Recombinant fusion protein targeting CD40 and CD47

Abstract

The present disclosure provides a recombinant fusion protein comprising (a) an anti-CD40 antibody or an antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region, and (b) a CD47-binding domain. The present disclosure provides a nucleic acid molecule encoding the recombinant fusion protein, a vector comprising the nucleic acid, and a host cell transformed or transfected with the vector or such nucleic acid. Further, the present disclosure provides a method for producing the recombinant fusion protein, a medical use of the recombinant fusion protein, and a kit comprising the recombinant fusion protein.

Description

Technical Field

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

[0002] All documents cited or referenced in this specification (including, but not limited to, all documentary documents, patents, and published patent applications cited in this specification) (the "three cited documents") are hereby incorporated by reference together with the manufacturer's instructions, descriptions, product specifications, and product sheets for any product described in the documents described or incorporated by reference herein and can be employed in the practice of the present invention. More specifically, all documents referenced are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequence referred to in this disclosure is incorporated by reference by taking the Genbank sequence as of the earliest effective filing date of this disclosure.

[0003] This disclosure generally relates to recombinant fusion proteins that bind to CD40 and CD47 with high affinity and functionality. Also provided are nucleic acid molecules encoding the recombinant fusion proteins, expression vectors, host cells, and methods for expressing the recombinant fusion proteins. This disclosure further provides pharmaceutical compositions that may contain the recombinant fusion proteins, as well as methods of treatment using the recombinant fusion proteins of this disclosure.

Background Art

[0004] CD40 and CD40L CD8+ cytotoxic T lymphocyte (CTL) responses play an important role in immunity against cancer and infections. However, an effective CTL response is triggered when an antigen-specific T cell receptor binds to a major histocompatibility complex (MHC) loaded with peptides on an antigen-presenting cell, followed by the involvement of co-stimulatory molecules (Zhu Y et al., (2011) Immunity. 34(4):466 - 478).

[0005] CD40 and its major ligand CD40L are such a pair of costimulatory molecules. CD40 is a type I transmembrane protein that was first identified as a cell surface marker on B lymphocytes and bladder tumor cells and was later found to be expressed in antigen-presenting cells such as macrophages, dendritic cells, and monocytes, while CD40L is mainly expressed in activated CD4+ T cells (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 its cytoplasmic domain, promoting signal transduction (Ma DY et al., (2009) Semin Immunol 21(5):265-272). CD 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, resulting in licensing of dendritic cells and replacement of the "CD4 + T cell help" required to drive the CD8+ T cell response (Ara A et al., (2018) Immunotargets Ther7:55-61). Immunostimulation by CD40 signals may be independent of innate immune receptors such as Toll-like receptors (Byrne KT & Vonderheide RH (2016) Cell Rep.15:2719-2732). It may also have the potential to convert cold tumors into hot tumors and enhance sensitivity to checkpoint inhibition therapy (Vonderheide RH (2020) Annu Rev Med.71:47-58).

[0006] The expression of CD40 has also been observed in tumor cells such as B-cell malignancies, melanoma, lung cancer, bladder cancer, gastric cancer, breast cancer, and ovarian cancer. Studies have shown that CD40-CD40L interaction may, for example, suppress the proliferation of tumor cells 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). Although there are also reports that CD40 signaling in tumor cells promotes tumor growth, it may at least enhance tumor antigen presentation.

[0007] Agonist antibodies against CD40 have been developed for the treatment of diseases. Selicrelumab (Pfizer and VLST) demonstrated clinical efficacy in patients with advanced melanoma in the first human single-dose trial (Vonderheide RH et al., (2007) J. Clin.Oncol.25:876-883; Bajor DL et al., (2014) Cancer Immunol.Res.2:1051-1058).

[0008] Biological agents that activate CD40 signaling have also been shown to be effective in the treatment of infectious diseases such as HIV-1 / AIDS, tuberculosis, and malaria (Elizabeth A Thompson, et al., (2015) J Immunol.195(3):1015-1024).

[0009] CD47 and SIRPα CD47 is a transmembrane protein expressed in many types of cells and plays roles in cell proliferation, migration, apoptosis, etc. (Ratnikova NM et al., (2017) Mol Biol.51(2):251-261). Its ligands also include signal regulatory protein α (SIRPα), also known as CD172a or Src homology 2 domain-containing phosphatase substrate 1, which is present in myeloid cells such as macrophages and dendritic cells. When binding to SIRPα, CD47 acts as a "don't eat me" signal to prevent phagocytosis of cells by macrophages. Cancer cells utilize such immune tolerance mechanisms to escape immune surveillance.

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

[0011] Studies have shown that by inhibiting CD47-SIRPα signaling, it induces the secretion of chemokines and cytokines that promote the recruitment of immune cells to tumors, induces macrophage-mediated phagocytosis of tumor cells, and antigen-specific CD8 +It has been shown that it may be possible to inhibit tumor growth by promoting the proliferation of T cells, reducing the number of regulatory T cells, enhancing antibody-dependent cytotoxicity such as NK cell-mediated cytotoxicity, and initiating apoptosis of tumor cells via a caspase-independent mechanism (Weiskopf K et al., (2016) J Clin Investig. 126:2610-2620; Tseng D et al., (2013) Proc Natl Acad Sci USA. 110:11103-11108; Kim MJ et al., (2008) Tumour Biol. 29:28-34). More specifically, blockade of the SIRPα-CD47 interaction promotes the phagocytosis of cancer cells by macrophages, and cancer cell-derived antigen peptides generated during phagocytosis may subsequently initiate an adaptive immune response (Chen, J. et al. (2017) 544:493-497).

[0012] Several CD47-targeted therapeutic agents, such as anti-CD47 antibodies and SIRPα-Fc fusion proteins, are currently in clinical trials as therapeutic agents for solid tumors and hematological tumors (Zhang W et al., (2020) supra). The SIRPα-Fc fusion protein can include full-length SIRPα or one or more of its extracellular immunoglobulin superfamily domains.

[0013] Increased expression of CD47 and its immunosuppressive role have also been confirmed in immune cells during viral and bacterial infections, and CD47 blockade with anti-CD47 antibodies promoted the activation and effector functions of macrophages, dendritic cells, and T cells (Cham LB et al., (2020) Antibodies (Basel) 9(3):44).

[0014] Bispecific or multispecific fusion proteins CD47 is widely expressed in human cells, especially at high levels in hematopoietic cells. Therefore, monospecific anti-CD47 antibodies or proteins can bind to normal cells, especially red blood cells that account for about half of the blood volume, and may cause harmful side effects such as severe anemia. For this reason, certain clinical trials have been terminated.

[0015] Bispecific or multispecific fusion proteins are designed to bind CD47 and another antigen (e.g., CD40), and by more precisely targeting tumor cells and / or immune cells in the microenvironment, it may be possible to reduce or eliminate off-target effects.

[0016] The citation or identification of any document in this application does not admit that such document is available as prior art for the present invention.

Summary of the Invention

[0017] The inventors of the present application designed and prepared a recombinant fusion protein that binds to both CD40 and CD47. Here, the recombinant fusion protein exhibits a low level of aggregation and has an equivalent or higher binding affinity for human and monkey CD40 proteins, an equivalent or higher blocking activity against CD40 - CD40L binding, an equivalent or higher blocking activity against CD47 - SIRPα binding, an equivalent or higher agonist activity against CD40 signaling, and an equivalent or higher ability to induce phagocytosis of CD47+ cells, compared to their monospecific counterparts and prior art antibodies such as cerulimumab.

[0018] The recombinant fusion protein of the present disclosure can be used in in vitro and in vivo assays and treatments for diseases related to CD40 and / or CD47 signaling such as tumors.

[0019] In a first aspect, the present disclosure provides a recombinant fusion protein that binds to both CD40 and CD47, which may comprise an anti-CD40 antibody or an antigen-binding portion thereof, and a CD47 binding domain.

[0020] An anti-CD40 antibody or an antigen-binding portion thereof may comprise a heavy chain variable region and a light chain variable region. In certain embodiments, the anti-CD40 antibody or an antigen-binding portion thereof may comprise two identical heavy chain variable regions and two identical light chain variable regions. In certain 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.

[0021] The anti-CD40 antibody or an antigen-binding portion thereof 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 an antigen-binding portion thereof may have agonist activity in CD40 signaling.

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

[0024] The light chain variable region of the anti-CD40 antibody or its antigen-binding portion may include VL CDR1, VL CDR2, and VL CDR3 that include the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. The light chain variable region may include 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 light chain variable region may include the amino acid sequence of SEQ ID NO: 8. The light chain constant region may be a κ light chain constant region or a λ light chain constant region. In certain embodiments, the light chain constant region may have, for example, the amino acid sequence of SEQ ID NO: 10.

[0025] The anti-CD40 antibody or its antigen-binding portion may include a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region includes VL CDR1, VL CDR2, and VL CDR3, where VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 may include the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. The heavy chain variable region may include 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 light chain variable region may include 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 heavy chain variable region may include the amino acid sequence of SEQ ID NO: 7. The light chain variable region may include the amino acid sequence of SEQ ID NO: 8.

[0026] In certain embodiments, the anti-CD40 antibody or its antigen-binding portion may include a heavy chain and a light chain. In certain embodiments, the anti-CD40 antibody or its antigen-binding portion may include two identical heavy chains and two identical light chains.

[0027] The heavy chain of an anti-CD40 antibody or an antigen-binding portion thereof may include a heavy chain variable region and a heavy chain constant region. Here, the heavy chain variable region and the heavy chain constant region may include the amino acid sequences described above. The heavy chain may include 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 may include the amino acid sequence of SEQ ID NO: 19.

