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

Reference Merging

[0001] This application claims priority to PCT application No. PCT / CN2022 / 091058, filed on May 6, 2022. This application is a divisional application of patent application No. 202380037741.4, filed on May 5, 2023, entitled "Recombinant Fusion Protein Targeting CD40 and CD47".

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

[0003] This disclosure relates to a recombinant fusion protein that binds CD40 and CD47 with high affinity and functionality. This 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. Furthermore, this disclosure provides a pharmaceutical composition comprising the recombinant fusion protein and a treatment method using the recombinant fusion protein. Background Technology CD40 and CD40L

[0004] CD8 + Cytotoxic T lymphocyte (CTL) responses play an important role in the immune defense against cancer and infection. An effective CTL response is triggered by the binding of antigen-specific T cell receptors to the 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 major ligand CD40L are a pair of co-stimulatory molecules. CD40 is a type I transmembrane protein initially identified as a cell surface marker on B lymphocytes and bladder tumor cells, and later found to be expressed on antigen-presenting cells such as macrophages, dendritic cells, and monocytes. CD40L is mainly composed of 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, it recruits TRAF1 to TRAF6 to their cytoplasmic domains to drive signal transduction (Ma DY et al., (2009) Semin Immunol 21(5):265-272). CD40 signal transduction in dendritic cells may lead to upregulation of MHC molecules and costimulatory molecules, as well as increased levels of T cell-stimulating cytokines (such as interleukin-12), thereby activating dendritic cells and replacing the driving CD8. + T cell responses require CD4+ + T-cell helper (Ara A et al., (2018) Immunotargets Ther. 7:55-61). CD40-mediated immune activation may be independent of innate immune receptors, such as Toll-like receptors (Byrne KT & Vonderheide RH (2016) Cell Rep. 15:2719-2732) and may transform 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 the interaction between CD40 and CD40L may inhibit tumor growth, for example in 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 has been reported that in some cases, CD40 signaling in tumor cells promotes tumor growth, but at least enhances antigen presentation in tumor cells.

[0007] Activating anti-CD40 antibodies have been developed for disease treatment. In the first 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] Biologics 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 various 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-1 containing the Src homology 2 domain, 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, preventing macrophages from phagocytosing cells. Cancer cells then use this immune tolerance mechanism to evade 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, squamous cell carcinoma of the head and neck, small cell lung cancer, non-small cell lung cancer, multiple myeloma, melanoma, hepatocellular carcinoma, liver cancer, cholangiocarcinoma, 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) FrontImmunol.11:18).

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

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

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

[0014] Because CD47 is widely 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, which make up half of the blood volume, thereby 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 (such as CD40) to more precisely target immune cells in the tumor and / or microenvironment, thereby reducing or eliminating off-target effects.

[0016] Any references or identifications in this application do not imply an admission that such documents are prior art to this invention. Invention Overview

[0017] The inventors of this application designed and prepared a recombinant fusion protein capable of binding to CD40 and CD47. This fusion protein exhibits low aggregation levels and, compared to single-specific counterparts and prior art antibodies (e.g., selicrelumab), possesses comparable or higher binding affinity for 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 of CD47-SIRPα binding. + The ability of cells to engulf.

[0018] The recombinant fusion protein described in this disclosure can be used in in vitro and in vivo experiments, as well as for the treatment of diseases related to CD40 and / or CD47 signaling, such as tumors.

[0019] In one aspect, this disclosure provides a recombinant fusion protein capable of binding 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 its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises two identical heavy chain variable regions and two identical light chain variable regions. In some embodiments, the heavy chain constant region is attached to the C-terminus of the heavy chain variable region, and optionally, the light chain constant region is attached to the C-terminus of the light chain variable region.

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

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

[0023] The heavy chain variable region of the anti-CD40 antibody or its antigen-binding fragment comprises VH CDR1, VH CDR2, and VHCDR3, 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 the 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 engineered to possess certain desired characteristics. 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 its antigen-binding fragment comprises VL CDR1, VL CDR2, and VLCDR3, wherein 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 may be a constant region of the κ and λ chains. In some embodiments, the light chain constant region has an amino acid sequence as shown in SEQ ID NO:10.

[0025] The anti-CD40 antibody or its antigen-binding fragment 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 light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3. The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 1, 2, 3, 4, 5, and 6. 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, 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 with SEQ ID NO:8. In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8.

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

[0027] The heavy chain of the anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain variable region and a heavy chain constant region, wherein the heavy chain variable region and the heavy chain constant region are composed of the above-described 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 its antigen-binding fragment 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 are composed of the amino acid sequence described above. 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 with 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 with SEQ ID NO:20. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown 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 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 the amino acid sequence 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 / capacity, having the amino acid sequence 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 may be linked 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 includes a heavy chain variable region, a light chain variable region, a heavy chain constant region linked to the C-terminus of the heavy chain variable region, a light chain constant region linked to the C-terminus of the light chain variable region, and a CD47 binding domain linked to either the N-terminus or the C-terminus of the heavy chain variable region or the light chain constant region. In some embodiments, the CD47 binding domain is linked to either the N-terminus of the heavy chain variable region or 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 includes a heavy chain and a light chain, and the CD47 binding domain is linked to either the N-terminus or the C-terminus of the heavy chain or the light chain. In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises two identical heavy chains and two identical light chains, with a CD47 binding domain attached to the N-terminus or C-terminus of each heavy chain or the C-terminus of each light chain.

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

[0033] In some embodiments, the recombinant fusion protein described in this 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) a 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 containing 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 containing the amino acid sequence shown in SEQ ID NO:18.

