: 26 was used as a control (reference K, Patel A et al., CD40 Enhances Type I Interferon Responses Downstream of CD47 Blockade, Bridging Innate and Adaptive Immunity. Cancer Immunol Res. 2020 Feb; 8(2): 230-245).

[0165] Briefly, 293T-NF-κB-Luc-CD40 cells in the logarithmic growth phase were resuspended in DMEM medium (Cat#:10566-016, Gibco) containing 10% FBS (Cat#:10099-141, Gibco) and added to 384-well plates (Cat#:3707, Corning) at 20 μL / well, with 5×10 3 Then, 20 μL of the recombinant fusion protein or control described in the present disclosure (initial concentration is 200 nM, 3-fold gradient dilution using culture medium) was added to each well and incubated at 37°C for 6 hours. Then, 30 μL of ONE-Glo was added to each well. TM Luciferase Assay Reagent, incubate at room temperature for 5 minutes. 200Pro was used to detect chemiluminescence. Graphpad Prism was used to analyze the data and calculate the EC 50 value.

[0166] The results are as follows Figure 8 shown.

[0167] It can be seen that BSI038×S-002, BSI038×S-004 and BSI038×S-005 exhibited CD40 agonistic activity comparable to that of HuCD40-C1H1-V2, and much higher than that of Selicrelumab and SIRPa-Fc-CD40L. 6.2 Ability to induce phagocytosis of cancer cells

[0168] SIRPα-isoform2-Fc, SIRPαV2D1M1-Fc (IgG2), SIRPαV2D1M2-Fc (IgG2), SIRPα-Fc-CD40L and wild-type SIRPαV2D1-Fc (IgG2) were used as controls to further detect the ability of the recombinant fusion protein described in the present disclosure to induce phagocytosis of cancer cells.

[0169] Briefly, EasySep without CD16 depletion was used according to the instructions. TM Monocytes were isolated from frozen human PBMCs using a human monocyte enrichment kit (Cat#: 19058, Stemcell) and plated in 6-well plates containing RPMI1640 + 10% FBS + 1% penicillin-streptomycin + 75 ng / ml human M-CSF for 6 days, with fresh cell culture medium added on day 3. Macrophages were isolated on day 6 and re-plated in 96-well plates and cultured overnight.

[0170] CFSE-labeled Jurkat cells were harvested and incubated at room temperature for 30 minutes with 50 μL of gradient dilutions of the recombinant fusion protein or control described in the present disclosure at a density of 400,000 / mL. The Jurkat cell / antibody mixture was then added to the above-collected macrophages at a ratio of effector cells: target cells = 1:2, and co-cultured at 37°C for 4 hours.

[0171] use All cells were collected from the plate using cell detachment buffer and washed once with FACS buffer. TM The cells were blocked with Fc receptor blocking solution (Cat#: 422302, Biolegend) and then stained with anti-human CD11b APC (Cat#: 301310, Biolegend). Flow cytometry was used to determine the phagocytic rate (%), i.e., CFSE + Groups and CD11b + The proportion of the population (containing phagocytic CFSE + CD11b + Macrophage count / CD11b + total number of macrophages).

[0172] The results are as follows Fig. 9 shown.

[0173] It can be seen that BSI038×S-002, BSI038×S-004, and BSI038×S-005 can all induce macrophages to phagocytize tumor cells, and their activity is better than SIRPα-Fc-CD40L. Example 7 Thermal Stability of Exemplary Recombinant Fusion Proteins