[0028] The light chain of an anti-CD40 antibody or an antigen-binding portion thereof may include a light chain variable region and optionally a light chain constant region. Here, the light chain variable region and the light chain constant region may include the amino acid sequences described above. The light chain may include 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 may include the amino acid sequence of SEQ ID NO: 20.

[0029] The heavy chain may include 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 light chain may include 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 certain embodiments, the heavy chain may include the amino acid sequence of SEQ ID NO: 19, and the light chain may include the amino acid sequence of SEQ ID NO: 20.

[0030] The CD47-binding domain can be human SIRPα or a part thereof. In some embodiments, the CD47-binding domain can be the first Ig-like extracellular domain of human SIRPα. In some embodiments, the CD47-binding domain is wild-type or a SIRPα isoform 2 (SIRPαV2) having a mutation. In certain embodiments, the CD47-binding domain is the first Ig-like extracellular domain of SIRPαV2 (SIRPαV2D1). In certain embodiments, SIRPαV2D1 is wild-type having, for example, the amino acid sequence of SEQ ID NO: 11 (X1 = V, X2 = K, X3 = S, X4 = K, X5 = F). In certain embodiments, SIRPαV2D1 is a variant having high CD47-binding affinity / ability and having, for example, the amino acid sequence of 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 an anti-CD40 antibody or an antigen-binding portion thereof. In certain embodiments, the anti-CD40 antibody or an antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, 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 light chain variable region, or the C-terminus of the heavy chain constant region or light chain constant region. In certain embodiments, the CD47-binding domain is linked to the N-terminus of the heavy chain variable region, or the C-terminus of the heavy chain constant region or light chain constant region. In certain embodiments, the anti-CD40 antibody or an antigen-binding portion thereof comprises 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, or the N-terminus or C-terminus of the light chain. In certain embodiments, the anti-CD40 antibody or an antigen-binding portion thereof comprises two identical heavy chains and two identical light chains, and the CD47-binding domain is linked to the N-terminus or C-terminus of each heavy chain, or the N-terminus or C-terminus of each light chain. In certain embodiments, the anti-CD40 antibody or an antigen-binding portion thereof comprises two identical heavy chains and two identical light chains, and the CD47-binding domain is linked to the N-terminus or C-terminus of each heavy chain, or the C-terminus of each light chain.

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

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

[0034] The recombinant fusion protein of the present disclosure may comprise: i) A first polypeptide chain and a second polypeptide chain comprising an anti-CD40 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 light chain variable region, and optionally a light chain constant region; or ii) A first polypeptide chain and a second polypeptide chain comprising an anti-CD40 heavy chain variable region and a heavy chain constant region, And a third polypeptide chain and a fourth polypeptide chain comprising an anti-CD40 light chain variable region, optionally a light chain constant region, and SIRPαV2D1, Wherein the heavy chain variable region in the first polypeptide chain and the light chain variable region in the third polypeptide chain bind to form a CD40 binding domain, Wherein the heavy chain variable region in the second polypeptide chain and the light chain variable region in the fourth polypeptide chain bind to form a CD40 binding domain, Wherein the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the second polypeptide chain are associated.

[0035] In certain embodiments, the recombinant fusion protein may comprise: i) A first polypeptide chain and a second polypeptide chain comprising an anti-CD40 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 light chain variable region, ii) A first polypeptide chain and a second polypeptide chain, respectively, comprising SIRPαV2D1, an anti-CD40 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, respectively, comprising an anti-CD40 light chain variable region, or iii) A first polypeptide chain and a second polypeptide chain from the N-terminus to the C-terminus, respectively, comprising an anti-CD40 heavy chain variable region and a heavy chain constant region, and a third polypeptide chain and a fourth polypeptide chain from the N-terminus to the C-terminus, respectively, comprising an anti-CD40 light chain variable region and SIRPαV2D1, wherein the heavy chain variable region in the first polypeptide chain and the light chain variable region in the third polypeptide chain bind to form a CD40 binding domain, wherein the heavy chain variable region in the second polypeptide chain and the light chain variable region in the fourth polypeptide chain bind to form a CD40 binding domain, characterized in that the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the second polypeptide chain are associated.

[0036] The recombinant fusion protein of the present disclosure may, in certain embodiments, comprise: i) A first polypeptide chain and a second polypeptide chain comprising an anti-CD40 heavy chain variable region, a heavy chain constant region, and SIRPαV2D1, and a third polypeptide chain and a fourth polypeptide chain from the N-terminus to the C-terminus, respectively, comprising an anti-CD40 light chain variable region and a light chain constant region, ii) A first polypeptide chain and a second polypeptide chain from the N-terminus to the C-terminus, respectively, comprising SIRPαV2D1, an anti-CD40 heavy chain variable region, and a heavy chain constant region, and a third polypeptide chain and a fourth polypeptide chain from the N-terminus to the C-terminus, respectively, comprising an anti-CD40 light chain variable region and a light chain constant region iii) A first polypeptide chain and a second polypeptide chain from the N-terminus to the C-terminus, respectively, comprising an anti-CD40 heavy chain variable region and a heavy chain constant region, a third polypeptide chain and a fourth polypeptide chain from the N-terminus to the C-terminus, respectively, comprising an anti-CD40 light chain variable region, a light chain constant region, and SIRPαV2D1, wherein the heavy chain variable region in the first polypeptide chain and the light chain variable region in the third polypeptide chain bind to form a CD40 binding domain, Here, the heavy chain variable region in the second polypeptide chain and the light chain variable region in the fourth polypeptide chain bind to form a CD40 binding domain, characterized in that the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the second polypeptide chain are associated.

[0037] In certain embodiments, the recombinant fusion protein of the present disclosure may include: i) a first polypeptide chain and a second polypeptide chain comprising an anti-CD40 heavy chain variable region, a heavy chain constant region, a linker, and SIRPαV2D1, and a third polypeptide chain and a fourth polypeptide chain each comprising an anti-CD40 light chain variable region and a light chain constant region, from N-terminus to C-terminus, ii) a first polypeptide chain and a second polypeptide chain from N-terminus to C-terminus comprising SIRPαV2D1, a linker, an anti-CD40 heavy chain variable region, and a heavy chain constant region, and a third polypeptide chain and a fourth polypeptide chain each comprising an anti-CD40 light chain variable region and a light chain constant region, from N-terminus to C-terminus iii) a first polypeptide chain and a second polypeptide chain each comprising an anti-CD40 heavy chain variable region and a heavy chain constant region, from N-terminus to C-terminus, a third polypeptide chain and a fourth polypeptide chain comprising an anti-CD40 light chain variable region, a light chain constant region, a linker, and SIRPαV2D1, wherein the heavy chain variable region in the first polypeptide chain and the light chain variable region in the third polypeptide chain bind to form a CD40 binding domain, wherein the heavy chain variable region in the second polypeptide chain and the light chain variable region in the fourth polypeptide chain bind to form a CD40 binding domain, characterized in that the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the second polypeptide chain are associated.

[0038] In certain embodiments, the recombinant fusion protein of the present disclosure may include: i) a first polypeptide chain and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 16, and a third polypeptide chain and a fourth polypeptide chain each comprising the amino acid sequence of SEQ ID NO: 20 ii) a first polypeptide chain and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17 (X1 = V, X2 = K, X3 = S, X4 = K, X5 = F; 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 each comprising the amino acid sequence of SEQ ID NO: 20 iii) a first polypeptide chain and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 19, and a third polypeptide chain and a fourth polypeptide chain each comprising the amino acid sequence of SEQ ID NO: 20

[0039] This application also provides a nucleic acid molecule encoding the recombinant fusion protein of the present disclosure, an expression vector that can constitute such a nucleic acid, and a host cell transformed or transfected with such an expression vector or such a nucleic acid. A method for preparing the recombinant fusion protein of the present disclosure using the host cell of the present disclosure is also provided, which may include (i) the step of expressing the recombinant fusion protein in the host cell, and (ii) the step of isolating the recombinant fusion protein from the host cell or its cell culture.

[0040] This application also provides a pharmaceutical composition that may include the recombinant fusion protein, nucleic acid molecule, expression vector, or host cell of the present disclosure, and a pharmaceutically acceptable carrier. The pharmaceutical composition may further include additional agents such as anti-tumor agents.

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

[0042] The disease can be cancer. The cancer can be 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 cancer, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, lung cancer (including small cell lung cancer and non-small cell lung cancer), multiple myeloma, melanoma, hepatocellular carcinoma, liver cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), and gastric cancer. In certain embodiments, the pharmaceutical compositions of the present disclosure can be used in combination with chemotherapy and checkpoint inhibition therapies, such as anti-PD-L1 antibodies, anti-PD-1 antibodies, or anti-CTLA-4 antibodies.

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

[0044] In certain embodiments, the subject is human.

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

[0046] In certain embodiments, the subject is human.

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

[0048] In certain embodiments, the subject is human.

[0049] Also provided is the use of the pharmaceutical compositions of the present disclosure in the treatment of diseases associated with CD40 and / or CD47 signaling, the enhancement of immune responses, and / or the restoration or reduction of immunosuppression.

[0050] In a fourth aspect, the present application provides a kit that may include the recombinant fusion protein, pharmaceutical composition, nucleic acid molecule, expression vector, or host cell of the present disclosure. The term "kit" refers to two or more components corresponding to any one of the recombinant fusion protein, pharmaceutical composition, nucleic acid molecule, expression vector, or host cell of the present disclosure being packaged together in a container, receptacle, or other manner. A kit can be described as a set of products and / or instruments sufficient to achieve a particular purpose and can be sold as a single unit.