[0034] The recombinant fusion protein described in this disclosure 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 an optional 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; In this configuration, 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 containing the anti-CD40 antibody heavy chain variable region, the heavy chain constant region, and SIRPαV2D1 from the N-terminus to the C-terminus, and a third polypeptide chain and a fourth polypeptide chain containing the anti-CD40 antibody light chain variable region. ii) A first polypeptide chain and a second polypeptide chain containing SIRPαV2D1, an anti-CD40 antibody heavy chain variable region and a heavy chain constant region from the N-terminus to the C-terminus, and a third polypeptide chain and a fourth polypeptide chain containing an anti-CD40 antibody light chain variable region; or iii) A first polypeptide chain and a second polypeptide chain containing an anti-CD40 antibody heavy chain variable region and a heavy chain constant region from the N-terminus to the C-terminus, and a third polypeptide chain and a fourth polypeptide chain containing an anti-CD40 antibody light chain variable region and SIRPαV2D1; In this configuration, 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 described in this disclosure comprises: i) A first and second polypeptide chain comprising the anti-CD40 antibody heavy chain variable region, the heavy chain constant region, and SIRPαV2D1 from the N-terminus to the C-terminus, and a third and fourth polypeptide chain comprising the anti-CD40 antibody light chain variable region and the light chain constant region from the N-terminus to the C-terminus; ii) A first and second polypeptide chain comprising SIRPαV2D1, the anti-CD40 antibody heavy chain variable region, and the heavy chain constant region from the N-terminus to the C-terminus, and a third and fourth polypeptide chain comprising the anti-CD40 antibody light chain variable region and the light chain constant region from the N-terminus to the C-terminus; iii) From the N-terminus to The C-terminus comprises a first polypeptide chain and a second polypeptide chain containing an anti-CD40 antibody heavy chain variable region and a heavy chain constant region, and a third polypeptide chain and a fourth polypeptide chain containing an anti-CD40 antibody light chain variable region, a light chain constant region, and SIRPαV2D1 from the N-terminus to the C-terminus; 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 described in this disclosure comprises: i) A first polypeptide chain and a second polypeptide chain containing the anti-CD40 antibody heavy chain variable region, heavy chain constant region, linker and SIRPαV2D1 from the N-terminus to the C-terminus, and a third polypeptide chain and a fourth polypeptide chain containing the anti-CD40 antibody light chain variable region and light chain constant region from the N-terminus to the C-terminus. ii) A first polypeptide chain and a second polypeptide chain containing SIRPαV2D1, a linker, an anti-CD40 antibody heavy chain variable region and a heavy chain constant region from the N-terminus to the C-terminus, and a third polypeptide chain and a fourth polypeptide chain containing an anti-CD40 antibody light chain variable region and a light chain constant region from the N-terminus to the C-terminus. iii) A first polypeptide chain and a second polypeptide chain containing the anti-CD40 antibody heavy chain variable region and heavy chain constant region from the N-terminus to the C-terminus, and a third polypeptide chain and a fourth polypeptide chain containing the anti-CD40 antibody light chain variable region, light chain constant region, linker and SIRPαV2D1 from the N-terminus to the C-terminus. In this configuration, 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 described in this 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 containing 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 a third polypeptide chain and a fourth polypeptide chain containing the amino acid sequence shown in SEQ ID NO:20; or iii) A first polypeptide chain and a second polypeptide chain containing the amino acid sequence shown in SEQ ID NO:19, and a third polypeptide chain and a fourth polypeptide chain containing the amino acid sequence shown in SEQ ID NO:18.

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

[0040] This application also provides a pharmaceutical composition comprising the recombinant fusion protein, nucleic acid molecule, expression vector or host cell described in this disclosure, and a pharmaceutically acceptable carrier. The pharmaceutical composition may also contain adjuvants, such as antitumor agents.

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

[0042] The disease may be cancer. The cancer may be a solid tumor or a hematologic malignancy, including but not limited to leiomyosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, colon cancer, kidney cancer, prostate cancer, cervical cancer, nasopharyngeal carcinoma, breast cancer, osteosarcoma, squamous cell carcinoma of the head and neck, 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 compositions described herein may 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. Such infectious diseases may be caused by bacterial, viral, or parasitic infections.

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

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

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

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

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

[0049] This application also provides the use of the pharmaceutical compositions described herein in treating diseases related to CD40 and / or CD47 signaling, enhancing immune responses, and / or reversing or reducing immunosuppression.

[0050] In a fourth aspect, this application provides a kit comprising the recombinant fusion protein, pharmaceutical composition, nucleic acid molecule, expression vector, or host cell described in this disclosure. The term "kit" refers to two or more components packaged in a container, 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 this disclosure. Therefore, a kit can be described as a group of products and / or tools capable of achieving a specific objective and can be marketed as a single unit.

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

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

[0053] Therefore, the object of this invention is not to include any previously known products, manufacturing processes of products, or methods of using products within the scope of this invention. The applicant therefore reserves the right and hereby declares the waiver of any previously known products, processes, or methods. Furthermore, it should be noted that this invention is not intended to include any products, processes, manufacturing processes, or methods of using products that do not meet the written description and enforceability requirements of the United States Patent and Trademark Office (USPTO) (35 U.S.SC § 112, paragraph 1) or the European Patent Office (EPO) (EPC Article 83). Therefore, the applicant reserves the right and hereby declares the waiver of any previously described products, manufacturing processes, or methods of using products. In the practice of this invention, compliance with Article 53(c) and Articles 28(b) and (c) of the European Patent Convention may be advantageous. All rights are expressly reserved to any embodiment of any granted patent involved that has been expressly rejected in a prior application by this application family or any other family or any third party. Nothing in this application shall be construed as an acceptance.

[0054] It is worth noting that in this disclosure, particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” have the meanings given to them by U.S. patent law. For example, they can mean “includes,” “included,” or “including.” Terms such as “primarily comprises” and “primarily includes” have the meanings given to them by U.S. patent law. For example, they allow the presence of elements not explicitly stated, but exclude elements found in the prior art or elements that affect the basic or novelty of the invention. Attached Figure Description

[0055] The following detailed description is given by way of example, but is not intended to limit the invention to the specific embodiments described. The best understanding can be obtained by referring to the accompanying drawings.

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

[0057] Figure 2 Flow cytometry was used to detect the binding ability of the recombinant fusion protein described herein 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 described herein to human CD47 expressed on the surface of 293F-CD47 cells.

[0059] Figure 4 A competitive ELISA assay was performed to detect the ability of the recombinant fusion protein described in this disclosure to block the binding of human CD40 to human CD40L.

[0060] Figure 5 Competitive ELISA assays were used to detect the ability of the recombinant fusion protein described in this 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 this 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 this disclosure to block the binding of human SIRPα to human CD47 on the cell surface.

[0063] Figure 8 Cell-based reporter gene assays were used to detect the CD40 agonist activity of the recombinant fusion protein described in this disclosure.

[0064] Figure 9Cell-based assays were conducted to detect the ability of the recombinant fusion protein described in this disclosure to induce macrophages to engulf human Jurkat cancer cells.

[0065] Figure 10 The binding affinity of the recombinant fusion protein described herein to human CD40 and CD47 was detected by double-antibody sandwich ELISA. Detailed Implementation

[0066] To better understand this disclosure, some terms are first defined. Other definitions are listed throughout the detailed description.

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

[0068] The term "human CD40" refers to a CD40 protein with an amino acid sequence derived from humans, such as the human CD40 amino acid sequence in NCBI number NP_001241.1 (Sasaki K et al., (2021) J Exp Clin Cancer Res 40(1):212). The term "monkey CD40" or "cynomolgus monkey CD40" refers to a CD40 protein with an amino acid sequence derived 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 spontaneously 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 derived from humans, such as the amino acid sequence of GenBank accession number AAH75849.1 (Strausberg RL et al., (2002) Proc. Natl. Acad. Sci. USA 99(26):16899-16903). In one embodiment, SIRPα contains a signal sequence; in other embodiments, SIRPα refers to the mature form of the protein. Ten human SIRPα alleles have been identified, and human SIRPα isoform 2 (or V2) has been reported to have reduced or minimal binding affinity to erythrocytes.

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

[0071] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, resulting in selective damage, destruction, or elimination of pathogens invading the body, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation.

[0072] The term "immunosuppression" refers to a decrease in the activity or efficacy of the immune system due to factors such as age, persistent illness, malnutrition, cancer, chemotherapy, or radiation therapy. Immunosuppression can be reversed under certain circumstances, such as by modulating specific pathways.

