CD47 / PD-L1-Targeting Protein Complexes and Methods of Use Thereof

Abstract

The present disclosure relates to protein complexes that target CD47, PD-L1, and / or TGFβ, and methods of use thereof. In one aspect, the protein complexes comprise one or more CD47 binding domains, each comprising all or a portion of the extracellular domain of SIRPα, one or more PD-L1 binding domains, each comprising all or a portion of the extracellular domain of PD-1, and optionally one or more TGFβ binding domains, each comprising all or a portion of the extracellular domain of TGFBR2. TIFF2025503731000003.tif107170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 300,440, filed January 18, 2022, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file titled 52246-0002WO1_SL_ST26.xml. The XML file was created on December 15, 2022 and is 24,297 bytes in size. The data within the XML file is incorporated herein by reference in its entirety.

[0003] Technical Field The present disclosure relates to protein complexes that target CD47 and PD-L1 and methods of use thereof. [Background technology]

[0004] background Signal regulatory protein alpha (SIRPα) is a regulatory membrane glycoprotein from the SIRP family. It is expressed primarily by myeloid cells, but also by stem cells or neurons. SIRPα acts as an inhibitory receptor and interacts with the widely expressed transmembrane protein CD47. This interaction negatively regulates the effector functions of innate immune cells, such as phagocytosis of host cells. SIRPα diffuses laterally on macrophage membranes and accumulates at the phagocytic synapse to bind CD47, which inhibits the cytoskeleton-intensive process of phagocytosis by macrophages. CD47 provides a "do not eat" signal by binding to the N-terminus of signal regulatory protein alpha (SIRPα). CD47 has been found to be overexpressed on many different tumor cells. Targeting CD47 and / or SIRPα may be useful for cancer immunotherapy. However, CD47 is also expressed on red blood cells (RBCs) and platelets, so inhibiting the CD47 / SIRPα interaction may cause phagocytosis of RBCs and platelets. Programmed death-1 (PD-1) is an immune checkpoint that protects against autoimmunity through a dual mechanism of promoting apoptosis of antigen-specific T cells in lymph nodes while reducing apoptosis in regulatory T cells. There is a need to develop therapies that target the CD47 / SIRPα and / or PD-1 / PD-L1 pathways. Summary of the Invention

[0005] overview The present disclosure relates to protein complexes that target CD47 and PD-L1, and methods of use thereof. In some cases, the protein complex may also target TGFβ.

[0006] In one aspect, the disclosure relates to a protein complex comprising: (a) an Fc; (b) a CD47-binding domain; and (c) a PD-L1 (programmed death ligand 1)-binding domain. In some embodiments, the protein complex described herein further comprises a TGFβ (transforming growth factor beta)-binding domain.

[0007] In some embodiments, the CD47 binding domain can bind to cells expressing CD47 (e.g., cancer cells) and / or block the interaction between CD47 and signal regulatory protein alpha (SIRPα). In some embodiments, the CD47 binding domain is or comprises a SIRPα extracellular domain (e.g., a human SIRPα extracellular domain). In some embodiments, the CD47 binding domain is an anti-CD47 antibody or an antigen-binding fragment thereof (e.g., an scFv or VHH). In some embodiments, the CD47 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6.

[0008] In some embodiments, the PD-L1 binding domain may bind to a cell expressing PD-L1 (e.g., a cancer cell) and / or block the interaction between PD-L1 and programmed cell death protein 1 (PD-1). In some embodiments, the PD-L1 binding domain is or comprises a PD-1 extracellular domain (e.g., a human PD-1 extracellular domain). In some embodiments, the PD-L1 binding domain is an anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., an scFv or VHH). In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5.

[0009] In some embodiments, the TGFβ binding domain may capture TGFβ, thereby increasing the immune response and / or improving the tumor microenvironment. In some embodiments, the TGFβ binding domain is or includes a TGFBR2 extracellular domain (e.g., a human TGFBR2 extracellular domain). In some embodiments, the TGFβ binding domain is an anti-TGFβ antibody or an antigen-binding fragment thereof (e.g., an scFv or VHH). In some embodiments, the TGFβ binding domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7.

[0010] In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the CD47 binding domain is linked to the N-terminus of the CH2 domain of the Fc, optionally via a hinge region. In some embodiments, the PD-L1 binding domain is linked to the N-terminus of the CD47 binding domain, optionally via a linker peptide. In some embodiments, the PD-L1 binding domain is linked to the N-terminus of the CH2 domain of the Fc, optionally via a hinge region. In some embodiments, the CD47 binding domain is linked to the N-terminus of the PD-L1 binding domain, optionally via a linker peptide. In some embodiments, the hinge region is a human IgG4 hinge region, optionally with a S228P mutation according to EU numbering. In some embodiments, the TGFβ binding domain is linked to the C-terminus of the CH3 domain of the Fc, optionally via a linker peptide.

[0011] In some embodiments, the protein complex comprises two or more CD47 binding domains.In some embodiments, the protein complex comprises two or more PD-L1 binding domains.In some embodiments, the protein complex comprises two or more TGFβ binding domains.