[0051] The kit may include one or more receptacles (vials, ampoules, containers, syringes, bottles, bags, etc.) of any suitable shape, size, and material (preferably waterproof, such as plastic or glass). The receptacle contains the disclosed recombinant fusion protein or pharmaceutical composition. The kit may further include instructions for use (such as a leaflet or a user manual).

[0052] Other features and advantages of the instant disclosure will become apparent from the following detailed description and examples, which should not be construed in a limiting sense. The contents of all documents, Genbank entries, patents, and published patent applications cited throughout this application are hereby expressly incorporated herein by reference.

[0053] Accordingly, it is an object of the present invention not to encompass any previously known product, method of manufacturing a product, or method of using a product, and the applicant reserves the right to disclaim any previously known product, process, or method and what is disclosed herein. Further, the present invention does not intend to include within the scope of the invention any product, process, or method of manufacturing a product or method of using a product that does not meet the written description requirements and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), and the applicant discloses and reserves the right to disclaim the previously described product, method of manufacturing a product, or method of using a product. In the practice of the invention, it may be advantageous to comply with Art. 53(c). It is advantageous in the practice of the invention to comply with 53(c) EPC as well as Rules 28(b) and (c) EPC. All rights to expressly deny embodiments that are subjects of patents granted to the applicant in the line of this application, other lines, or prior applications of third parties are expressly reserved. Nothing in this document shall be construed as a commitment.

[0054] It should be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as "comprises," "composed," "comprising," etc. can have the meaning ascribed in U.S. patent law. For example, they mean "includes," "is included," "contains," etc., and terms such as "consisting essentially of," "consisting essentially of," etc. have the meaning ascribed in U.S. patent law. For example, they allow elements not expressly recited, but exclude elements found in the prior art or elements that affect the basic or novel characteristics of the present invention.

Brief Description of the Drawings

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

[0056]

Figure 1

[0057]

Figure 2

[0058]

Figure 3

[0059]

Figure 4

[0060]

Figure 5

[0061]

Figure 6

[0062]

Figure 7

[0063]

Figure 8

[0064]

Figure 9

[0065]

Figure 10

BEST MODE FOR CARRYING OUT THE INVENTION

[0066] To make the present disclosure more readily understandable, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0067] The term "CD40" refers to differentiation antigen 40. The term "CD40" includes variants, isoforms, homologs, orthologs and paralogs. For example, an antibody specific for the human CD40 protein may in certain cases cross-react with CD40 proteins from non-human species such as monkeys. In other embodiments, an antibody specific for the human CD40 protein is completely specific for the human CD40 protein and does not show cross-reactivity with other species or other types, or may cross-react with CD40 from certain other species but not with all other species.

[0068] The term "human CD40" refers to a CD40 protein having an amino acid sequence derived from a human, such as the amino acid sequence of human CD40 having NCBI reference number NP_001241.1 (Sasaki K et al., (2021) J Exp Clin Cancer Res 40(1):212). The term "monkey CD40" or "dog CD40" refers to a CD40 protein having an amino acid sequence derived from the monkey species.

[0069] The term "SIRPα" refers to wild-type signal regulatory protein α, or a recombinant or non-recombinant polypeptide having the amino acid sequence of wild-type signal regulatory protein α, or a natural or naturally occurring allelic variant of signal regulatory protein α, or an artificial variant of signal regulatory protein α. In one embodiment, SIRPα is wild-type mammalian SIRPα, but in a preferred embodiment, SIRPα is wild-type human SIRPα. The term "human SIRPα" refers to an SIRPα protein having an amino acid sequence derived from a human, such as the amino acid sequence having GenBank accession number: AAH75849.1 (Strausberg R.L. et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99(26):16899-16903). In one embodiment, SIRPα includes a signal sequence, and in another embodiment, SIRPα refers to the mature form of the protein. Ten human SIRPα alleles have been discovered to date, and human SIRPα isoform 2 (or V2) has been reported to have a reduced or minimal binding affinity for erythrocytes.

[0070] As used herein, the term "bispecific" refers to a fusion protein and a binding molecule comprising at least first and second binding domains, wherein the first binding domain is capable of binding to one antigen or target, and the second binding domain is capable of binding to another antigen or target. Thus, the bispecific fusion protein according to the present application includes binding specificities for at least two different antigens or targets and is at least bispecific. The "bispecific fusion protein" of the present application also includes multispecific binding molecules such as trispecific binding molecules, the latter of which includes three binding domains. In addition, it is also contemplated that the bispecific fusion protein of the present application has additional functions in addition to the function of binding to the target molecules CD40 and CD47.

[0071] The term "immune response" refers to, for example, the action of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, as a result of which invading pathogens, cells or tissues infected with pathogens, cancer cells, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues are selectively damaged, destroyed, or eliminated from the human body.

[0072] The term "immunosuppression" or "immunosuppressive" refers to a decrease in the activation or efficacy of the immune system caused by, for example, aging, persistent diseases, malnutrition, cancer, chemotherapy, or radiotherapy. Immunosuppression can, in certain situations, be reversed, for example, by manipulating some of the pathways.

[0073] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as CD40, through at least one antigen-binding site, where the antigen-binding site is typically within the variable region of the immunoglobulin molecule. As used herein, this term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-chain Fv (scFv) antibodies, heavy-chain antibodies (HCAbs), light-chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monovalent antibodies, fusion proteins containing the antigen-binding site of an antibody, and any other modified immunoglobulin molecule containing an antigen-binding site, and further, as long as the antibody exhibits the desired biological activity, it is an antigen-binding site (e.g., a dual variable domain immunoglobulin molecule). Also included in the antibodies are, but not limited to, mouse antibodies, chimeric antibodies, humanized antibodies, and human antibodies. The antibody can be any of the following five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Based on the identity of the heavy-chain constant domains called alpha, delta, epsilon, gamma, and mu. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional configurations. The antibody may be naked or conjugated to other molecules such as toxins or radioisotopes. Unless explicitly indicated otherwise, the term "antibody" as used herein includes the "antigen-binding portion" of an intact antibody. Conventional IgG is a glycoprotein in which two identical heavy chains (H) and two identical light chains (L) are linked by disulfide bonds. Each heavy chain may be composed of a heavy-chain variable region (hereinafter abbreviated as V H and a heavy-chain constant region C H in some cases. The heavy-chain constant region may be composed of three domains, CH1, CH2, and CH3. Each light chain may be composed of a light-chain variable region (hereinafter abbreviated as V L and a light-chain constant region C L in some cases. The light-chain constant region may be composed of one domain, CL. V H and V LThe region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and highly conserved regions called framework regions (FRs). V H and V L each consist of three CDRs and four FRs, and are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In the variable regions of the heavy and light chains, there are binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The "functional fragment" of the heavy chain constant region refers to a part of the constant region that maintains desired properties such as binding affinity to Fc receptors and / or complement system proteins.

[0074] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody means one or more fragments of the antibody that retain the ability to specifically bind to an antigen (e.g., CD40 protein). It has been shown that the antigen-binding function of an antibody can be exerted by fragments of the full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include: (i) monovalent Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, divalent fragments that can include two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of VH domains (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); (viii) nanobodies, heavy chain variable regions containing one variable domain and two constant domains. Furthermore, the two domains of an Fv fragment, V L and VH are encoded by separate genes, which can be joined using recombinant methods by a synthetic linker that causes the V L and V H regions to pair and form a single protein chain that makes a monovalent molecule (known as a single-chain Fv (scFv); e.g., 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 intended to be included within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques well known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0075] An "agonist" anti-CD40 antibody or antigen-binding portion, or "agonist activity" in CD40 signaling, refers to an anti-CD40 antibody or antigen-binding portion that binds to CD40 and activates / induces CD40 signaling to promote, for example, activation and proliferation of immune cells, and production of cytokines and chemokines. An agonist anti-CD40 antibody or antigen-binding portion thereof may enhance the natural and acquired immune responses against tumors in cancer subjects via, for example, increased antigen-presenting ability of APCs, activation of tumor-specific CD4 + and CD8 + T cells, secretion of cytokines and chemokines by lymphocytes and monocytes, enhanced ability of cytotoxic lymphocytes and NK cells to kill tumor cells, and the like.

[0076] As used herein, a recombinant fusion protein or bispecific fusion protein that "specifically binds to CD40 (such as human CD40)" refers to a fusion protein that binds to CD40 proteins (human CD40 and CD40 proteins derived from one or more non-human species), but does not substantially bind to non-CD40 proteins. Similarly, a fusion protein that "specifically binds to CD47 (e.g., human CD47)" refers to a fusion protein that binds to CD47 proteins of human or other non-human species, but does not bind to proteins other than CD47. Desirably, the fusion protein binds to CD40 or CD47 with "high affinity", i.e., KD is 5.0 x 110 -8 M or less, more desirably 1.0 x 10 -8 M or less.

[0077] As used herein, the term "does not substantially bind to" a protein or cell means that there is no binding to the protein or cell, or binding with low affinity, i.e., KD is 1.0×10 -6 M or more, more preferably 1.0×10 -5 M or more, still more preferably 1.0×10 -4 M or more, still more preferably 1.0×10 -3 M or more, even more preferably 1.0×10 -2 M or more, which means that there is binding to the protein or cell with a KD of 1.0×10

[0078] The term "high affinity" means that the KD for the target antigen is 1.0×10 -6 M or less, more preferably 1.0×10 -8 M or less, even more preferably 1.0×10 -9 M or less, even more preferably 1.0×10 -9 M or less.