[0073] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., CD40) through at least one antigen-binding site, where the antigen-binding site is typically located within the variable region of the immunoglobulin molecule. 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 containing an antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site (e.g., a dual-variable-domain immunoglobulin molecule), provided that the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be any of the five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated alpha, delta, epsilon, gamma, and mu based on the characteristics of their heavy chain constant regions. Different types of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies can be naked or bound to other molecules, including but not limited to toxins and radioisotopes. Unless otherwise explicitly stated, the term “antibody” as used herein includes the “antigen-binding portion” of a complete antibody. Conventional IgG is a glycoprotein consisting of two identical heavy chains (H) and two identical light chains (L) linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region contains a single domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, also known as complement-determining regions (CDRs), separated by more conserved framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The antibody constant region mediates 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. The “functional fragment” of the heavy chain constant region refers to the portion of the constant region that retains desired properties, such as binding affinity for Fc receptors and / or complement system proteins.

[0074] As used herein, the term "antigen-binding fragment" (or simply "antibody fragment") of an antibody 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 achieved by fragments of a full-length antibody. Examples of binding fragments covered by the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains in one arm of an antibody; (v) dAb fragments consisting of VH domains (Ward et al., (1989) Nature 341:544-546); (vi) separated complementarity-determining regions (CDRs); and (viii) nanobodies, i.e., heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked together via a synthetic linker to form a single-chain protein using recombination methods, where the VL and VH regions pair to form a monovalent molecule (called a 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 antibodies. 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 their antigen-binding fragments, or the "agonistic activity" of CD40 signaling, refer to the binding of anti-CD40 antibodies or their antigen-binding fragments to CD40 and the activation / induction of CD40 signaling, such as promoting the activation and proliferation of immune cells and the production of cytokines and chemokines. Agonistic anti-CD40 antibodies or their antigen-binding fragments can enhance the antigen-presenting ability of APCs and activate tumor-specific CD40. + and CD8 + T cells, lymphocytes, and monocytes secrete cytokines and chemokines, enhance the killing of tumor cells by cytotoxic lymphocytes and NK cells, and promote the innate and adaptive immune responses of cancer patients to tumors.

[0076] As described herein, a recombinant fusion protein that "specifically binds to CD40 (e.g., human CD40)" or a bispecific fusion protein refers to a protein that binds to CD40 (human CD40 and possibly CD40 proteins from one or more non-human species), but substantially does not 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 CD47 proteins but does not bind to non-CD47 proteins. Preferably, the fusion protein binds to both 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 "basically non-binding" protein or cell means that it does not bind to said protein or cell, or does not bind to said protein or cell with high affinity, i.e., K-cells that bind to the protein or cell. D 1.0×10 -6 M or higher, preferably 1.0 × 10 -5 M or higher, more preferably 1.0 × 10 -4 M or higher, more preferably 1.0 × 10 -3 M or higher, or even more preferably 1.0 × 10 -2 M or higher.

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

[0079] The term "K" used in this article assoc "or "K a "K" refers to the binding rate of a specific protein-protein interaction, such as antibody-antigen or receptor-ligand interactions. The term "K" is used in this article. dis "or "K d "K" refers to the dissociation rate of a specific protein-protein interaction. The term "K" is used in this article. D "" refers to the dissociation constant, which is determined by K d With K a The ratio (i.e., Kd / Ka) is obtained and expressed as molar concentration (M). K D The value can be determined using methods known in the art. A preferred assay antibody K DThe method uses surface plasmon resonance, and more preferably uses biosensor systems, such as Biacore. TM system.

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

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

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

[0083] The term "therapeutic effective amount" refers to the amount of the fusion protein described in this disclosure that is sufficient to prevent or improve symptoms associated with a disease or condition (e.g., cancer) and / or reduce the severity of the disease or condition. Therapeutic effective amount should be understood as being relevant to the condition being treated, and the actual effective amount can be readily determined by those skilled in the art.

[0084] The "identity" percentage of two or more nucleic acids or peptides used herein refers to the fact that two or more sequences or subsequences are completely identical or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned (with gaps introduced if necessary) to achieve maximum correspondence, and conserved amino acid substitutions may or may not be considered part of sequence consistency. The identity percentage can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining amino acid or nucleotide sequence alignment results are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GGWisconsin Package, and variants thereof. In some embodiments, the two nucleic acids or peptides of this disclosure are substantially identical, meaning that when compared and aligned using sequence comparison algorithms or by visual inspection to achieve 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 at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity.

[0085] It is worth noting that, unless the context clearly indicates otherwise, the singular form used herein encompasses plural references. Thus, for example, reference to "a reagent" includes one or more such different reagents, and reference to "the method" includes equivalent steps and methods known to those skilled in the art, which may be modified or substituted for the methods described herein.

[0086] Several aspects of this disclosure are further described in detail below.

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

[0088] The recombinant fusion protein described in this disclosure exhibits low aggregation levels and, compared to single-specific counterparts and existing antibodies (e.g., Selicrelumab), possesses comparable or higher binding affinity for human and 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 of CD47-SIRPα binding. + The ability of cells to engulf (e.g., cancer cells, including Jurkat cells or HL-60 cells).

[0089] The recombinant fusion protein described in this application comprises three components: a CD47 binding domain, a linker, and an anti-CD40 antibody or its antigen-binding fragment. Those skilled in the art will recognize that these three components can be designed in various ways. The CD47 binding domain can be linked to, for example, the N-terminus or C-terminus of an anti-CD40 antibody or its antigen-binding fragment. Preferably, human sequences are used in human cancer treatment because the strong immunogenicity of proteins or peptides derived from non-human animals can lead to allergic reactions and other adverse effects. However, depending on the specific application, other animal proteins or peptides may also be used in this application and, where appropriate, humanized.

[0090] The CD47 binding domain can be any protein or peptide that binds CD47, such as SIRPα, SIRPα variants, or affinity-optimized SIRPα variants. A “variant” of SIRPα is defined as one or more amino acids whose amino acid sequence is altered compared to wild-type SIRPα. Variants can have “conserved” changes, where the substituted amino acids have similar structural or chemical properties, such as replacing leucine with isoleucine. More rarely, variants can have “non-conserved” 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 wild-type SIRPαV2D1 or a SIRPαV2D1 variant engineered to have higher CD47 binding affinity / ability. In some embodiments, SIRPαV2D1 may be wild-type, comprising an amino acid sequence having at least 95% identity with SEQ ID NO:11 (X1=V, X2=K, X3=S, X4=K, X5=F). In some embodiments, SIRPαV2D1 may be 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 can be a variant of SIRPαV2D1 comprising an amino acid sequence having at least 95% identity with 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 can be a variant of SIRPαV2D1 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 having one or more amino acid changes in the amino acid sequence of SIRPαV2D1 compared to wild-type SIRPαV2D1. Variants can have “conserved” changes, where the substituted amino acids have similar structural or chemical properties, for example, replacing leucine with isoleucine. More rarely, variants may have “non-conserved” 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 higher sequence identity with wild-type SIRPαV2D1.