[0012] In one aspect, the disclosure relates to a protein complex comprising: (a) a first polypeptide comprising, from N-terminus to C-terminus, a first PD-L1 binding domain, an optional first linker peptide, a first CD47 binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and an optional first TGFβ binding domain; and (b) a second polypeptide comprising, from N-terminus to C-terminus, a second PD-L1 binding domain, an optional third linker peptide, a second CD47 binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and an optional second TGFβ binding domain. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain comprise a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO:5. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:6. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:7. In some embodiments, the first hinge region and / or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO:16. In some embodiments, the first Fc region and / or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO:17, optionally with a lysine or alanine residue added to the C-terminus. In some embodiments, the first linker peptide and / or the third linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO:8. In some embodiments, the second linker peptide and / or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the first polypeptide and / or the second polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO: 1.

[0013] In some embodiments, the first polypeptide further comprises a third PD-L1 binding domain and the second polypeptide further comprises a fourth PD-L1 binding domain. In some embodiments, the third PD-L1 binding domain and / or the fourth PD-L1 binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:5. In some embodiments, the third PD-L1 binding domain is linked to the N-terminus of the first PD-L1 binding domain, optionally via a fifth linker peptide, and in some embodiments the fourth PD-L1 binding domain is linked to the N-terminus of the second PD-L1 binding domain, optionally via a sixth linker peptide. In some embodiments, the fifth linker peptide and / or the sixth linker peptide comprise a sequence that is at least 80% identical to SEQ ID NO:10. In some embodiments, the first polypeptide and / or the second polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:3.

[0014] In one aspect, the disclosure relates to a protein complex comprising: (a) a first polypeptide comprising, from N-terminus to C-terminus, a first CD47 binding domain, an optional first linker peptide, a first PD-L1 binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and an optional first TGFβ binding domain; and (b) a second polypeptide comprising, from N-terminus to C-terminus, a second CD47 binding domain, an optional third linker peptide, a second PD-L1 binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and an optional second TGFβ binding domain. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain comprise a sequence at least 80%, 90%, 95% or 100% identical to SEQ ID NO:6. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain comprise a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO:5. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain comprise a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO:7. In some embodiments, the first hinge region and / or the second hinge region comprise a sequence that is at least 80% identical to SEQ ID NO:16. In some embodiments, the first Fc region and / or the second Fc region comprise a sequence that is at least 80% identical to SEQ ID NO:17, optionally with a lysine or alanine residue added to the C-terminus. In some embodiments, the first linker peptide and / or the third linker peptide comprise a sequence that is at least 80% identical to SEQ ID NO:8. In some embodiments, the second linker peptide and / or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the first polypeptide and / or the second polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:2.

[0015] In some embodiments, the first polypeptide further comprises a third PD-L1 binding domain and the second polypeptide further comprises a fourth PD-L1 binding domain. In some embodiments, the third PD-L1 binding domain and / or the fourth PD-L1 binding domain comprise a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO:5. In some embodiments, the third PD-L1 binding domain is fused between the first PD-L1 binding domain and the first hinge region, optionally via a fifth linker peptide between the first PD-L1 binding domain and the third PD-L1 binding domain, and in some embodiments the fourth PD-L1 binding domain is fused between the second PD-L1 binding domain and the second hinge region, optionally via a sixth linker peptide between the second PD-L1 binding domain and the fourth PD-L1 binding domain. In some embodiments, the fifth linker peptide and / or the sixth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the first polypeptide and / or the second polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:4.

[0016] In one aspect, the present disclosure relates to a nucleic acid comprising a polynucleotide encoding a protein complex described herein. In some embodiments, the nucleic acid is DNA (e.g., cDNA) or RNA (e.g., mRNA).

[0017] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0018] In one aspect, the disclosure relates to a cell comprising the vector described herein. In some embodiments, the cell is a CHO cell.

[0019] In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0020] In one aspect, the disclosure relates to a method of producing a protein complex, the method comprising: (a) culturing a cell described herein under conditions sufficient for the cell to produce the protein complex; and (b) harvesting the protein complex produced by the cell.

[0021] In one aspect, the present disclosure relates to a protein conjugate comprising a protein complex described herein covalently linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0022] In one aspect, the disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a protein complex as described herein or a protein conjugate as described herein. In some embodiments, the subject has cancer cells that express CD47 and / or PD-L1. In some embodiments, the cancer is breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, diffuse large B-cell lymphoma, mesothelioma, lung cancer, ovarian cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial carcinoma, stomach cancer, cholangiocarcinoma, head and neck cancer, hematological cancer, or a combination thereof.

[0023] In one aspect, the disclosure relates to a method of reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a protein complex as described herein or a composition comprising a protein conjugate as described herein.

[0024] In one aspect, the disclosure relates to a method of killing a tumor cell, the method comprising contacting the tumor cell with an effective amount of a composition comprising a protein complex as described herein or a protein conjugate as described herein.