[0079] As used herein, the term "K assoc " or "K a " is intended to refer to the association rate of a specific protein-protein interaction such as an antibody-antigen or receptor-ligand, while the term "K dis " or "Kd The term " " is intended to refer to the dissociation rate of a specific protein-protein interaction. As used herein, "K" D The term " " means the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is intended to be expressed as molar concentration (M). K D values can be determined using methods well established in the art. K D A preferred method for determining is a method using surface plasmon resonance, preferably a method using a biosensor system such as a Biacore® system.

[0080] "EC" 50 The term " " is also called the half-maximal effective concentration and refers to the concentration of the fusion protein of the present disclosure that induces a response intermediate between the baseline maximum value after a specific exposure time.

[0081] "IC" 50 The term " " is also called the half-inhibitory concentration and refers to the concentration of the fusion protein of the present disclosure that inhibits a specific biological or biochemical function by 50% relative to the absence of the fusion protein.

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

[0083] The term "therapeutically effective amount" refers to the amount of the fusion protein of the present disclosure sufficient to prevent or ameliorate symptoms associated with a disease or condition (such as cancer) and / or reduce the severity of the disease or condition. A therapeutically effective amount is understood in the context of the condition being treated, and the actual effective amount can be readily identified by one of ordinary skill in the art.

[0084] As used herein, the term "percent identity" in the context of two or more nucleic acids or polypeptides refers to the specified percentage of identical sequences or subsequences, or nucleotides or amino acid residues, that are identical when compared and aligned (with gaps inserted as appropriate) to maximize matches, either with or without considering maximum matches. Percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software available for obtaining alignments of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the present disclosure are substantially identical, which means that when compared and aligned using a sequence comparison algorithm or by visual inspection to obtain maximum matches, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity.

[0085] It should be noted that as used herein, the singular forms of "a", "an", and "the" include the plural forms as well, unless the context clearly dictates otherwise. Thus, for example, the expression "a reagent" includes one or more different reagents, and the expression "the method" includes equivalent steps and methods known to those skilled in the art that are modifications or alternatives to the methods described herein.

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

[0087] The recombinant fusion proteins of the present disclosure may include the following: (a) an anti-CD40 antibody or a binding portion of such an antibody, and (b) a CD47 binding domain. The CD47 binding domain may be optionally bound to the anti-CD40 antibody or its antigen-binding portion via a linker.

[0088] The recombinant fusion proteins of the present disclosure exhibit low levels of aggregation and have equal or higher binding affinity for human and monkey CD40 proteins, equal or higher blocking activity against CD40-CD40L binding, equal or higher blocking activity against CD47-SIRPα binding, equal or higher agonist activity against CD40 signaling, and equal or higher ability to induce phagocytosis of CD47+ cells (e.g., cancer cells including Jurkat cells or HL-60 cells), compared to their single specific counterparts and prior art antibodies such as cetuximab.

[0089] The three components in the recombinant fusion proteins of the present application are a CD47 binding domain, a linker, and an anti-CD40 antibody or its antigen-binding portion. One skilled in the art will recognize that there are many design options in selecting the above three components. The CD47 binding domain may be linked, for example, to the N-terminus or C-terminus of the anti-CD40 antibody or antigen-binding portion. Preferably, human-derived sequences are used for human cancer treatment because the strong immunogenicity of proteins or peptides derived from non-human animals may cause allergies and other side effects. However, based on different application purposes, other animal proteins or peptides, and humanized ones if appropriate, can also be used in the present application.

[0090] The CD47 binding domain can be any protein or peptide capable of binding to CD47, such as SIRPα, a SIRPα variant, or an affinity-optimized variant of SIRPα. A "variant" of SIRPα is defined as an amino acid sequence of SIRPα in which one or more amino acids are changed compared to wild-type SIRPα. The variant may have "conservative" changes, where the substituted amino acid has similar structural or chemical properties, such as the substitution of leucine with isoleucine. More rarely, the variant may have "non-conservative" changes, such as the substitution of glycine with tryptophan. Similar minor changes may also include deletions or insertions of amino acids, 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 more sequence identity.

[0091] In certain embodiments, the CD47 binding domain can be the first Ig-like extracellular domain of human SIRPα isoform 2 (SIRPαV2D1). SIRPαV2D1 can be wild-type SIRPαV2D1, or a SIRPαV2D1 variant modified to enhance its binding affinity / ability to bind CD47. In certain embodiments, SIRPαV2D1 can be wild-type and contain an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 (X1 = V, X2 = K, X3 = S, X4 = K, X5 = F). In certain embodiments, SIRPαV2D1 can be wild-type and contain the amino acid sequence of SEQ ID NO: 11 (X1 = V, X2 = K, X3 = S, X4 = K, X5 = F). In certain embodiments, SIRPαV2D1 can be a SIRPαV2D1 variant containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 (X1 = I, X2 = R, X3 = T, X4 = K, X5 = F; or X1 = I, X2 = R, X3 = T, X4 = R, X5 = V). In certain embodiments, SIRPαV2D1 can be a SIRPαV2D1 variant containing the amino acid sequence of 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 an amino acid sequence of SIRPαV2D1 in which one or more amino acids have changed as compared to wild-type SIRPαV2D1. The variant may have "conservative" changes, where the substituted amino acid has similar structural or chemical properties, such as substitution of leucine with isoleucine. More rarely, the variant may have "non-conservative" changes, such as substitution of glycine with tryptophan. Similar minor changes may also include amino acid deletions or insertions, or both. In one embodiment, the SIRPαV2D1 variant contains a wild-type SIRPαV2D1 polypeptide having at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity.

[0092] The linker mainly functions as a spacer between the CD47 binding site and the anti-CD40 antibody or antigen-binding site. The linker is composed of amino acids linked by peptide bonds and preferably consists of 5 to 30, 10 to 30, 10 to 20, or 15 amino acids, and the amino acids are selected from the 20 naturally occurring amino acids. As will be understood by those skilled in the art, 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 configured such that most of the amino acids have no steric hindrance, such as glycine and alanine. Exemplary linkers include polyglycine (especially (Glys, poly(Gly-Ala)), and -GGGGS- (SEQ ID NO: 12), -GGGGSGGGGS- (SEQ ID NO: 13), -GGGGSGGGGSGGGGS- (SEQ ID NO: 14), and -GGGGSGGGGSGGGGSGGGGS- (SEQ ID NO: 15). The linker may be a non-peptide linker. For example, alkyl linkers such as -NH-, -(CH 2 )s-C(O)- (s = 2 - 20) can be used. These alkyl linkers may further be substituted with a non-steric hindrance group such as lower alkyl (e.g., C 1-4 ) lower acyl, halogen (e.g., CI, Br), CN, NH 2 , phenyl, etc. In other embodiments, the recombinant fusion protein of the present application can be assembled in the absence of a linker.

[0093] The CD40 antibody can be an isolated monoclonal antibody as disclosed in WO2021 / 197335. The anti-CD40 antibody or antigen-binding portion thereof of the present disclosure can be humanized.

[0094] The anti-CD40 antibody or antigen-binding portion thereof of the present disclosure may include a heavy chain variable region and a light chain variable region. In certain embodiments, the anti-CD40 antibody or antigen-binding portion thereof may include two identical heavy chain variable regions and two identical light chain variable regions. In certain 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 portion thereof of the present disclosure may include a heavy chain and a light chain. In certain embodiments, the anti-CD40 antibody or antigen-binding portion thereof of the present disclosure includes 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. The light chain may include a light chain variable region and optionally a light chain constant region.

[0096] The heavy chain variable region of the present disclosure may include VH CDR1, VH CDR2, VH CDR3, each including the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. The heavy chain variable region may include 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 a human IgG1, IgG2, or IgG4 heavy chain constant region and may optionally be genetically engineered to have altered functional properties, such as changes in Fc receptor binding affinity. The heavy chain constant region may include 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% sequence identity with SEQ ID NO: 9. In certain embodiments, the heavy chain constant region may include the amino acid sequence of SEQ ID NO: 9.

[0098] The heavy chain variable region of the present disclosure may include VL CDR1, VL CDR2, and VL CDR3 that include the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. The light chain variable region may include 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 light chain constant region may be a human κ light chain constant region or a λ light chain constant region. The light chain constant region may include 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 certain embodiments, the light chain constant region may include the amino acid sequence of SEQ ID NO: 10.

[0099] The heavy chain variable region may include VH CDR1, VH CDR2, and VH CDR3, each containing the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. The light chain variable region may include VL CDR1, VL CDR2, and VL CDR3, each containing the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. The heavy chain variable region may include 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 light chain variable region may include 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 certain embodiments, the heavy chain variable region may include the amino acid sequence of SEQ ID NO: 7. The light chain variable region may include the amino acid sequence of SEQ ID NO: 8. The heavy chain constant region may include 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. The light chain constant region may include 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 certain embodiments, the heavy chain constant region may include the amino acid sequence of SEQ ID NO: 9. The light chain constant region may include the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the heavy chain variable region may include the amino acid sequence of SEQ ID NO: 7. The light chain variable region may include the amino acid sequence of SEQ ID NO: 7. The heavy chain variable region may include the amino acid sequence of SEQ ID NO: 8. The light chain constant region may include the amino acid sequence of SEQ ID NO: 9.