[0092] The linker primarily 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 peptide-linked amino acids, preferably 5 to 30, 10 to 30, 10 to 20, or 15 peptide-linked amino acids, wherein the amino acids are selected from 20 natural amino acids. Those skilled in the art will understand that one or more of these amino acids may be glycosylated. In one embodiment, the 5 to 30 amino acids may be selected from glycine, alanine, proline, asparagine, glutamine, serine, and lysine. In one embodiment, the linker is primarily composed of sterically unhindered amino acids, such as glycine and alanine. Exemplary linkers are polyglycine (especially (Glys, poly(Gly-Ala)) and polyalanine, such as -GGGGS- (SEQ ID NO:12), -GGGGSGGGGS- (SEQ ID NO:13), -GGGGSGGGGSGGGGS- (SEQ ID NO:14), and -GGGGSGGGGSGGGGSGGGS- (SEQ ID NO:15). Linkers can also be non-peptide linkers. For example, alkyl linkers such as -NH-, -(CH2)sC(O)-, where s = 2-20, can be used. These alkyl linkers can also be replaced by any sterically unhindered group such as lower alkyl groups (e.g., C). 1-4 Substitution with lower acyl groups, halogens (e.g., Cl, Br), CN, NH2, phenyl, etc. In other embodiments, the recombinant fusion protein of this application can be assembled without a linker.

[0093] Anti-CD40 antibodies can be isolated monoclonal antibodies, as disclosed in WO2021 / 197335. The anti-CD40 antibodies or antigen-binding fragments thereof described in this disclosure can be humanized.

[0094] The anti-CD40 antibody or its antigen-binding fragment described in this disclosure comprises a heavy chain variable region and a light chain variable region. In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises two identical heavy chain variable regions and two identical light chain variable regions. In some embodiments, the heavy chain constant region is linked to the C-terminus of the heavy chain variable region, and optionally, the light chain constant region is linked to the C-terminus of the light chain variable region.

[0095] The anti-CD40 antibody or antigen-binding fragment thereof described in this disclosure comprises a heavy chain and a light chain. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof described in this disclosure comprises two identical heavy chains and two identical light chains. The heavy chain may include a heavy chain variable region and a heavy chain constant region, and the light chain may include a light chain variable region and an optional light chain constant region.

[0096] The heavy chain variable region described in this disclosure comprises VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: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 may be the heavy chain constant region of human IgG1, IgG2, or IgG4, and may optionally be 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 with 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 regions described in this disclosure include VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1, VL CDR2, and VL CDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 5, and 6. The light chain variable regions contain amino acid sequences 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 regions may be constant regions of the human κ or λ 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 with 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 contain the amino acid sequences shown in SEQ ID NO: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 contain the amino acid sequences shown in SEQ ID NO: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 with 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 with 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. The heavy chain constant region contains 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:9, and the light chain constant region contains 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: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 described in this disclosure 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 an optional 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; In this configuration, 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 linked to the N-terminus or C-terminus of an anti-CD40 antibody or its antigen-binding fragment. In some embodiments, the anti-CD40 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain constant region is linked to the C-terminus of the heavy chain variable region, the light chain constant region is linked to the C-terminus of the light chain variable region, and the CD47 binding domain is linked 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 includes a heavy chain and a light chain, and the CD47 binding domain is linked 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 herein can be modified by altering one or more residues within one or both variable regions (i.e., VH and / or VL) of an anti-CD40 antibody or its antigen-binding fragment, such as one or more residues within one or more CDR regions and / or one or more frame regions. Alternatively, the recombinant fusion protein can be modified by altering residues within the constant regions of the anti-CD40 antibody or its antigen-binding fragment, for example, to change the effector function of the recombinant fusion protein. Furthermore, the recombinant fusion protein can be modified at residues in the CD47 binding domain to alter its binding affinity for CD47 or other functional properties.

[0103] In some implementations, CDR transplantation can be used to modify the variable region of an anti-CD40 antibody or its antigen-binding fragment. Antibodies primarily interact with target antigens through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. Therefore, the amino acid sequences within the individual antibody's CDRs are more diverse than those outside the CDRs. Since the CDR sequence is responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific natural antibody can be expressed by constructing expression vectors in which the CDR sequence of a specific natural antibody is transplanted into the backbone 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 this disclosure relates to a recombinant fusion protein comprising a heavy chain variable region and a light chain variable region of an anti-CD40 antibody, wherein the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 sequences as described above in this disclosure, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 sequences as described above in this disclosure. While these fusion proteins contain the CDR sequences of the heavy chain and light chain variable regions of the anti-CD40 antibody of this disclosure, they may contain different framework sequences.

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

[0106] Using Gapped BLAST (Altschul et al., (1997), a sequence similarity retrieval method well known to those skilled in the art), the antibody protein sequence was compared with a compiled protein sequence database.

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

[0108] Furthermore, the anti-CD40 antibody or its antigen-binding fragment of the recombinant fusion protein described in this disclosure can be engineered to include modifications within the Fc region, or as an alternative to modifications within the frame or CDR region, typically to alter one or more functional properties of the recombinant fusion protein, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cytotoxicity. Additionally, the recombinant fusion protein described in this 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 to alter the number of cysteine ​​residues in the hinge region, for example, by increasing or decreasing them. 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 and heavy chains, or increase or decrease antibody stability.

[0110] In another embodiment, the Fc-hinge region of the anti-CD40 antibody or its antigen-binding fragment in the recombinant fusion protein of this disclosure is mutated to alter 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, such that the binding affinity of the fusion protein to Staphylococcus aureus protein A (SpA) is weakened relative to the binding affinity of the native Fc hinge region to SpA. This method is described in more detail in U.S. Patent No. 6,165,745.

[0111] Amino acid alterations near the junction of the Fc and non-Fc regions can significantly increase the serum half-life of the Fc fusion protein. Therefore, the junction region of the recombinant fusion protein of this disclosure can be altered relative to the naturally occurring immunoglobulin heavy chain sequence, preferably within about 10 amino acids at the junction. These amino acid alterations can lead to increased hydrophobicity. In one embodiment, the C-terminal lysine residue of the constant region 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 its antigen-binding fragment in the recombinant fusion protein described in this disclosure can be modified. For example, the affinity of the anti-CD40 antibody or its antigen-binding fragment for an antigen can be increased by modifying the glycosylation. Such glycosylation can be achieved, for example, by altering one or more glycosylation sites within the sequence of the anti-CD40 antibody or its antigen-binding fragment. For example, one or more amino acid substitutions can be performed to eliminate one or more variable region framework glycosylation sites, thereby deglycosylating the site. This deglycosylation can increase the antibody's affinity for the antigen. See US Pat. Nos. 5,714,350 and 6,350,861.

[0113] Another modification of the anti-CD40 antibody or its antigen-binding fragment in the recombinant fusion protein described in this disclosure is PEGylation. For example, antibodies can be PEGylated to increase their biological (e.g., serum) half-life. The term "polyethylene glycol" as used herein is intended to cover any form of PEG used to produce other proteins, such as mono(C1-C1) PEG. 10 Alkyl- or aryl-hydroxy-polyethylene glycol or polyethylene glycol-maleimide. Methods for PEGylating proteins are known in the art and can be applied to the antibodies described in this disclosure. See EP 0,154,316 and EP 0,401,384.