[0025] In one aspect, the present disclosure relates to a pharmaceutical composition comprising a protein complex described herein and a pharma- ceutically acceptable carrier.

[0026] As used herein, the term "protein complex" or "protein construct" refers to a complex having one or more polypeptides. In some embodiments, a protein complex has two or more polypeptides, and the polypeptides may associate with each other to form dimers or multimers.

[0027] As used herein, the term "CD47 binding domain" refers to a protein domain capable of binding to CD47. In some embodiments, the CD47 binding domain may be an anti-CD47 antibody, an antigen-binding fragment thereof (e.g., scFv or VHH), or a CD47 binding protein or a portion thereof. In some embodiments, the CD47 binding domain may have one or more self-stabilizing domains. In some embodiments, the CD47 binding domain comprises or consists of a SIRPα extracellular domain. SIRPα may be wild-type SIRPα, human SIRPα, a polypeptide derived from wild-type SIRPα (e.g., having a mutation), or a portion thereof (e.g., the extracellular region of SIRPα, or the IgV domain of SIRPα). In some embodiments, the polypeptide derived from wild-type SIRPα may have one or more mutations. In some embodiments, the SIRPα extracellular domain comprises or consists of substantially the entire extracellular region of SIRPα or a variant thereof. In some embodiments, the SIRPα extracellular domain comprises or consists of the IgV domain of SIRPα or a variant thereof. In some embodiments, the IgV domain comprises one or more mutations.In some embodiments, the SIRPα extracellular domain comprises one or more mutations.

[0028] As used herein, the term "PD-L1 binding domain" refers to a protein domain capable of binding to PD-L1. In some embodiments, the PD-L1 binding domain may be an anti-PD-L1 antibody, an antigen-binding fragment thereof (e.g., scFv or VHH), or a PD-L1 binding protein or portion thereof. In some embodiments, the PD-L1 binding domain may have one or more auto-stabilizing domains. In some embodiments, the PD-L1 binding domain comprises or consists of the PD-1 extracellular domain. PD-1 may be wild-type PD-1, human PD-1, a polypeptide derived from wild-type PD-1 (e.g., having a mutation), or a portion thereof (e.g., the extracellular domain of PD-1). In some embodiments, the polypeptide derived from wild-type PD-1 may have one or more mutations. In some embodiments, the PD-1 extracellular domain comprises or consists of substantially the entire extracellular domain of PD-1 or a variant thereof. In some embodiments, the PD-1 extracellular domain comprises or consists of a portion of the extracellular domain of PD-1 or a variant thereof. In some embodiments, the PD-1 extracellular domain comprises one or more mutations.

[0029] As used herein, the term "cancer" refers to cells capable of autonomous growth. Examples of such cells include cells with an abnormal state or condition characterized by rapidly proliferating cell proliferation. The term is meant to include cancerous growths, e.g., tumors, oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematological, nervous, hepatic, gastrointestinal, and endocrine systems, as well as adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell carcinomas of the lung, and cancer of the small intestine. "Spontaneously occurring" cancer includes any cancer that is not experimentally induced by the implantation of cancer cells into a subject, including, for example, spontaneously occurring cancers, cancers caused by exposure of a patient to a carcinogen, cancers caused by the insertion of a transgenic cancer gene or knockout of a tumor suppressor gene, and cancers caused by infection, e.g., viral infection. The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissue. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to cancers derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to malignant tumors of mesenchymal origin. The term "hematopoietic neoplastic disorder" includes diseases involving hyperplasia / tumor cells of hematopoietic origin. Hematopoietic neoplastic disorders can arise from the myeloid, lymphoid, or erythroid lineages, or from their precursor cells. Hematologic cancers are cancers that arise in hematopoietic tissues, such as bone marrow, or in cells of the immune system. Examples of hematologic cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0030] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification to describe an animal, human, or non-human to which treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated in this disclosure. A human patient can be an adult human or a juvenile human (e.g., a human under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, miniature pigs), equines, canines, felines, bovines, and other domestic, livestock, and zoo animals.

[0031] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.