[0100] The recombinant fusion protein of the present disclosure may include the following: i) A first polypeptide chain and a second polypeptide chain comprising an anti-CD40 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 light chain variable region and optionally a light chain constant region; or ii) A first polypeptide chain and a second polypeptide chain comprising an anti-CD40 heavy chain variable region and a heavy chain constant region, and a third polypeptide chain and a fourth polypeptide chain comprising an anti-CD40 light chain variable region, optionally a light chain constant region, and SIRPαV2D1, wherein the heavy chain variable region in the first polypeptide chain and the light chain variable region in the third polypeptide chain combine to form a CD40 binding domain, wherein the heavy chain variable region in the second polypeptide chain and the light chain variable region in the fourth polypeptide chain combine to form a CD40 binding domain, characterized in that the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the second polypeptide chain are associated.

[0101] The CD47 binding domain may be linked to the N-terminus or C-terminus of the anti-CD40 antibody, or its antigen-binding portion. In certain embodiments, the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain variable region, a light chain variable region, 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 the C-terminus of the heavy chain constant region or the light chain constant region. In certain embodiments, the anti-CD40 antibody or its antigen-binding portion comprises 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 portion, or the N-terminus or C-terminus of the light chain.

[0102] The recombinant fusion protein of the present disclosure is, for example, within one or more CDR regions and / or within one or more framework regions of an anti-CD40 antibody or its antigen-binding portion, one or both variable regions (i.e., V H and / or V LIt can be modified by modifying one or more residues within . Further or alternatively, the recombinant fusion protein can be engineered by modifying residues within the constant region of, for example, an anti-CD40 antibody or an antigen-binding portion thereof to modify the effector function of the recombinant fusion protein. Further, residues of the CD47-binding domain can be modified to alter the binding affinity or other functional properties for CD47.

[0103] In certain embodiments, CDR grafting can be used to engineer the variable region of an anti-CD40 antibody or an antigen-binding portion thereof. Antibodies interact primarily with a target antigen via amino acid residues located in the six heavy and light chain complementarity-determining regions (CDRs). Thus, the amino acid sequences within the CDRs are more diverse among individual antibodies compared to the sequences outside the CDRs. Since the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a parental antibody by constructing an expression vector that contains CDR sequences derived from the parental antibody grafted into framework sequences from different antibodies having different properties (see, for example, Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad. USA 86:10029-10033; U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0104] Accordingly, another embodiment of the present disclosure relates to a recombinant fusion protein that may include a VH CDR1, VH CDR2, and VH CDR3 sequence that may include the sequences of the present disclosure as described above, and / or an anti-CD40 heavy chain variable region that may include a VL CDR1, VL CDR2, and VL CDR3 sequence that may include the sequences of the present disclosure as described above. These fusion proteins include the VH and VL CDR sequences of the anti-CD40 antibodies of the present disclosure, but they can include different framework sequences.

[0105] Such framework sequences can be obtained from publicly available DNA databases or the published literature that includes germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database. As another example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database.

[0106] Antibody protein sequences are compared to a compiled protein sequence database using one of the sequence similarity search methods known to those skilled in the art as gapped BLAST (Altschul et al., (1997), supra).

[0107] Preferred framework sequences for use in the antibodies of the present disclosure are those that are structurally similar to the framework sequences used by the antibodies of the present disclosure. The VH CDR1, VH CDR2, and VH CDR3 sequences can be transplanted into a framework region having the same sequences as those found in the germline immunoglobulin genes from which the framework sequence is derived, or the CDR sequences can be transplanted into a framework region that contains one or more mutations as compared to the germline sequences. For example, in some cases, it has been found beneficial to mutate residues within the framework region in order to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370). Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0108] In addition, or as an alternative to the modifications made within the framework or CDR regions, the anti-CD40 antibody or antigen-binding portion thereof in the recombinant fusion proteins of the present disclosure can typically be engineered to include modifications within the Fc region in order to modify one or more functional properties of the recombinant fusion protein such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cell cytotoxicity. Further, the recombinant fusion proteins of the present disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the fusion protein), or modified to change its glycosylation, again changing one or more functional properties.

[0109] In one embodiment, the hinge region of C H1 is modified such that the number of cysteine residues within the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of C H1 is altered, for example, to facilitate the assembly of the light and heavy chains, or to increase or decrease the stability of the antibody.

[0110] In another embodiment, the Fc hinge region or antigen-binding portion thereof of the anti-CD40 antibody in the recombinant fusion protein of the present disclosure is mutated, and the biological half-life of the recombinant fusion protein is changed. Specifically, in another embodiment, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the fusion protein has impaired Staphylococcus protein A (SpA) binding compared to native Fc hinge domain SpA binding. This method is described in more detail in U.S. Patent No. 6,165,745.

[0111] Changes in amino acids near the junction of the Fc portion and the non-Fc portion can significantly increase the serum half-life of the Fc fusion protein. Thus, the junction region of the recombinant fusion protein of the present disclosure may preferably contain changes within about 10 amino acids from the junction relative to the sequence of the naturally occurring immunoglobulin heavy chain. These amino acid changes can result in an increase in hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which the C-terminal lysine residue is substituted. Preferably, the C-terminal lysine residue of the IgG sequence is substituted with a non-lysine amino acid (e.g., alanine or leucine) to further increase the serum half-life.

[0112] In yet another embodiment, the glycosylation of the anti-CD40 antibody or antigen-binding portion thereof in the recombinant fusion protein is modified. The glycosylation can be modified, for example, to enhance the affinity of the anti-CD40 antibody or antigen-binding portion thereof for the antigen. Such sugar chain modifications can be achieved, for example, by modifying one or more of the glycosylation sites within the sequence of the anti-CD40 antibody or antigen-binding portion thereof. For example, one or more amino acid substitutions can be made to effect removal of glycosylation sites in one or more variable region frameworks so as to eliminate glycosylation at that site. Such aglycosylation can enhance the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861. Nos. 5,714,350 and 6,350,861.

[0113] The antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. As used herein, the term "polyethylene glycol" is intended to include any of the forms of PEG that have been used to derivatize other proteins, such as mono(C 1 -C 10 )alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. Methods of pegylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See EP 0 154 316 and EP 0 401 384.

[0114] The CD47-binding domain of the recombinant fusion protein of the present disclosure may be modified to have a higher CD47-binding affinity. In some embodiments, the SIRPαV2D1 variant contains one or more mutations in the SIRPαV2D1 domain as compared to wild-type SIRPαV2D1. Surface plasmon resonance (SPR) can be used to determine the CD47-binding affinity of the SIRPαV2D1 variant. As an alternative method for determining the CD47-binding affinity of the SIRPαV2D1 variant, a cell-binding assay can be used. When mutations are introduced into SIRPαV2D1 or the recombinant fusion protein of the present disclosure, the resulting variant or fusion protein generally has sufficient SIRPα biological activity to be useful as a therapeutic protein. In some embodiments, the biological activity of the SIRPαV2D1 variant is at least 0.01-fold, 0.03-fold, 0.06-fold, 0.1-fold, 0.3-fold, 0.6-fold, 1-fold, 3-fold, 5-fold, 6-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold or 100-fold that of wild-type SIRPαV2D1 or a fusion protein comprising wild-type SIRPαV2D1. The biological activity of the SIRPαV2D1 variant can be tested in in vitro or in vivo assays. In vitro assays for determining the biological activity of SIRPαV2D1 in cells expressing CD47 are well established in the art. For example, the biological activity can be determined in a leukocyte migration assay as described by Liu et al. (J. Mol. Bio., 365:680, 2007).

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

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

[0117] The present disclosure also provides an expression vector comprising the nucleic acid molecule of the present disclosure. Examples of vectors include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transformation-competent artificial chromosomes (TACs), mammalian artificial chromosomes (MACs), human artificial episomal chromosomes (HAECs), and the like. The present disclosure further provides a host cell transformed or transfected with the expression vector or the nucleic acid according to the present disclosure. Suitable host cells include Escherichia coli, yeast, and other eukaryotes. In one embodiment, the DNA encoding the polypeptide chains forming each recombinant fusion protein of the present disclosure is inserted into one or more expression vectors such that the gene is operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" means that the antibody gene is linked to the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the recombinant fusion protein gene.

[0118] The term "control / regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of nucleotides. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus, such as the adenovirus major late promoter (AdMLP), and promoters and / or enhancers derived from polyoma virus enhancers. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or the β-globin promoter, can also be used. Furthermore, regulatory elements consisting of sequences from different sources, such as the SRα promoter system (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472) containing sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1, etc., can be mentioned. Expression vectors and expression control sequences are selected to be compatible with the expression host cell to be used.

[0119] In addition to the recombinant fusion proteins encoding nucleotides and regulatory sequences, the expression vectors of the present disclosure can carry additional sequences such as sequences that regulate the replication of the vector in host cells (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically, the selectable marker gene confers resistance to agents such as G418, hygromycin or methotrexate on the host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr− host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0120] For the expression of the peptide chains constituting the recombinant fusion protein, an expression vector encoding the peptide chains is transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to include a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, transfection with DEAE-dextran, and the like. Although it is theoretically possible to express the recombinant fusion proteins of the present disclosure in either prokaryotic cells or eukaryotic host cells, the expression of antibodies in eukaryotic cells, particularly mammalian host cells, is most preferred because such eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete properly folded immunologically active recombinant fusion proteins than prokaryotic cells.