[0174] The thermal stability of the recombinant fusion protein was tested. Briefly, GloMelt TM The Thermal Shift Protein Stability Assay Kit performs protein thermal shift analysis to determine the Tm value (melting temperature). TM The dye was thawed and returned to room temperature, and the vial containing the dye was vortexed and centrifuged. Then, 10× dye was prepared by adding 5 μL 200× dye to 95 μL PBS. Then 2 μL 10× dye and 10 μg of the recombinant fusion protein or control described in the present disclosure were added, and PBS was added to a total reaction volume of 20 μL. Finally, the tube containing the dye and fusion protein or control was briefly centrifuged and placed in a real-time PCR thermal cycler (Roche, LightCycler 480II), and the melting curve program was set according to the parameters of Table 4. Table 4 Melting curve program parameters Procedure temperature gradient Keep Time Initial temperature 25℃ NA 30s Melting curve 25-99℃ 0.1℃ / s NA Table 5 Melting temperature of recombinant fusion protein

[0175] The results are shown in Table 5, and the recombinant fusion protein will be stable in the human body.

[0176] Although the present disclosure has been described above in conjunction with one or more embodiments, it should be understood that the present disclosure is not limited to these embodiments, and is intended to cover all alternatives, modifications and equivalents included in the spirit and scope of the appended claims. All references cited herein are incorporated by reference in their entirety.

[0177] The sequences in this application are summarized below. ***

[0178] Having described in detail several preferred embodiments of the present invention, it should be understood that the invention defined in the preceding paragraphs is not limited to the specific details set forth in the above description, since many obvious changes can be made without departing from the spirit or scope of the present invention.

Claims

1. A recombinant fusion protein comprising: (a) an anti-CD40 antibody or an antigen-binding fragment thereof, and (b) a CD47 binding domain, wherein the CD47 binding domain is the first Ig-like extracellular domain of human SIRPα isoform 2 (SIRPαV2D1).

2. The recombinant fusion protein according to claim 1, wherein the anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the VH CDR1, VH CDR2 and VH CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3, and the VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

3. A recombinant fusion protein according to claim 2, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, and / or the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

8. 4 . The recombinant fusion protein according to claim 2 , wherein a heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the heavy chain constant region is a heavy chain constant region of human IgG1, IgG2 or IgG4. The recombinant fusion protein according to claim 4 , wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

9.

6. The recombinant fusion protein according to any one of claims 2 to 5, wherein the light chain constant region is connected to the C-terminus of the light chain variable region.

7. The recombinant fusion protein according to claim 6, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

10.

8. The recombinant fusion protein according to any one of claims 1 to 7, wherein the CD47 binding domain is linked to the N-terminus or C-terminus of the anti-CD40 antibody or antigen-binding fragment thereof.

9. The recombinant fusion protein according to any one of claims 1 to 8, wherein the CD47 binding domain is linked to the N-terminus of the heavy chain variable region or the light chain variable region, or is linked to the C-terminus of the heavy chain constant region or the light chain constant region.

10. The recombinant fusion protein according to claim 8, wherein the CD47 binding domain is connected to the anti-CD40 antibody or antigen-binding fragment thereof via a linker.

11. The recombinant fusion protein according to claim 9, wherein the CD47 binding domain is connected to the N-terminus of the heavy chain variable region or the light chain variable region through a linker, or is connected to the C-terminus of the heavy chain constant region or the light chain constant region through a linker. 12 . The recombinant fusion protein according to claim 10 , wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 12, 13, 14 or 15. 13 . The recombinant fusion protein according to any one of claims 1 to 12 , wherein the CD47 binding domain is wild-type SIRPαV2D1 or a SIRPαV2D1 variant.

14. The recombinant fusion protein according to claim 13, wherein the CD47 binding domain comprises the amino acid sequence shown in SEQ ID NO:11, wherein X1=V, X2=K, X3=S, X4=K, X5=F of SEQ ID NO:11; or X1=I, X2=R, X3=T, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=R, X5=V.