[0032] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art may also be used. The materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0034] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0035] [Figure 1-1] 1A to 1D show schematic structures of HCB301-4, HCB301-3, HCB301-2, and HCB301-1, respectively. [Figure 1-2] See description of Figure 1-1. [Diagram 2] 1 shows the results of total cell binding of HCB301 protein to transfected CHO-S cells expressing CD47. [Diagram 3] Figure 1 shows the results of total cellular binding of HCB301 protein to transfected CHO-S cells expressing PD-L1. [Figure 4] 4 shows the results of total cell binding of HCB301 protein to CD47-expressing FaDu cells. [Figure 5A] Figures 5A-5B show the results of RBC binding of HCB301 protein, and Figure 5B shows the results without a curve for Hu5F9-G4. [Figure 5B] See legend to Figure 5A. [Figure 6A] Figures 6A-6B show platelet binding results for HCB301 protein. Figure 6B shows the results without a curve for Hu5F9-G4. [Figure 6B] See legend to Figure 6A. [Figure 7A] The results and EC50 values ​​of human TGFβ1 binding of HCB301 protein are shown. IgG4-TGFβ trap was used as a positive control. [Figure 7B] The results and EC50 values ​​of human TGFβ2 binding of HCB301 protein are shown. IgG4-TGFβ trap was used as a positive control. [Figure 7C] The results and EC50 values ​​of human TGFβ3 binding of HCB301 protein are shown. IgG4-TGFβ trap was used as a positive control. [Figure 8A] Shown is the percentage binding of HCB301 protein at a concentration of 32 pM to non-transfected OE19 cells and transfected OE19 cells expressing PD-L1. [Figure 8B] Figures 8B-8G show the binding curves of SIRPα_G4, PD1_G4, HCB301-3, HCB301-4, HCB301-1, and HCB301-2 to untransfected OE19 cells and transfected OE19 cells expressing PD-L1. [Figure 8C] See legend to Figure 8B. [Figure 8D] See legend to Figure 8B. [Figure 8E] See legend to Figure 8B. [Figure 8F] See legend to Figure 8B. [Figure 8G] See legend to Figure 8B. [Figure 9] 4 shows the results of RBC hemagglutination of HCB301 protein. [Figure 10] 1 shows the results of SIRPα / CD47 blockade of HCB301 protein on transfected CHO-S cells expressing CD47. [Figure 11] Figure 2 shows the results of PD-1 / PD-L1 blockade of HCB301 protein on transfected CHO-S cells expressing PD-L1. Biotin-PD1-ECD-Fc represents biotinylated PD1_G4. [Figure 12A] 1 shows TGFβ-1-mediated smad2 reporter activity inhibited by HCB301 protein. [Figure 12B] 1 shows TGFβ-2-mediated smad2 reporter activity inhibited by HCB301 protein. [Figure 12C] 1 shows TGFβ-3-mediated smad2 reporter activity inhibited by HCB301 protein. [Figure 13]1 shows the phagocytic activity of HCB301 protein induced by Raw264.7 mouse macrophages against CD47-expressing Jurkat cells. [Figure 14A] FIG. 1 shows HCB301 protein-induced cell proliferation in an MLR assay in the presence of TGFβ1. [Figure 14B] FIG. 1 shows HCB301 protein-induced IL-2 secretion in an MLR assay in the presence of TGFβ1 as measured by ELISA. [Figure 14C] FIG. 1 shows HCB301 protein-induced IFN-γ secretion in an MLR assay in the presence of TGFβ1 as measured by ELISA. [Figure 15A] 15A-15B show a table summarizing the results of the in vitro assays. [Figure 15B] See legend to Figure 15A. [Figure 16-1] FIG. 16 lists the sequences discussed in this disclosure. [Figure 16-2] See description of Figure 16-1. [Figure 16-3] See description of Figure 16-1. [Figure 16-4] See description of Figure 16-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description The present disclosure provides protein complexes that bind to CD47 and PD-L1. These protein complexes can be used to simultaneously target the CD47 / SIRPα pathway and the PD-1 / PD-L1 pathway. The results show that the protein complexes can effectively bind to CD47-expressing cancer cells, block the interaction between endogenous SIRPα and CD47, and thereby induce innate immune responses (e.g., phagocytosis of cancer cells by macrophages). Meanwhile, the protein complexes showed minimal binding to RBC cells or platelets, thereby inhibiting host cell clearance, as observed by the anti-CD47 antibody magrolimab. In addition, the results show that the protein complexes can selectively bind to PD-L1-expressing cancer cells and block the interaction between endogenous PD-1 and PD-L1. Furthermore, the protein complexes can also contain a TGFβ trap, thereby inhibiting TGFβ-induced immunosuppressive responses and increasing T cell proliferation and cytokine secretion.

[0037] Thus, the protein complexes described herein may be used for cancer therapy involving enhanced tumor immunogenicity and antigen presentation via increased phagocytosis by macrophages (e.g., by inactivating CD47-mediated inhibition of phagocytosis), and enhanced T cell activation via inhibition of the PD-1 / PD-L1 and TGFβ signaling pathways.

[0038] SIRPα extracellular domain Signal regulatory protein alpha (SIRPα, SIRPa, Sirpa, or CD172A) is a transmembrane protein. It has an extracellular region that contains three Ig-like domains, and a cytoplasmic region that contains an immunoreceptor tyrosine-based inhibitory motif that mediates binding of the protein tyrosine phosphatases SHP1 and SHP2. Tyrosine phosphorylation of SIRPα is regulated by various growth factors and cytokines, as well as by integrin-mediated cell adhesion to extracellular matrix proteins. SIRPα is particularly abundant in myeloid cells such as macrophages and dendritic cells, but is expressed only at low levels in T cells, B cells, NK cells, and NKT cells.