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

[0115] On the other hand, this disclosure provides nucleic acid molecules encoding the recombinant fusion protein described herein. In some embodiments, the nucleic acid molecules provided herein encode: 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) a 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:17. 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 containing 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 containing the amino acid sequence shown in SEQ ID NO:18. Nucleic acids can be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “isolated” or “substantially pure” after being purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) using standard techniques. The nucleic acids described in this disclosure can 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 this disclosure can be obtained using standard molecular biology techniques. For example, the nucleic acid molecules described in this disclosure can be chemically synthesized.

[0117] This disclosure also provides expression vectors comprising the nucleic acid molecules described herein. Vectors include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), transformant artificial chromosomes (TAC), mammalian artificial chromosomes (MAC), and human artificial attached chromosomes (HAEC). This disclosure also provides host cells transformed or transfected with the expression vectors or nucleic acids described herein. Suitable host cells include *Escherichia coli*, yeast, and other eukaryotes. In one embodiment, DNA encoding a polypeptide chain of the recombinant fusion protein described herein is inserted into one or more expression vectors, thereby operatively linking these genes to transcriptional and translational regulatory sequences. Hereinafter, the term "operatively linked" means that the encoding nucleotide is linked to the vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the recombinant fusion protein gene.

[0118] The term "regulatory sequence" refers to a sequence containing a promoter, an enhancer, and other expression control elements that regulate nucleotide transcription or translation (e.g., polyadenylation signaling). Such regulatory sequences have been described in the article by Goeddel et al. (GeneExpression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that guide high-level protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), and adenoviruses, such as the adenovirus major late promoter (AdMLP) and polyomavirus enhancers. 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 be used, such as the SRα promoter system, which contains sequences from the SV40 early promoter and long terminal repeat sequences from 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 vectors of this disclosure may also carry other sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selective marker genes. Selective marker genes facilitate the selection of host cells into which the vector has been introduced (see US Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, selective marker genes typically confer resistance to drugs (e.g., G418, hygromycin, or methotrexate) in host cells into which the vector has been introduced. Preferred selective marker genes include the dihydrofolate reductase (DHFR) gene (for the selection / amplification of methotrexate in DHFR-host cells) and the neo gene (for G418 selection).

[0120] To express the peptide chain that constitutes the recombinant fusion protein, the expression vector encoding the peptide chain is transfected into host cells using standard techniques. The term "transfection" encompasses various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-glucan transfection. Although the recombinant fusion protein described herein can theoretically be expressed in prokaryotic or eukaryotic host cells, expression in eukaryotic cells, particularly mammalian host cells, is most preferred. This is because eukaryotic cells, especially mammalian cells, are more likely than prokaryotic cells to assemble and secrete correctly folded and immunologically active recombinant fusion proteins.

[0121] Preferred mammalian host cells for expressing the recombinant fusion protein described herein 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 DHFR selective markers, for example, see RJ Kaufman and PASharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. For the use of NSO myeloma cells in particular, another preferred expression system is the GS gene expression system, described in WO 87 / 04462, WO 89 / 01036, and EP 338,841. The recombinant fusion protein is prepared by culturing the host cells for a sufficient period of time to allow expression of the recombinant fusion protein in the host cells, or more preferably, to allow secretion of the recombinant fusion protein into the culture medium in which the host cells grow. Recombinant fusion proteins can be recovered from the culture medium using standard protein purification methods.

[0122] In another aspect, this disclosure provides a pharmaceutical composition comprising the recombinant fusion protein, nucleic acid molecule, expression vector, and / or host cell described herein, 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 pharmaceutically active ingredients, such as another antibody or drug, such as an antitumor drug.

[0123] Pharmaceutical compositions may contain any number of excipients. Permissible 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 appropriate excipients are taught in the 20th edition of Remington: Pharmaceutical Science and Practice (Lippincott Williams & Wilkins 2003), edited by Gennaro, the contents of which are 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 may be encapsulated in a material to protect it from acids and other natural conditions that could inactivate it. The phrase "parenteral administration" as used herein refers to a route of administration that is typically administered by injection, other than enteral and local administration, including but not limited to intravenous, intramuscular, intra-articular, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Additionally, the pharmaceutical compositions of this disclosure may be administered via non-parenteral routes, such as local, epidermal, or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0125] The pharmaceutical composition may be in the form of a sterile aqueous solution or dispersion, or it may be formulated as a microemulsion, liposome, or other ordered structure suitable for high drug concentrations.

[0126] The "therapeutic effective dose" of the recombinant fusion protein, nucleic acid molecule, expression vector, or host cell described in this disclosure is preferably a dose that reduces the severity of disease symptoms, increases the frequency and duration of symptom-free periods, or prevents damage or disability caused by the disease. For example, in the treatment of a subject with a tumor, the "therapeutic effective dose" is preferably a dose 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 an untreated subject. Therapeutic effective doses of the therapeutic recombinant fusion protein, nucleic acid molecule, expression vector, or host cell described in this disclosure can reduce tumor size or otherwise improve the symptoms of a subject, 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, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, *Continuous and Controlled Release Drug Delivery Systems*, edited by J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

[0128] The therapeutic composition can be administered via medical devices, such as (1) needle-free subcutaneous injection devices (US Pat. No. 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 (US Pat. No. 4,487,603); (3) transdermal devices (US Pat. No. 4,486,194); (4) infusion devices (US Pat. No. 4,447,233 and 4,447,224); and (5) osmosis devices (US Pat. No. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0129] Pharmaceutical compositions comprising the recombinant fusion protein, nucleic acid molecule, expression vector or host cell described herein have a variety of in vitro and in vivo uses, relating to, for example, the treatment of diseases associated with CD40 and / or CD47 signaling, such as tumors or infectious diseases.

[0130] This disclosure provides a method for treating diseases related to CD40 signaling and / or CD47 signaling, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition described in this disclosure.

[0131] The disease may be a tumor or cancer. The tumors include, but are not limited to, leiomyosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, colon cancer, kidney cancer, prostate cancer, cervical cancer, nasopharyngeal carcinoma, breast cancer, osteosarcoma, squamous cell carcinoma of the head and neck, 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 human being.

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

[0133] On the other hand, this disclosure provides a method for modulating or enhancing an immune response in a subject in need, comprising administering the pharmaceutical composition described in this disclosure to the subject such that the subject's immune response is modulated / enhanced.

[0134] This disclosure also provides a method for reversing or alleviating immunosuppression in subjects in need, comprising administering the pharmaceutical composition described in this disclosure to the subject.