[0121] Preferred mammalian host cells for expressing the fusion proteins of the present disclosure include Chinese hamster ovary (CHO cells) (Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. When used particularly in NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036 and EP338,841. When a recombinant expression vector is introduced into a mammalian host cell, the recombinant fusion protein is produced by culturing the host cell for a period sufficient to allow expression of the recombinant fusion protein in the host cell, or more preferably, by secretion of the recombinant fusion protein into the medium in which the host cell is grown. The recombinant fusion protein can be recovered from the medium using standard protein purification methods.

[0122] In another aspect, the present disclosure provides a pharmaceutical composition that can include a recombinant fusion protein, nucleic acid molecule, expression vector, and / or host cell of the present disclosure formulated with a pharmaceutically acceptable carrier. The recombinant fusion protein, nucleic acid molecule, expression vector, and / or host cell can be administered separately when the pharmaceutical composition contains two or more recombinant fusion proteins, nucleic acid molecules, expression vectors, or host cells. The pharmaceutical composition may optionally include one or more additional pharmaceutically active ingredients, such as another antibody or a drug such as an anti-tumor drug.

[0123] The pharmaceutical composition can be composed of any number of excipients. Excipients that can be used include carriers, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweetening agents, preservatives, and combinations thereof. The selection and use of appropriate excipients are taught in Gennaro, Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0124] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient can be coated with a material to protect it from the action of acids and other natural conditions that can inactivate it. As used herein, the term "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intraarticular, intracapsular, subarachnoid, intraspinal, epidural and intraperiosteal injections and infusions. Alternatively, the pharmaceutical compositions of the present disclosure can be administered by non-parenteral routes, such as topical, epidermal, mucosal routes, such as nasal, oral, vaginal, rectal, sublingual, topical, etc.

[0125] The pharmaceutical composition can be in the form of a sterile aqueous solution or dispersion. It can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high-concentration drugs.

[0126] The "therapeutically effective dosage" of the recombinant fusion protein, nucleic acid molecule, expression vector, or host cell of the present disclosure preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or prevention of impairment or decline in body function due to the disease. For example, for the treatment of cancer-bearing subjects, the "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to untreated subjects. The therapeutically effective amount of the therapeutic recombinant fusion protein, nucleic acid molecule, expression vector, or host cell of the present disclosure can reduce the size of the tumor or improve the symptoms of a subject, who is usually human or may also be another mammal.

[0127] The pharmaceutical composition can be a controlled-release formulation, including implants, transdermal patches, and microencapsulation delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0128] The therapeutic composition can be administered via a medical device. For example, (1) needleless subcutaneous injection devices (e.g., U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556). (2) Microinfusion pumps (U.S. Patent No. 4,487,603); (3) transdermal devices (U.S. Patent No. 4,486,194); (4) infusion devices (U.S. Patent Nos. 4,447,233 and 4,447,224). (5) Osmotic devices (U.S. Patent Nos. 4,439,196 and 4,475,196). The disclosures of these are incorporated herein by reference.

[0129] The pharmaceutical composition comprising the recombinant fusion protein, nucleic acid molecule, expression vector, or host cell of the present disclosure has numerous in vitro and in vivo applications, such as the treatment of diseases related to CD40 and / or CD47 signaling, such as tumors and infectious diseases.

[0130] The present disclosure provides a method for treating a disease related to CD40 signaling and / or CD47 signaling, which may include administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure to a subject.

[0131] The disease may be a tumor or cancer. 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 cancer, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, lung cancer (including small cell lung cancer and non-small cell lung cancer), multiple myeloma, melanoma, hepatocellular carcinoma, liver cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), and gastric cancer. In certain embodiments, the subject is human.

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

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

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

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

[0136] The present disclosure is further illustrated by the following examples, which should not be construed as being more limiting. The contents of all figures, and all documents, Genbank sequences, patents, and published patent applications cited throughout this application are hereby expressly incorporated by reference into this specification.

[0137] Examples Example 1 Construction of an exemplary recombinant fusion protein The recombinant fusion protein was constructed by linking the first Ig-like extracellular domain of human SIRPα isoform 2 (SIRPαV2D1) to the N-terminus or C-terminus of the heavy chain, optionally via a linker, or to the C-terminus of the light chain of a humanized anti-CD40 antibody named HuCD40-C1H1-V2 as described in WO2021 / 197335, using the human IgG2 heavy chain constant region. SIRPαV2D1 has the amino acid sequence of wild type (X1 = V, X2 = K, X3 = S, X4 = K, X5 = F) of SEQ ID NO: 11, or a specific mutant (X1 = I, X2 = R, X3 = T, X4 = K, X5 = F; or X1 = I, X2 = R, X3 = T, X4 = R, X5 = V) designed to enhance the binding affinity for CD having the amino acid sequence of SEQ ID NO: 11. The anti-CD40 antibody HuCD40-C1H1-V2 is an IgG antibody having 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. 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 of SEQ ID NOs: 7, 8, 9 and 10, respectively. Here, the heavy chain variable region contains VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region contains VL CDR1, VL CDR2 and VL CDR3 having the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.

[0138] Specific structures and sequence IDs of exemplary recombinant fusion proteins are described in Table 1 below and shown in Figure 1.

Table 1

[0139] Example 2 Expression and Identification of Exemplary Recombinant Fusion Proteins Briefly described, nucleic acids encoding the polypeptide chains constituting each recombinant fusion protein were synthesized and then inserted into the pTT5 vector, respectively. Thereafter, plasmid DNA extracts of the above vectors were transfected into mammalian cells (CHO cells), and the exemplary recombinant fusion proteins of the present disclosure were expressed and secreted in CHO cells. Next, the recombinant fusion proteins were purified using a Protein A affinity chromatography column and subjected to SDS-PAGE and SEC-HPLC analysis, and the results are shown in Tables 2-1 and 2-2.

Table 2-1

Table 2-2

[0140] From the results, it can be seen that the recombinant fusion proteins of the present disclosure showed high purity, and most of the fusion proteins existed as monomers.

[0141] Example 3 Using BIACORE surface plasmon resonance technology Measurement of the binding affinity of exemplary recombinant fusion proteins The purified recombinant fusion proteins were used to measure their binding affinity and binding kinetics using a Biacore T200 system (GE healthcare, Pittsburgh, PA, USA). Anti-CD40 antibody HuCD40-C1H1-V2 (having the heavy and light chains of SEQ ID NOs: 19 and 20), seribulimab (agonist anti-CD40 antibody prepared in-house using the heavy and light chains of SEQ ID NOs: 27 and 28, Roche Inc.), and 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), SIRPα-isoform 2-Fc (SEQ ID NO: 25) were used as positive controls.

[0142] For the measurement of binding affinity, a Protein A chip (Cat#: 29-1275-56, GE healthcare) was used. The recombinant fusion proteins of the present disclosure and the positive control were each flowed onto the chip at a concentration of 2 μg / ml and a flow rate of 10 μl / min. Subsequently, the serially diluted recombinant human CD40-his protein (Cat#: CD0-H5228, Acro biosystems), human CD47-his protein (Cat#: CD7-H5227, Acro biosystems), cynomolgus CD40-his protein (self-made bioreation, SEQ ID NO: 29) or cynomolgus CD47-his protein (Cat#: CD7-C52H1, Acro biosystems) was serially diluted in HBS-EP+ buffer with an initial concentration of 200 nM and flowed onto the chip at a flow rate of 30 μl / min. The association kinetics was followed for 2 minutes and the dissociation kinetics was followed for 10 minutes. The association and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore evaluation software. K D , K a and K d values were determined and summarized in Tables 3-1, 3-2, 3-3 and 3-4 below.

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0143] All recombinant fusion proteins of the present disclosure specifically bound to human CD40 and cyno CD40 with high binding affinity.

[0144] Furthermore, the recombinant fusion proteins of the present disclosure also specifically bind to human CD47 and cyno CD47, and the CD47 binding affinities of BSI038×S-004 and BSI038×S-005 were comparable to those of SIRPαV2D1M1-Fc (IgG2) and SIRPαV2D1M2-Fc (IgG2), and were better than that of BSI038×S-002.

[0145] Example 4 Binding Activity of Exemplary Recombinant Fusion Proteins The binding activity of the recombinant fusion proteins of the present disclosure to human CD40 was further measured by flow cytometry (FACS) using 293T-CD40 cells prepared in-house at Biosion that stably express the full length of human CD40 (uniprot#P25942-1). The 293T-CD40 cells were prepared by transfecting 293T cells according to the instructions of Lipofectamine 3000 transfection reagent (Thermo Fisher) using a pCMV-T-P plasmid with DNA encoding human CD40 (uniprot#P25942-1) inserted between the EcoRI and XbaI sites.

[0146] Briefly, 293T-CD40 cells were recovered from the cell culture flask, washed twice, and then resuspended in phosphate buffered saline (PBS) containing 2% v / v fetal bovine serum (FACS buffer). Then, 1 x 10 5Individual 293T-CD40 cells were added and incubated on ice for 40 minutes together with 100 μl of the disclosed serial-diluted recombinant fusion protein or control (starting from 10 μg / mL serially diluted 5-fold with FACS buffer). The cells were washed twice with FACS buffer, and 100 μl / well of goat anti-human IgG(Fab)-PE (diluted 1:1000 with FACS buffer, Cat#:109-116-097, Jackson ImmunoResearch) was added. After incubation in the dark at 4 °C for 40 minutes, the cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument and plotted against the concentration of the recombinant fusion protein or control. The data were analyzed using Graphpad Prism, and the EC 50 value was reported. The results are shown in Figure 2.