[0039] The extracellular region of SIRPα can interact with its ligand CD47. Interaction of SIRPα on macrophages with CD47 on erythrocytes prevents phagocytosis of Ig-opsonized erythrocytes by macrophages in vitro and in vivo. Ligation of SIRPα on phagocytes by CD47 expressed on neighboring cells results in phosphorylation of the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibitory motif, leading to recruitment of SHP-1 and SHP-2 phosphatases. One resulting downstream effect is the prevention of accumulation of myosin-IIA at the phagocytic synapse and the resulting inhibition of phagocytosis. Thus, CD47-SIRPα interaction functions as a negative immune checkpoint to send a "don't eat me" signal to ensure that healthy self-cells are not inappropriately phagocytosed. However, overexpression of CD47 is also found in almost all types of tumors, some of which include acute myeloid leukemia, non-Hodgkin's lymphoma, bladder cancer, and breast cancer. Such negative regulation of macrophages can be minimized by blocking the binding of CD47 to SIRPα. Thus, agents that block CD47 / SIRPα interaction can promote both antibody-dependent cellular phagocytosis (ADCP) and, in some cases, cause antibody-dependent cellular cytotoxicity (ADCC), and thus can be used to treat various cancers.

[0040] Blocking CD47 / SIRPα interaction can promote cell phagocytosis and therefore can be used to treat various cancers. It causes the recognition and elimination of cancer cells by innate immunity. Agents that target CD47 or SIRPα can be used to treat various tumors and cancers, such as solid tumors, hematological malignancies (e.g., relapsed or refractory hematological malignancies), acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, bladder cancer, ovarian cancer, and small cell lung cancer tumors.

[0041] In addition, SIRPα acts to inhibit in vivo clearance of CD47-expressing host cells, including red blood cells and platelets, by macrophages. CD47-SIRPα interaction also appears to be essential for hematopoietic stem cell engraftment. Blocking CD47 / SIRPα interaction can cause accidental death of normal red blood cells, resulting in anemia and causing inflammation. Therefore, it is important to regulate the interaction of SIRPα-targeting agents with CD47, for example, to limit or control their effects on red blood cells.

[0042] A detailed description of SIRPα and its functions can be found, for example, in Yanagita et al. “Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy” JCI insight 2.1 (2017) and Seiffert et al. “Signal-regulatory protein α (SIRPα) but not SIRPβ is involved in T-cell activation, binds to CD47 with high affinity, and is expressed on immature CD34+CD38-hematopoietic cells” Blood 97.9 (2001): 2741-2749, which are incorporated by reference in their entireties.

[0043] Human SIRPα is a member of the signal regulatory proteins (SIRPs). Signal regulatory proteins are cell surface Ig superfamily proteins that mediate essential cell surface protein interactions and signal transduction. All SIRPs contain an N-terminal extracellular region, a single transmembrane domain, and a C-terminal intracellular region.

[0044] The extracellular region of human SIRPα (UniProt identifier: P78324) contains an IgV domain, an Ig-like C1-1 type domain, and an Ig-like C1-2 type domain. These correspond to amino acids 32-137, 148-247, and 254-348 of the human SIRPα protein (SEQ ID NO: 23, NP_542970.1). Amino acids 1-30 are the signal peptide. Human SIRPα also contains a long intracellular domain that contains two putative immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Activation of SIRPα ITIMs delivers inhibitory signals that negatively regulate cellular responses.

[0045] In some embodiments, the protein complex comprises one or more CD47 binding domains. In some embodiments, the CD47 binding domain comprises or consists of a SIRPα extracellular domain. As used herein, "SIRPα extracellular domain" refers to all or a portion of the extracellular region of SIRPα or a variant thereof, a portion of the extracellular region can bind to CD47. The SIRPα extracellular domain can have one or more protein domains that can fold independently and form a self-stabilizing structure. In some embodiments, the SIRPα extracellular domain comprises or consists of one or more domains selected from an IgV domain, an Ig-like C1-1 type domain, and an Ig-like C1-2 type domain. In some embodiments, the SIRPα extracellular domain comprises or consists of an IgV domain. In some embodiments, the SIRPα extracellular domain comprises or consists of an IgV domain and an Ig-like C1-1 type domain. In some embodiments, the SIRPα extracellular domain comprises or consists of an IgV domain, an Ig-like C1-1 type domain, and an Ig-like C1-2 type domain.

[0046] In some embodiments, a SIRPα extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 31-148 of the human SIRPα protein (NCBI Accession No.: AAH26692.1, SEQ ID NO: 12). In some embodiments, a CD47 binding domain or a SIRPα domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, a CD47 binding domain or a SIRPα extracellular domain described herein comprises an IgV domain of a human SIRPα protein. In some embodiments, a CD47 binding domain or a SIRPα extracellular domain described herein comprises an IgV domain of a mouse SIRPα protein.

[0047] PD-1 extracellular domain PD-1 (programmed death-1) is an immune checkpoint that protects against autoimmunity through a dual mechanism: promoting apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes while reducing apoptosis in regulatory T cells (anti-inflammatory, suppressor T cells).