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

[0136] This disclosure is further illustrated by the following examples, which should not be construed as further limitations. All figures and 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 was constructed by optionally linking the first Ig-like extracellular domain (SIRPαV2D1) of human SIRPα isoform 2 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 (as described in WO2021 / 197335, named HuCD40-C1H1-V2) having a constant region of the human IgG2 heavy chain. The SIRPαV2D1 was wild-type, having the amino acid sequence shown in SEQ ID NO:11 (X1=V,X2=K,X3=S,X4=K,X5=F); or a mutant mutated to increase its binding affinity to CD47, having the amino acid sequence 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 with two identical heavy chains and two identical light chains. Each heavy chain contains a heavy chain variable region and a heavy chain constant region from the N-terminus to the C-terminus, and each light chain contains a light chain variable region and a light chain constant region from the N-terminus to the C-terminus. The heavy chain variable region, light chain variable region, heavy chain constant region, and light chain constant region contain the amino acid sequences shown in SEQ ID NO:7, 8, 9, and 10, respectively. The heavy chain variable region contains VH CDR1, VH CDR2, and VH CDR3, which contain the amino acid sequences shown in SEQ ID NO:1, 2, and 3, respectively. The light chain variable region contains VLCDR1, VL CDR2, and VL CDR3, which contain the amino acid sequences shown in SEQ ID NO:4, 5, and 6, respectively.

[0138] The specific structures and sequence IDs of exemplary recombinant fusion proteins are shown in Table 1 below. Figure 1 As shown. Table 1. Molecular structure and amino acid sequence ID of exemplary recombinant fusion proteins Example 2: Expression and Identification of Exemplary Recombinant Fusion Protein

[0139] In short, nucleic acids encoding the individual polypeptide chains of the recombinant fusion protein were first synthesized and inserted into the pTT5 vector. Then, the plasmid DNA of the vector was transfected into mammalian cells (CHO cells), which expressed and secreted the exemplary recombinant fusion protein of this disclosure. The recombinant fusion protein was then purified using a protein A affinity chromatography column and analyzed by SDS-PAGE and SEC-HPLC. 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] The results show that the recombinant fusion protein described in this disclosure has high purity, and most of the fusion protein exists in monomeric form. Example 3 uses BIACORE surface plasmon resonance to detect the binding affinity of an exemplary recombinant fusion protein.

[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 (with heavy and light chains as shown in SEQ ID NO:19 and 20), Selicrelumab (an anti-CD40 antibody agonist from Roche, prepared in-house, with heavy and light chains as shown in SEQ ID NO: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] Affinity assays were performed using a Protein A chip (Cat#:29-1275-56, GE Healthcare). The recombinant fusion protein of this disclosure at a concentration of 2 μg / ml and the positive control were respectively flowed through the chip at a flow rate of 10 μL / min. Then, with an initial concentration of 200 nM, HBS-EP was used. + Recombinant human CD40-his protein (Cat#:CD0-H5228, Acro biosystems), human CD47-his protein (Cat#:CD7-H5227, Acro biosystems), cynomolgus macaque CD40-his protein (Biosion in-house, SEQ ID NO:29), or cynomolgus macaque CD47-his protein (Cat#:CD7-C52H1, Acro biosystems) diluted 2-fold in buffer (provided by Biacore) was flowed through the chip at a flow rate of 30 μl / min. Binding kinetics were tracked for 2 minutes, and dissociation kinetics for 10 minutes. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore evaluation software to determine K.D 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 bind specifically to human CD40 and cynomolgus monkey CD40 with high binding affinity.

[0144] Furthermore, the recombinant fusion proteins described in this disclosure 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 protein

[0145] Using Biosion's self-made 293T-CD40 cells stably expressing full-length human CD40 (uniprot#P25942-1), the binding activity of the recombinant fusion protein described herein to human CD40 was further determined by flow cytometry (FACS). Following the instructions of the Lipofectamine 3000 transfection reagent (Thermo Fisher), pCMV-TP plasmid containing 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] In short, 293T-CD40 cells were collected from cell culture flasks, washed twice, and resuspended in phosphate-buffered saline (PBS) (FACS buffer) containing 2% v / v fetal bovine serum. Then, 1 × 10⁻⁶ cells were added to each well of a 96-well plate. 5Two 293T-CD40 cells were incubated on ice for 40 minutes with 100 μL of serially diluted recombinant fusion protein or control (starting concentration 10 μg / mL, serially diluted 5-fold with FACS buffer). Cells were washed twice with FACS buffer, and 100 μL of goat anti-human IgG (Fab)-PE (1:1000 dilution with FACS buffer, Cat#:109-116-097, Jackson ImmunoResearch) was added to each well. After incubation at 4°C in the dark for 40 minutes, cells were washed three times with FACS buffer and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument, and plotted against recombinant fusion protein or control concentrations. Data were analyzed using Graphpad Prism, and EC50 was calculated. 50 Value. Result as follows Figure 2 As shown.

[0147] Using Biosion's self-made 293F-CD47 cells stably expressing full-length human CD47 (NCBI#NP_942088.1), the binding activity of the recombinant fusion protein described herein to human CD47 was further determined by flow cytometry (FACS). Following the instructions of the Lipofectamine 3000 transfection reagent (Thermo Fisher), pCMV-TP plasmid containing DNA encoding human CD47 (NP_942088.1) inserted between the EcoRI and XbaI sites was transfected into 293F cells to prepare 293F-CD47 cells (Thermo Fisher).

[0148] 293F-CD47 cells were collected from cell culture flasks, washed twice, and resuspended in phosphate-buffered saline (PBS) (FACS buffer) containing 2% v / v fetal bovine serum. Then, 1 × 10⁻⁶ cells were added to each well of a 96-well plate. 5Two 293T-CD40 cells were incubated on ice for 40 minutes with 100 μL of serially diluted recombinant fusion protein or control (starting concentration 10 μg / mL, serially diluted 5-fold with FACS buffer). Cells were washed twice with FACS buffer, and 100 μL of goat anti-human IgG (Fc)-PE (1:1000 dilution with FACS buffer, Cat#:109-115-098, Jackson Immuno Research) was added to each well. After incubation at 4°C in the dark for 50 minutes, cells were washed three times with FACS buffer and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument, and mean fluorescence intensity plots were generated based on the recombinant fusion protein or control concentration. Data were analyzed using a Graphpad Prism to derive EC50. 50 Value. Result as follows Figure 3 As shown.

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

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

[0151] The binding activity of the recombinant fusion protein to human CD40 and CD47 was detected using a double-antibody sandwich ELISA. In short, human CD40-Fc protein (Cat#:CD0-H5253, ACRO Biosystems) was diluted to 0.2 μg / mL with PBS solution, and 100 μL was coated per well in a 96-well plate and incubated overnight at 2–8 °C. The next day, the 96-well plate was washed three 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 plate was washed three times with washing buffer.

[0152] The recombinant fusion protein or control described herein was serially diluted with blocking buffer (initial concentration 100 nM, 6-fold serial dilution) and added to 100 μL per well of a 96-well plate coated with CD40-Fc. The plate was incubated at 37°C for 1 hour. The 96-well plate was washed three times with washing buffer, and then 100 μL of 3 μg / mL human CD47-his protein (Cat#: CD7-H5227, ACRO Biosystems) was added to each well. The plate was 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. The plate was incubated at 37°C for 1 hour. The 96-well plate was washed again with washing buffer. Finally, TMB was added to develop the color of the 96-well plate, and the reaction was stopped by adding 1 M H2SO4. The absorbance at 450 nm was read using a microplate reader, and then a curve was plotted between the OD(450) value and the concentration of the recombinant fusion protein or control. The data were analyzed using Graphpad Prism to derive the EC50. 50 Value. Result as follows Figure 10 As shown.