15. The recombinant fusion protein according to any one of claims 1 to 14, comprising: i) an anti-CD40 antibody heavy chain variable region-heavy chain constant region-linker-SIRPαV2D1 polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 16; and an anti-CD40 antibody light chain variable region-light chain constant region polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 20; ii) SIRPαV2D1-linker-anti-CD40 antibody heavy chain variable region-heavy chain constant region polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 17, wherein X1=V, X2=K, X3=S, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=R, X5=V of SEQ ID NO: 17; and an anti-CD40 antibody light chain variable region-light chain constant region polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 20; or iii) an anti-CD40 antibody heavy chain variable region-heavy chain constant region polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 19; and an anti-CD40 antibody light chain variable region-light chain constant region-linker-SIRPαV2D1 polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:

18.

16. The recombinant fusion protein according to any one of claims 1 to 15, comprising: i) a first polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody heavy chain variable region, a heavy chain constant region, a linker, and SIRPαV2D1, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 16; a second polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody heavy chain variable region, a heavy chain constant region, a linker, and SIRPαV2D1, wherein the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 16; a third polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody light chain variable region and a light chain constant region, the third polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 20; and a fourth polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody light chain variable region and a light chain constant region, the fourth polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 20; ii) a first polypeptide chain, comprising, from N-terminus to C-terminus, SIRPαV2D1, a linker, an anti-CD40 antibody heavy chain variable region and a heavy chain constant region, the first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 17, wherein X1=V, X2=K, X3=S, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=R, X5=V of SEQ ID NO: 17; A second polypeptide chain, comprising, from N-terminus to C-terminus, SIRPαV2D1, a linker, an anti-CD40 antibody heavy chain variable region and a heavy chain constant region, the second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 17, wherein X1=V, X2=K, X3=S, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=K, X5=F; or X1=I, X2=R, X3=T, X4=R, X5=V of SEQ ID NO: 17; a third polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody light chain variable region and a light chain constant region, the third polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 20; and a fourth polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody light chain variable region and a light chain constant region, the fourth polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 20; iii) a first polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody heavy chain variable region and a heavy chain constant region, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 19; a second polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody heavy chain variable region and a heavy chain constant region, the second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 19; a third polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody light chain variable region, a light chain constant region, a linker, and SIRPαV2D1, wherein the third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 18; and a fourth polypeptide chain, comprising, from N-terminus to C-terminus, an anti-CD40 antibody light chain variable region, a light chain constant region, a linker, and SIRPαV2D1, wherein the fourth polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 18; wherein the heavy chain variable region of the first polypeptide chain and the light chain variable region of the third polypeptide chain associate to form a CD40 binding domain; the heavy chain variable region of the second polypeptide chain and the light chain variable region of the fourth polypeptide chain associate to form a CD40 binding domain; and the heavy chain constant region of the first polypeptide chain and the heavy chain constant region of the second polypeptide chain associate together.

17. A nucleic acid encoding the recombinant fusion protein according to any one of claims 1 to 16. An expression vector comprising the nucleic acid of claim 17 .

19. A host cell transformed or transfected with the nucleic acid of claim 17 or the expression vector of claim 18.

20. A pharmaceutical composition comprising the recombinant fusion protein of any one of claims 1 to 16, the nucleic acid of claim 17, the expression vector of claim 18 or the host cell of claim 19, and a pharmaceutically acceptable carrier.

21. Use of the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating diseases related to CD40 and / or CD47 signaling.

22. The use according to claim 21, wherein the disease is cancer.

23. The use according to claim 22, wherein the cancer is a solid tumor or a hematological tumor.

24. The method of claim 23, wherein the cancer is leiomyosarcoma, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, colon cancer, kidney cancer, prostate cancer, cervical cancer, nasopharyngeal cancer, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, lung cancer (including small cell lung cancer and non-small cell lung cancer), multiple myeloma, melanoma, hepatocellular carcinoma, liver cancer, bile duct cancer, ovarian cancer, bladder cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma) or gastric cancer.

25. A kit comprising the recombinant fusion protein of any one of claims 1 to 16, the nucleic acid of claim 17, the expression vector of claim 18, the host cell of claim 19 or the pharmaceutical composition of claim 20.

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