[0048] PD-1 is mainly expressed on the surface of T cells and primary B cells, and its two ligands (PD-L1 and PD-L2) are widely expressed on antigen-presenting cells (APCs). The interaction between PD-1 and its ligand plays an important role in the negative regulation of immune response. Inhibition of the binding between PD-1 and its ligand can expose tumor cells to the killing effect of the immune system, thus achieving the effect of killing tumor tissue and treating cancer.

[0049] PD-L1 is expressed on tumor cells in many different cancers. By binding to and resulting in the inhibition of PD-1 on T cells, PD-L1 expression is a major mechanism by which tumor cells can evade immune attack. Overexpression of PD-L1 can conceptually be attributed to two mechanisms: intrinsic and adaptive. The inherent expression of PD-L1 on cancer cells is associated with cellular / genetic abnormalities in these tumor cells. Activation of cell signaling, including the AKT and STAT pathways, leads to increased PD-L1 expression. In primary mediastinal B-cell lymphoma, genetic fusion of MHC class II transactivator (CIITA) with PD-L1 or PD-L2 occurs, leading to overexpression of these proteins. Amplification of chromosome 9p23-24, where PD-L1 and PD-L2 are located, leads to increased expression of both proteins in classical Hodgkin's lymphoma. The adaptive mechanism is associated with the induction of PD-L1 expression in the tumor microenvironment. PD-L1 can be induced on tumor cells in response to interferon-gamma. In microsatellite unstable colon cancer, PD-L1 is expressed primarily on myeloid cells within the tumor and thus suppresses cytotoxic T cell function.

[0050] The use of PD-1 blockade to enhance antitumor immunity stems from observations in chronic infection models where preventing PD-1 interactions reversed T cell exhaustion. Similarly, blockade of PD-1 prevents T cell PD-1 / tumor cell PD-L1 or T cell PD-1 / tumor cell PD-L2 interactions, resulting in restoration of T cell-mediated antitumor immunity.

[0051] For a detailed description of PD-1 and the use of anti-PD-1 antibodies to treat cancer, see, for example, Topalian, Suzanne L., et al. "Safety, activity, and immune correlates of anti-PD-1 antibody in cancer." New England Journal of Medicine 366.26(2012):2443-2454; Hirano, Fumiya, et al. "Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity." Cancer research 65.3(2005):1089-1096; Raedler, Lisa A. "Keytruda (pembrolizumab): first PD-1 inhibitor approved for previously treated unresectable or metastatic melanoma." American health & drug benefits 8.Spec Feature(2015):96; Kwok, Gerry, et al. al. "Pembrolizumab (Keytruda)." (2016): 2777-2789, US2017 / 0247454, US9,834,606(B), and US8,728,474, each of which is incorporated by reference in its entirety.

[0052] According to the UniProt identifier Q15116, the extracellular region of human PD-1 corresponds to amino acids 24-170 of SEQ ID NO:11, the transmembrane region of human PD-1 corresponds to amino acids 171-191 of SEQ ID NO:11, and the cytoplasmic region of human PD-1 corresponds to amino acids 192-288 of SEQ ID NO:11. The PD-1 extracellular region also has an IgV domain corresponding to amino acids 35-145 of the human PD-1 protein (SEQ ID NO:11, NP_005009.2). The signal peptide corresponds to amino acids 1-23 of SEQ ID NO:11. The cytoplasmic region of human SIRPα also has an immunoreceptor tyrosine-based inhibition motif (ITIM, corresponding to amino acids 221-226 of SEQ ID NO:11) and an immunoreceptor tyrosine-based switch motif (ITSM, corresponding to amino acids 241-251 of SEQ ID NO:11).

[0053] In some embodiments, the protein complex comprises one or more PD-L1 binding domains. In some embodiments, the PD-L1 binding domain comprises or consists of the PD-1 extracellular domain. As used herein, "PD-1 extracellular domain" refers to all or a portion of the extracellular region of PD-1, or a variant thereof, a portion of the extracellular region that can bind to PD-L1. The PD-1 extracellular domain can have one or more protein domains that can fold independently and form a self-stabilizing structure. In some embodiments, the PD-1 extracellular domain comprises or consists of an IgV domain. In some embodiments, the PD-1 extracellular domain does not comprise a signal peptide.

[0054] In some embodiments, the PD-1 extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-170 of the human PD-1 protein (NCBI Accession No. NP_005009.2, SEQ ID NO: 11). In some embodiments, the PD-L1 binding domain or PD-1 extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the PD-L1 binding domain or SIRPα extracellular domain described herein comprises the IgV domain of the human PD-1 protein. In some embodiments, the PD-L1 binding domain or SIRPα extracellular domain described herein comprises all or a portion of the extracellular domain of the mouse PD-L1 protein.