[0153] from Figure 10 It can be seen that the recombinant fusion protein described in this disclosure specifically binds to human CD40 and CD47, and has higher binding activity compared to SIRPα-Fc-CD40L. Example 5: Exemplary recombinant fusion protein's activity in blocking CD40-CD40L or CD47-SIRPα binding. 5.1 Detection of the inhibitory effect on CD40-CD40L binding using ligand blocking ELISA

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

[0155] The recombinant fusion protein or control described herein was serially diluted with PBST containing 2.5% w / v skim milk (starting concentration 10 μg / mL, 5-fold serial dilutions) at 100 μL per well in a 96-well plate coated with CD40-Fc, and incubated at 37°C for 40 min. The plate was washed four times with wash buffer, and then 100 μL of biotin-labeled human CD40L-his protein (Cat#:10239-H08E, Sino Biological) at a concentration of 95 ng / mL was added to each well, and the plate was incubated at 37°C for 40 min. The plate was washed again with wash buffer, and then 100 μL of HRP-conjugated streptavidin (diluted 1:10000 with PBST buffer, Cat#:016-030-084, Jackson ImmunoResearch) was added to each well, and the plate was incubated at 37°C for 40 min, followed by washing again with wash buffer. Finally, TMB was added to develop the color in the 96-well plate, and then 1M H2SO4 was added to terminate the reaction. The absorbance was read using a microplate reader in dual-wavelength mode, with TMB at 450 nm and the reference wavelength at 630 nm. A graph was then plotted with OD(450-630) on the ordinate and the concentration of the recombinant fusion protein or control on the x-axis. The data were analyzed using Graphpad Prism to determine the IC50. 50 Value. Result as follows Figure 4 As shown. 5.2 Detection of the inhibitory effect on CD47-CD172a binding using ligand blocking ELISA

[0156] The ability of the recombinant fusion protein described herein to block CD47-CD172a binding was detected using a competitive ELISA. Briefly, 2 μg / mL of human CD172a-Fc protein (internally prepared, amino acid sequence SEQ ID NO:24) prepared in coating buffer (carbonate / bicarbonate buffer) was coated onto 96-well plates at 100 μL per well and incubated overnight at 4°C. The next day, the plates were washed once with washing buffer (PBS + 0.05% v / v Tween-20, PBST), then blocked at 37°C for 2 hours with 5% w / v skim milk prepared in PBST, followed by washing four times with washing buffer.

[0157] The recombinant fusion protein or control described herein was serially diluted (starting at 100 nM, 5-fold serial dilutions) and mixed with 20 ng / mL biotin-labeled human CD47-Fc (Biosion, SEQ ID NO: 23) in PBST containing 2.5% w / v skim milk. After incubation at 37°C for 40 minutes, 100 μL was added to each well of a 96-well plate coated with CD172a-Fc. After incubation at 37°C for 40 minutes, the plate was washed four times with washing buffer. Then, 100 μL of HRP-conjugated streptavidin (diluted 1:10000 with PBST buffer, Cat#: 016-030-084, Jackson ImmunoResearch) was added to each well, and the plate was incubated at 37°C for 40 minutes. The plate was washed again with washing buffer. Finally, TMB was added to develop the color of the 96-well plate, and the reaction was terminated by adding 1 M H2SO4. Absorbance was read in dual-wavelength mode on a microplate reader, with TMB at 450 nm and reference wavelength at 630 nm. A graph was then plotted with OD(450-630) as the ordinate and the concentration of the recombinant fusion protein or control as the abscissa. The data were analyzed using GraphpadPrism to determine the IC50. 50 Values. 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. Results are as follows. Figure 5 As shown. 5.3 Detection of the inhibitory effect on CD47-SIRPα binding using ligand blocking ELISA

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

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

[0160] The recombinant fusion protein or control described herein was serially diluted with PBS (FACS buffer) containing 2% v / v fetal bovine serum (initial concentration 100 nM, 3-fold serial dilution). Simultaneously, 293F-CD47 cells in logarithmic growth phase were harvested, washed twice with FACS buffer, centrifuged, and collected. Cells were then divided into 1×10⁻⁶ cells per well. 5Cells were seeded at a density of 100 μL / well of diluted antibody or control solution in 96-well plates and incubated at 4°C for 60 min. The plates were washed twice, and then 100 μL of 148 ng / mL biotin-labeled human CD172a-Fc protein (internally prepared, amino acid sequence SEQ ID NO:24) prepared with FACS buffer was added to each well, and the plates were incubated at 4°C for 60 min. The plates were washed twice with FACS buffer, and then 100 μL of R-phycoerythrin-streptavidin (diluted 1:500 with FACS buffer, Cat#:016-110-084, Jackson Immuno Research) was added to each well, and the plates were incubated at 4°C in the dark for 40 min. The cells were washed twice and resuspended in FACS buffer. Fluorescence was detected using a Becton Dickinson FACS Canto II-HTS instrument, and a curve was plotted with MFI (mean fluorescence intensity) on the ordinate and the concentration of recombinant fusion protein or control on the x-axis. Analyze the data using Graphpad Prism and derive the IC. 50 Value. Result as follows Figure 6 and Figure 7 As shown.

[0161] from Figure 4 It can be seen that the recombinant fusion protein described in this disclosure can 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 activities are higher than those of the positive controls SIRPαV2D1M1-Fc(IgG2) and SIRPαV2D1M2-Fc(IgG2).

[0163] also, Figure 6 and Figure 7 The recombinant fusion proteins described in this disclosure are shown to block the binding of human SIRPα to human CD47 on the cell surface, with most exhibiting blocking activity comparable to the positive control. Furthermore, BSI038×S-004 and BSI038×S-005 exhibited higher CD47-SIRPα blocking activity than BSI038×S-002. Example 6: Functional Detection of Exemplary Recombinant Fusion Protein Based on Cells 6.1 Detection of CD40 agonist activity in cell-based reporter gene assays

[0164] The CD40 agonistic activity of the recombinant fusion protein described herein was detected using 293T-NF-κB-Luc-CD40 reporter cells stably expressing full-length human CD40 (uniprot No. P25942-1). Following the instructions of the Lipofectamine 3000 transfection reagent (Thermo Fisher), pCMV-TP plasmid containing DNA encoding human CD40 inserted between the EcoRI and XbaI sites was transfected into 293F cells to prepare 293T-NF-κB-Luc-CD40 cells. When these cells were exposed to a CD40 agonist, CD40 signaling was activated, and luciferase expression was upregulated by the NF-κB transcription factor, which could be measured by fluorescence analysis. The recombinant fusion protein SIRPα-Fc-CD40L with the amino acid sequence shown in SEQ ID NO:26 was used as a control (see 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] In short, 293T-NF-κB-Luc-CD40 cells in logarithmic growth phase were resuspended in DMEM medium (Cat#:10566-016, Gibco) containing 10% FBS (Cat#:10099-141, Gibco) and added at 20 μL / well to 384-well plates (Cat#:3707, Corning), with 5 × 10⁶ cells per well. 3 Cells were then added to each well. 20 μL of serially diluted recombinant fusion protein of this disclosure or a control (initial concentration 200 nM, serially diluted 3-fold using culture medium) was then added to each well, and the cells were incubated at 37°C for 6 hours. Then 30 μL of ONE-Glo was added to each well. TM The luciferase assay reagent was incubated at room temperature for 5 minutes. Tecan was used. Chemiluminescence was detected using a 200Pro. Data were analyzed using a Graphpad Prism to derive EC. 50 value.