[0147] Also, the binding activity of the gene recombinant fusion protein to human CD47 on the cell membrane was tested by flow cytometry (FACS) using 293F-CD47 cells prepared in-house at Biosion that express full-length human CD47 (NCBI #NP_942088.1). The 293F-CD47 cells were prepared by transfecting 293F cells according to the instructions of Lipofectamine 3000 transfection reagent (Thermo Fisher) using a pCMV-T-P plasmid with DNA encoding human CD47 (NP_942088.1) inserted between the EcoRI and XbaI sites.

[0148] The 293F-CD47 cells were recovered from the cell culture flask, washed twice, and then resuspended in phosphate-buffered saline (PBS) containing 2% v / v fetal bovine serum (FACS buffer). Then, 1 x 10 5Individual 293F-CD47 cells were added and incubated on ice for 40 minutes with 100 μl of the disclosed serial-diluted recombinant fusion protein or control (starting from 10 μg / mL serially diluted 5-fold with FACS buffer). The cells were washed twice with FACS buffer, and 100 μl / well of goat anti-human IgG(Fc)-PE (diluted 1:1000 with FACS buffer, Cat#:109-115-098, Jackson ImmunoResearch) was added. After incubation in the dark at 4°C for 50 minutes, the cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument, and the MFI (mean fluorescence intensity) was plotted against the recombinant fusion protein or control. The data were analyzed using Graphpad Prism, and the EC 50 value was reported. The results are shown in Figure 3.

[0149] As can be seen from Figure 2, the recombinant fusion protein of the present disclosure specifically binds to human CD40.

[0150] According to Figure 3, the recombinant fusion protein of the present disclosure specifically binds to human CD47. In particular, the binding activity of BSI038×S-002 to human CD47 was equivalent to that of the positive control SIRPa-isoform2-Fc.

[0151] The binding activities of the recombinant fusion protein to human CD40 and CD47 were also tested by a dual-binding ELISA assay. Specifically, 100 μl / well of human CD40-Fc protein (Cat#:CD0-H5253, ACRO Biosystems) was dissolved in PBS at a concentration of 0.2 μg / mL and coated onto a 96-well plate, and incubated overnight at 2-8°C. The next day, the plate was washed three times with wash buffer (PBS + 0.05% v / v Tween-20, PBST), and then blocked with blocking buffer (PBS + 1% w / v BSA) at 37°C for 3 hours. Thereafter, the plate was washed three times with wash buffer.

[0152] The recombinant fusion protein of the disclosure or control was serially diluted with blocking buffer (starting from 100 nM with 6-fold serial dilution), 100 μL of which was added to each well of the CD40-Fc binding plate and incubated at 37°C for 1 hour. The plate was washed three times with washing buffer, and 100 μl / well of 3 μg / mL human CD47-his protein (Cat#: CD7-H5227, ACRO Biosystems) was added and incubated at 37°C for 1 hour. The plate was washed again with washing buffer, and 100 μl / well of anti-histidine tag antibody (HRP) (diluted 1:2000 with blocking buffer, Cat#: 105327-MM02T-H, Sino Biological) was added and incubated at 37°C for 1 hour. The plate was washed again with washing buffer. Finally, TMB was added to develop the plate, and 1M H2SO4 was added to stop the reaction. The absorbance was measured with a microplate reader set at 450 nm, and the OD(450) values were plotted against the concentration of the recombinant fusion protein or control. The data was analyzed using Graphpad Prism, and the EC 50 value was reported. The results are shown in Figure 10.

[0153] It can be seen from Figure 10 that the recombinant fusion protein of the present disclosure specifically binds to human CD40 and CD47 and shows higher binding activity than SIRPα-Fc-CD40L.

[0154] Example 5 Blocking Activity of Exemplary Recombinant Fusion Proteins that Inhibit CD40-CD40L or CD47-SIRPα Binding 5.1 Inhibitory Effect on CD40-CD40L Binding Tested by Ligand Blocking ELISA The ability of the recombinant fusion protein of the present disclosure to inhibit CD40-CD40L binding was measured by a competitive ELISA assay. Briefly, 100 μl / well of human CD40-Fc protein (prepared in-house with the amino acid sequence of SEQ ID NO: 22) was prepared at 2 μg / mL with coating buffer (carbonate / bicarbonate buffer), coated on a 96-well microplate, and incubated overnight at 4°C. The next day, the plate was washed once with wash buffer (PBS + 0.05% v / v Tween-20, PBST), and then blocked with 5% w / v non-fat milk in PBST at 37°C for 2 hours. Then, the plate was washed 4 times with wash buffer.

[0155] The recombinant fusion protein of the disclosure or a control (serial 5-fold dilutions from 10 μg / mL) was added to the CD40-Fc binding plate at 100 μl / well each with PBST supplemented with 2.5% w / v non-fat milk, and incubated at 37°C for 40 minutes. The plate was washed 4 times with the washing solution, 100 μl / well of 95 ng / mL biotinylated human CD40L-his protein (Cat#: 10239-H08E, Sinobiological) was added, and incubated at 37°C for 40 minutes. The plate was washed again with the washing solution, streptavidin-conjugated HRP (diluted 1:10000 with PBST buffer, Cat#: 016-030-084, Jackson ImmunoResearch) was added at 100 μl / well each, and incubated at 37°C for 40 minutes. The plate was washed again with wash buffer. Finally, TMB was added to develop the plate, and 1M H2SO4 was added to stop the reaction. The absorbance was read using a microplate reader in 2-wavelength mode with TMB at 450 nm and reference wavelength 630 nm, and the OD(450-630) values were plotted against the concentration of the recombinant fusion protein or the control. The data was analyzed using Graphpad Prism, and the IC 50 value was reported. The results are shown in Figure 4.

[0156] 5.2 Inhibitory effect on CD47-CD172a binding tested by ligand blocking ELISA The ability of the recombinant fusion proteins of the present disclosure to inhibit CD47-CD172a binding was measured in a competitive ELISA assay. Briefly, 100 μl / well of human human CD172a-Fc protein (prepared in-house with the amino acid sequence of SEQ ID NO: 24) was prepared at 2 μg / mL with coating buffer (carbonate / bicarbonate buffer), coated onto a 96-well microplate, and incubated overnight at 4°C. The next day, the plate was washed once with wash buffer (PBS + 0.05% v / v Tween-20, PBST), and then blocked with 5% w / v non-fat milk in PBST for 2 hours at 37°C. Thereafter, the plate was washed 4 times with wash buffer.

[0157] The recombinant fusion proteins of the present disclosure or controls were serially diluted (starting from 100 nM with 5-fold serial dilutions) and mixed with 20 ng / ml biotinylated human CD47-Fc (manufactured in-house at Biosion, SEQ ID NO: 23) in PBST supplemented with 2.5% w / v non-fat milk. The mixture was incubated at 37°C for 40 minutes, and 100 μl aliquots were added to the CD172a-Fc coated plates. The plates were incubated at 37°C for 40 minutes, washed 4 times with wash solution, then 100 μl / well of streptavidin-conjugated HRP (diluted 1:10000 in PBST buffer, Cat#: 016-030-084, Jackson ImmunoResearch) was added, incubated at 37°C for 40 minutes, and washed again with wash solution. Finally, TMB was added to develop the plates, and 1M H 2 SO 4 was added to stop the reaction. The plates were read on a microplate reader using the two-wavelength mode with TMB at 450 nm and a reference wavelength of 630 nm, and the OD(450 - 630) values were plotted against the concentration of the recombinant fusion protein or control. The data was analyzed using Graphpad Prism, and IC 50 values were reported. Wild-type SIRPα V2D1-Fc (IgG2) (SEQ ID NO: 21, X1 = V, X2 = K, X3 = S, X4 = K, X5 = F) was used as a positive control. The results are shown in Figure 5.

[0158] 5.3 Inhibitory effect on CD47-SIRPα binding tested by cell-based ligand blocking FACS The activity of the recombinant fusion proteins of the present disclosure to inhibit the binding of SIRPα to cell surface CD47 was evaluated by flow cytometry (FACS) using in-house prepared 293F-CD47 cells.

[0159] Briefly, 293F-CD47 cells were prepared by transfecting 293F cells with a pCMV-T-P plasmid containing DNA encoding human CD47 (NP_942088.1) inserted between the EcoRI and XbaI sites according to the instructions of Lipofectamine 3000 transfection reagent (Thermo Fisher).

[0160] The recombinant fusion proteins or controls of the present disclosure were diluted by three-fold serial dilution starting from 100 nM in PBS containing 2% v / v fetal bovine serum (FACS buffer). On the other hand, 293F-CD47 cells in the logarithmic growth phase were harvested, washed twice with FACS buffer, and collected by centrifugation. Then, 1x10 5 cells were suspended in 100 μl of diluted antibody or control per well of a 96-well plate and incubated at 4°C for 60 minutes. The plate was washed twice, and 100 μL / well of 148 ng / mL biotinylated human CD172a-Fc protein (prepared in-house with the amino acid sequence SEQ ID NO: 24) was incubated in FACS buffer for 60 minutes at 4°C. After washing the plate twice with FACS buffer, 100 μl / well of R-Phycoerythrin streptavidin (diluted 1:500 in FACS buffer, Cat#: 016-110-084, Jackson Immunoresearch) was added and incubated at 4°C in the dark for 40 minutes. The cells were washed twice and then resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument, and the MFI (mean fluorescence intensity) was plotted against the recombinant fusion proteins or controls. The data were analyzed using Graphpad Prism, and IC 50The value was reported. The results are shown in FIGS. 6 and 7.