[0055] TGFBR2 extracellular domain TGF-beta receptor type 2 (TGFBR2) is the ligand-binding receptor for all members of the TGF-beta family and is expressed in virtually all cell types, including fibroblasts. Ligand-induced cellular responses are mediated through either canonical Smad-dependent or non-canonical Smad-independent signaling pathways, such as the c-Jun N-terminal kinase, Akt, Src, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase pathways. Ligand binding to TGFBR2 leads to receptor dimerization and autophosphorylation, which then binds to TGF-beta receptor type 1 (TGFBR1) or type 3 (TGFBR3). The newly formed heterotetrameric complex then phosphorylates the regulatory SMAD (SMAD2 or SMAD3), which in its phosphorylated state binds to the co-SMAD molecule SMAD4. The regulatory SMAD / co-SMAD complex translocates to the nucleus and acts as a transcription factor to regulate target gene expression.

[0056] A detailed description of TGFβ, TGFBR2, and the use of TGFβ traps to treat cancer are described, for example, in Bierie, B., et al. “TGFβ: the molecular Jekyll and Hyde of cancer.” Nature Reviews Cancer 6.7(2006):506-520, Kim, B., et al. “Novel therapies emerging in oncology to target the TGF-β pathway.” Journal of Hematology & Oncology 14.1(2021):1-20, and Lind, H., et al. “Dual targeting of TGF-β and PD-L1 via a bifunctional anti-PD-L1 / TGF-βRII agent: status of preclinical and clinical advances.” Journal for immunotherapy of cancer 8.1(2020), each of which is incorporated by reference in its entirety.

[0057] According to the UniProt identifier P37173, the extracellular domain of human TGFBR2 corresponds to amino acids 23 to 166 of SEQ ID NO: 13, the transmembrane domain of human TGFBR2 corresponds to amino acids 167 to 187 of SEQ ID NO: 13, and the cytoplasmic domain of human TGFBR2 corresponds to amino acids 188 to 567 of SEQ ID NO: 13. The signal peptide corresponds to amino acids 1 to 22 of SEQ ID NO: 13.

[0058] In some embodiments, the protein complex comprises one or more TGFβ binding domains. In some embodiments, the TGFβ binding domain comprises or consists of the TGFBR2 extracellular domain. As used herein, "TGFBR2 extracellular domain" refers to all or a portion of the extracellular region of TGFBR2 or a variant thereof, a portion of the extracellular region can bind to TGFβ. The TGFBR2 extracellular domain can have one or more protein domains that can fold independently and form a self-stabilizing structure. In some embodiments, the TGFBR2 extracellular domain does not include a signal peptide.

[0059] In some embodiments, a TGFBR2 extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 24-159 of the human TGFBR2 protein (NCBI Accession No.: NP_003233.4, SEQ ID NO: 13). In some embodiments, a TGFβ binding domain or TGFBR2 extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, a TGFβ binding domain or TGFBR2 extracellular domain described herein comprises all or a portion of the human TGFBR2 extracellular domain. In some embodiments, a TGFβ binding domain or TGFBR2 extracellular domain described herein comprises all or a portion of the extracellular domain of a mouse PD-L1 protein.

[0060] Protein complexes that target CD47, PD-L1, and / or TGFβ The present disclosure provides protein complexes that can specifically bind to CD47. In some embodiments, these protein complexes can block the CD47 / SIRPα signaling pathway and thus increase the immune response. In some embodiments, these protein complexes can initiate phagocytosis.

[0061] The present disclosure also provides protein complexes that can specifically bind to PD-L1. In some embodiments, these protein complexes can block the PD-1 / PD-L1 signaling pathway and thus increase the immune response. In some embodiments, these protein complexes can induce T cell activation, proliferation, and / or cytokine release.

[0062] In one aspect, the disclosure provides a protein complex or protein construct comprising or consisting of an Fc, one or more CD47 binding domains, one or more PD-L1 binding domains, and optionally one or more TGFβ binding domains. As used herein, the term "Fc" refers to a fragment crystallizable region of an antibody (e.g., IgG, IgE, IgM, IgA, or IgD). The term "Fc region" or "Fc region sequence" refers to the heavy chain constant domains (e.g., CH2 and CH3) in the heavy chain peptide that forms the Fc region. In some embodiments, the protein complex or protein construct comprises 1, 2, 3, 4, 5, or 6 CD47 binding domains. In some embodiments, the protein complex or protein construct comprises 1, 2, 3, 4, 5, or 6 PD-L1 binding domains. In some embodiments, the protein complex or protein construct comprises 1, 2, 3, 4, 5, or 6 TGFβ binding domains.

[0063] In some embodiments, the protein complex or protein construct comprises or consists of Fc, a first domain that specifically binds cluster of differentiation 47 (CD47), and a second domain that specifically binds programmed death-ligand 1 (PD-L1).

[0064] In some embodiments, the first domain may bind to a cell expressing CD47 (e.g., a cancer cell) and / or block the interaction between CD47 and signal regulatory protein alpha (SIRPα). In some embodiments, the first domain comprises all or a portion of the extracellular region of SIRPα. In some embodiments, the SIRPα is human SIRPα. In some embodiments, the first domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6.