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

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

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

[0169] In short, according to the instructions, use EasySep without removing CD16. TM Human monocyte enrichment kit (Cat#:19058, Stemcell) was used to isolate monocytes from frozen human PBMCs and seed them in 6-well plates containing RPMI 1640 + 10% FBS + 1% penicillin-streptomycin + 75 ng / ml human M-CSF for 6 days. Fresh cell culture medium was added on day 3. On day 6, macrophages were isolated and reseeded in 96-well plates and cultured overnight.

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

[0171] use Cell separation medium was used to collect all cells from the plate, and the cells were washed once with FACS buffer. Subsequently, Human TruStain FcX was used. TM Cells were blocked with Fc receptor blocking solution (Cat#:422302, Biolegend) and then stained with anti-human CD11b APC (Cat#:301310, Biolegend). Phagocytosis rate (%), or CFSE, was determined by flow cytometry. + Groups and CD11b + The proportion of the group (including those that phagocytose CFSE) + CD11b of cells + Macrophage count / CD11b + (Total number of macrophages).

[0172] The results are as follows Figure 9 As shown.

[0173] It can be seen that BSI038×S-002, BSI038×S-004, and BSI038×S-005 can all induce macrophages to phagocytose tumor cells, and their activity is superior to that of SIRPα-Fc-CD40L. Example 7: Thermal stability of an exemplary recombinant fusion protein

[0174] The thermal stability of the recombinant fusion protein was tested. In short, GloMelt was used. TM The thermal displacement protein stability assay kit is used to analyze the thermal displacement of proteins to determine the Tm value (melting temperature). In short, let GloMelt... TM The dye was thawed and brought to room temperature, then the vial containing the dye was vortexed and centrifuged. Next, 10× dye was prepared by adding 5 μL of 200× dye to 95 μL of PBS. Then, 2 μL of 10× dye and 10 μg of the recombinant fusion protein or control described herein 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), with the melting curve program set according to the parameters in Table 4. Table 4 Melting Curve Program Parameters Procedure Steps temperature gradient Duration initial temperature 25℃ NA 30s Melting curve 25-99℃ 0.1℃ / s NA Table 5 Melting temperatures of recombinant fusion proteins

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

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

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

[0178] Several preferred embodiments of the present invention have been described in detail above. It should be understood that the invention as defined in the above paragraphs is not limited to the specific details set forth in the above description, as 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) Anti-CD40 antibody or its antigen-binding fragment, and (b) CD47 binding domain, wherein the CD47 binding domain is the first Ig-like extracellular domain (SIRPαV2D1) of human SIRPα isoform 2.

2. The recombinant fusion protein according to claim 1, wherein 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 variable region comprises VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, 2 and 3, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, 5 and 6.

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

8.

4. The recombinant fusion protein according to claim 2 or 3, wherein the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2 or IgG4.

5. 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 2-5, 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 1-7, wherein the CD47 binding domain is linked to the N-terminus or C-terminus of the anti-CD40 antibody or its antigen-binding fragment.

9. The recombinant fusion protein according to any one of claims 1-8, wherein 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.

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

11. The recombinant fusion protein of 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 via a linker, or to the C-terminus of the heavy chain constant region or the light chain constant region via a linker.

12. The recombinant fusion protein according to claim 10 or 11, 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 1-12, 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; 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 1-14, comprising: i) Anti-CD40 antibody heavy chain variable region-heavy chain constant region-linker-SIRPαV2D1 polypeptide chain, which contains the amino acid sequence shown in SEQ ID NO:16; and anti-CD40 antibody light chain variable region-light chain constant region polypeptide chain, which contains the amino acid sequence shown in SEQ ID NO:

20. ii) A 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; 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 containing 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 containing the amino acid sequence shown in SEQ ID NO:

18.

16. The recombinant fusion protein according to any one of claims 1-15, comprising: i) A first polypeptide chain comprising, from the N-terminus to the C-terminus, a variable region of the anti-CD40 antibody heavy chain, a constant region of the heavy chain, a linker, and SIRPαV2D1, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:16; The second polypeptide chain comprises, from the N-terminus to the C-terminus, a variable region of the anti-CD40 antibody heavy chain, a constant region of the heavy chain, a linker, and SIRPαV2D1, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:

16. The third polypeptide chain comprises a variable region of the anti-CD40 antibody light chain and a constant region of the light chain from the N-terminus to the C-terminus, and the third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:20; and The fourth polypeptide chain, from the N-terminus to the C-terminus, includes a variable region of the anti-CD40 antibody light chain and a constant region of the light chain, and the fourth polypeptide chain contains the amino acid sequence shown in SEQ ID NO:20; ii) A first polypeptide chain comprising, from the N-terminus to the C-terminus, SIRPαV2D1, a linker, a variable region of the anti-CD40 antibody heavy chain, and a constant region of the heavy chain, wherein the first polypeptide chain comprises 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; The second polypeptide chain, from the N-terminus to the C-terminus, comprises SIRPαV2D1, a linker, a variable region of the anti-CD40 antibody heavy chain, and a constant region of the heavy chain. The second polypeptide chain comprises 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. The third polypeptide chain comprises a variable region of the anti-CD40 antibody light chain and a constant region of the light chain from the N-terminus to the C-terminus, and the third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:20; and The fourth polypeptide chain, from the N-terminus to the C-terminus, includes a variable region of the anti-CD40 antibody light chain and a constant region of the light chain, and the fourth polypeptide chain contains the amino acid sequence shown in SEQ ID NO:20; iii) A first polypeptide chain comprising a variable region and a constant region of the anti-CD40 antibody heavy chain from the N-terminus to the C-terminus, the first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:19; The second polypeptide chain includes a variable region and a constant region of the anti-CD40 antibody heavy chain from the N-terminus to the C-terminus, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:19; The third polypeptide chain, from the N-terminus to the C-terminus, includes a variable region of the anti-CD40 antibody light chain, a constant region of the light chain, a linker, and SIRPαV2D1, and the third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:18; and The fourth polypeptide chain, from the N-terminus to the C-terminus, includes a variable region of the anti-CD40 antibody light chain, a constant region of the light chain, a linker, and SIRPαV2D1, and the fourth polypeptide chain contains the amino acid sequence shown in SEQ ID NO:18; Specifically, 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 1-16.

18. 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 1-16, the nucleic acid of claim 17, the expression vector of claim 18, or the host cell of claim 19, and a pharmaceutically acceptable vector.

21. Use of the pharmaceutical composition of 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 hematoma.

24. The use according to claim 23, wherein the cancer is leiomyosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, colon cancer, kidney cancer, prostate cancer, cervical cancer, nasopharyngeal carcinoma, breast cancer, osteosarcoma, squamous cell carcinoma of the head and neck, 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 1-16, 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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