[0161] From FIG. 4, it can be seen that the recombinant fusion protein of the present disclosure has an activity equivalent to that of the positive control and can block the binding of human CD40 and human CD40L.

[0162] From FIG. 5, it can be seen that BSI038×S-002 has a higher activity than wild-type SIRPαV2D1-Fc(IgG2) and can block the binding of human CD47 and human CD172a. Furthermore, BSI038×S-004 and BSI038×S-005 can block the binding of human CD47 and human CD172a and showed higher activities than the positive controls SIRPαV2D1M1-Fc(IgG2) and SIRPαV2D1M2-Fc(IgG2).

[0163] Furthermore, according to FIGS. 6 and 7, the recombinant fusion protein of the present disclosure can inhibit the binding of human SIRPα to human CD47 on the cell surface, and most of them had equivalent blocking activity compared to the positive control. Furthermore, BSI038×S-004 and BSI038×S-005 showed higher CD47-SIRPα blocking activity than BSI038×S-002.

[0164] Example 6 Cell-based functional assay of exemplary recombinant fusion proteins 6.1 CD40 agonist activity tested in a cell-based reporter assay The recombinant fusion protein of the present disclosure was further tested for agonist activity in CD40 signaling using the CD40-expressing reporter cell line 293T-NF-κB-Luc-CD40 that stably expresses full-length human CD40 (uniprot No. P25942-1). 293T-NF-κB-Luc-CD40 cells were transfected with the pGL4.32 [luc2P / NF-κB-RE / Hygro] vector (Promega, GenBank® Accession Number: EU581860) into 293T cells according to the instructions of Lipofectamine 3000 transfection reagent (Thermo Fisher), and then a pCMV-T-P plasmid inserted with DNA encoding human CD40 between EcoRI and XbaI was inserted. When a CD40 agonist is brought into contact with these cells, the CD40 signal is activated and the expression of luciferase is upregulated by the NF-κB transcription factor. The expression of luciferase can be measured by a luminescence assay. CD40L having the amino acid sequence of SEQ ID NO: 26 described in the reference "K, Patel A, et al. CD40 enhances the type I interferon response downstream of CD47 blockade and bridges innate and adaptive immunity. Cancer Immunology Research. February 2020;8(2):230-245" was used as a control.

[0165] Briefly, 5×10 in the logarithmic growth phase in 20 μL of DMEM medium (Cat#: 10566-016, Gibco) supplemented with 10% FBS (Cat#: 10099-141, Gibco) 3293T-NF-κB-Luc-CD4 cells were plated into each well of a 384-well cell culture plate (Cat#: 3707, Corning). To the plate, 20 μl each of the disclosed recombinant fusion protein or control (starting at 200 nM and serially diluted 3-fold in culture medium) was sequentially added and incubated at 37 °C for 6 hours. Thereafter, the reagent of ONE-Glo® Luminescence Assay System (30 μl / well, Cat#: E6120, Promega) was added to the plate and incubated at room temperature for 5 minutes. Chemiluminescence was measured using a Tecan Infinite® 200 Pro device. Data were analyzed using Graphpad Prism, and the EC 50 value was reported.

[0166] The results are shown in Figure 8.

[0167] BSI038×S-002, BSI038×S-004, and BSI038×S-005 showed CD40 agonist activity, which was comparable to that of HuCD40-C1H1-V2 and much higher than that of celecoxib and SIRPa-Fc-CD40L.

[0168] 6.2 Ability to induce phagocytosis of cancer cells The recombinant fusion proteins of the present disclosure were further tested for their ability to induce phagocytosis of cancer cells, using recombinant proteins SIRPα-isoform 2-Fc, SIRPαV2D1M1-Fc (IgG2), SIRPαV2D1M2-Fc (IgG2), SIRPα-Fc-CD40L, and wild-type SIRPαV2D1-Fc (IgG2) as controls.

[0169] Briefly, monocytes were isolated from cryopreserved human PBMCs using the EasySep® Human Monocyte Enrichment Kit (Cat#: 19058, Stemcell) without performing CD16 depletion according to the manual, seeded into 6-well plates supplemented with RPMI1640 + 10% FBS + 1% penicillin streptomycin + 75 ng / ml human M-CSF, and fresh cell culture medium was added on day 3. Macrophages were detached from the plates, re-seeded into 96-well plates on day 6, and cultured overnight.

[0170] CFSE-labeled Jurkat cells were harvested and incubated at room temperature for 30 minutes at a density of 1 million cells per 0.4 ml of serial diluted recombinant fusion protein of the disclosure or control at 50 μl. Subsequently, the Jurkat cell / antibody mixture was added to the macrophages harvested above at an effector cell:target cell ratio of 1:2 and co-cultured at 37°C for 4 hours.

[0171] All cells were harvested from the plates with Accutase® cell detachment solution and washed once with FACS buffer. Subsequently, the cells were blocked with Human TruStain FcX® Fc Receptor Blocking Solution (Cat#: 422302, Biolegend) and stained with anti-human CD11b APC (Cat#: 301310, Biolegend). The phagocytosis rate (%) was determined by flow cytometry as the ratio of the CFSE+ population to the CD11b + population (CD11b containing phagocytosed CFSE+ cells + number of macrophages / counted CD11b + total number of macrophages).

[0172] The results are shown in Figure 9.

[0173] BSI038×S-002, BSI038×S-004, BSI038×S-005 were able to induce phagocytosis of tumor cells by macrophages and showed stronger activity than SIRPα-Fc-CD40L.

[0174] Example 7 Thermal Stability of Exemplary Recombinant Fusion Proteins The thermal stability of the recombinant fusion protein was tested. Briefly, the melting temperature (Tm) was measured by a protein thermal shift assay using the GloMelt® Thermal Shift Protein Stability Kit (Cat# 33022-T, Biotium). Briefly, the GloMelt® dye was thawed and returned to room temperature. The vial containing the dye was vortexed and centrifuged. Next, 5 μL of the dye diluted 200-fold in 95 μL of PBS was added to prepare a 10-fold diluted dye. Next, 2 μL of the 10-fold diluted dye and 10 μg of the disclosed or control recombinant fusion protein were added, and PBS was added to make the total reaction volume 20 μL. The tubes containing the dye and the fusion protein or control were gently centrifuged and set in a real-time PCR thermocycler (Roche, LightCycler 480 II) with the melt curve program set with the parameters in Table 4. [Table 4] [Table 5]

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

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

[0177] The sequences in this application are summarized below. JPEG2025517619000011.jpg172105JPEG2025517619000012.jpg171104JPEG2025517619000013.jpg171105JPEG2025517619000014.jpg255156JPEG2025517619000015.jpg171104JPEG2025517619000016.jpg11153

[0178] Thus, while the preferred embodiments of the present invention have been described in detail, many obvious modifications can be made without departing from the spirit or scope of the present invention, and it is believed that the invention defined by the above paragraphs is not limited to the specific details shown in the above description.

Claims

**Claim 1** A recombinant fusion protein comprising: (a) an anti-CD40 antibody or an antigen-binding portion thereof, and (b) a CD47-binding domain, wherein the CD47-binding domain is the first Ig-like extracellular domain of human SIRPα isoform 2 (SIRPαV2D1). **Claim 2** The recombinant fusion protein according to claim 1, wherein the anti-CD40 antibody or an antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region. Here, the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 each comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 each comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. **Claim 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. **Claim 4** The recombinant fusion protein according to claim 2 or 3, wherein a heavy chain constant region is linked to the C-terminus of the heavy chain variable region, and the heavy chain constant region is a human IgG1, IgG2, or IgG4 heavy chain constant region. **Claim 5** The recombinant fusion protein according to claim 4, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

9. **Claim 6** The recombinant fusion protein according to claim 6, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO:

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

10. **Claim 8** The recombinant fusion protein according to any one of claims 1 to 7, wherein the CD47-binding domain is linked to the N-terminus or C-terminus of the anti-CD40 antibody or an antigen-binding portion thereof. **Claim 9** The recombinant fusion protein according to any one of claims 1 to 8, wherein the CD47-binding domain is linked to the N-terminus of the heavy chain variable region or light chain variable region, or the C-terminus of the heavy chain constant region or light chain constant region. **Claim 10** The recombinant fusion protein according to claim 8, wherein the CD47 binding domain is linked to the anti-CD40 antibody or an antigen-binding portion thereof via a linker.

11. The recombinant fusion protein according to claim 9, wherein the CD47 binding domain is linked via a linker 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.

12. The recombinant fusion protein according to claim 10 or 11, wherein the linker comprises the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15.

13. The recombinant fusion protein according to any one of claims 1 to 12, wherein the CD47 binding domain is wild-type SIRPαV2D1 or a SIRPαV2D1 variant.

14. The CD13 binding domain comprises the amino acid sequence of SEQ ID NO: 11, wherein X1 = V, X2 = K, X3 = S, X4 = K, X5 = F; X1 = I, X2 = R, X3 = T, X4 = K, X5 = F; or X1 = I, X2 = R, X3 = T, X4 = R, X5 = V.

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

18.

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

17. A nucleic acid encoding the recombinant fusion protein according to any one of claims 1 to 16.

18. An expression vector containing the nucleic acid according to claim 17.

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

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

21. Use of the pharmaceutical composition according to claim 20 in the preparation of a therapeutic agent for a disease related to CD40 and / or CD47 signal transduction.

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 cancer or a hematological cancer.

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 cancer, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, lung cancer (including small cell lung cancer and non-small cell lung cancer), multiple myeloma, melanoma, hepatocellular carcinoma, liver cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), or gastric cancer.

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

Images