[0065] In some embodiments, the second domain may bind to cells expressing PD-L1 (e.g., cancer cells) and / or stimulate T cell activation and proliferation. In some embodiments, the second domain comprises all or a portion of the extracellular region of programmed cell death protein 1 (PD-1). In some embodiments, the PD-1 is human PD-1. In some embodiments, the second domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5.

[0066] In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the first domain is linked to the N-terminus of the CH2 domain in the Fc, optionally via a hinge region. In some embodiments, the second domain is linked to the N-terminus of the CH2 domain in the Fc, optionally via a hinge region. In some embodiments, the hinge region is a human IgG4 hinge region, optionally having a S228P mutation according to EU numbering.

[0067] In some embodiments, the protein complex or protein construct further comprises a third domain that specifically binds transforming growth factor beta (TGFβ). In some embodiments, the third domain is linked to the C-terminus of the CH3 domain of the Fc, optionally via a linker peptide.

[0068] In some embodiments, the protein complex comprises two or more first domains. In some embodiments, the protein complex comprises two or more second domains. In some embodiments, the protein complex comprises two or more third domains.

[0069] In some embodiments, the CD47 binding domain, the PD-L1 binding domain, and the TGFβ binding domain are linked to the Fc region via any of the linker peptide or hinge region sequences described herein.

[0070] Several embodiments of protein complexes are shown in Figures 1A-1D and are described in detail below.

[0071] HCB301-4 In one aspect, the disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide comprises, preferably from N-terminus to C-terminus, a first PD-L1 binding domain, an optional first linker peptide, a first CD47 binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and an optional first TGFβ binding domain. The second polypeptide comprises, preferably from N-terminus to C-terminus, a second PD-L1 binding domain, an optional third linker peptide, a second CD47 binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and an optional second TGFβ binding domain. A schematic structure of an exemplary protein complex having the HCB301-4 format is shown in Figure 1A.

[0072] In some embodiments, the first and / or second PD-L1 binding domain comprises all or a portion of the extracellular domain of PD-1, e.g., amino acids 26-170 of the human PD-1 protein (NCBI Accession No.: NP_005009.2, SEQ ID NO: 11), or SEQ ID NO: 5. In some embodiments, the first and / or second PD-L1 binding domains are identical. In some embodiments, the first and / or second PD-L1 binding domains are different. In some embodiments, the first and / or second PD-L1 binding domains comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5.

[0073] In some embodiments, the first, second, third, and / or fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs:8-10. In some embodiments, the first, second, third, and / or fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO:18) or GSGGSG (SEQ ID NO:19).

[0074] In some embodiments, the first and / or second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1.

[0075] HCB301-2 In one aspect, the disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide comprises, preferably from N-terminus to C-terminus, a third PD-L1 binding domain, an optional fifth linker peptide, a first PD-L1 binding domain, optionally a first linker peptide, a first CD47 binding domain, optionally a first hinge region, a first Fc region, optionally a second linker peptide, and optionally a first TGFβ binding domain. The second polypeptide comprises, preferably from N-terminus to C-terminus, a fourth PD-L1 binding domain, optionally a sixth linker peptide, a second PD-L1 binding domain, optionally a third linker peptide, a second CD47 binding domain, optionally a second hinge region, a second Fc region, optionally a fourth linker peptide, and optionally a second TGFβ binding domain. A schematic structure of an exemplary protein complex having the HCB301-2 format is shown in Figure 1C.

[0076] In some embodiments, the first, second, third, and / or fourth PD-L1 binding domains comprise all or a portion of the extracellular domain of PD-1, e.g., amino acids 26-170 of the human PD-1 protein (NCBI Accession No.: NP_005009.2, SEQ ID NO: 11), or SEQ ID NO: 5. In some embodiments, the first, second, third, and / or fourth PD-L1 binding domains are identical. In some embodiments, the first, second, third, and / or fourth PD-L1 binding domains are different. In some embodiments, the first, second, third, and / or fourth PD-L1 binding domains comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5.

[0077] In some embodiments, the first, second, third, fourth, fifth, and / or sixth linker peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 8-10. In some embodiments, the first, second, third, fourth, fifth, and / or sixth linker peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO: 18) or GSGGSG (SEQ ID NO: 19).

[0078] In some embodiments, the first and / or second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:3.

[0079] HCB301-3 In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide comprises, preferably from N-terminus to C-terminus, a first CD47 binding domain, an optional first linker peptide, a first PD-L1 binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and an optional first TGFβ binding domain. The second polypeptide comprises, preferably from N-terminus to C-terminus, a second CD47 binding domain, an optional third linker peptide, a second PD-L1 binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and an optional second TGFβ binding domain. A schematic structure of an exemplary protein complex having the HCB301-4 format is shown in FIG. 1B.

[0080] In some embodiments, the first and / or second PD-L1 binding domain comprises all or a portion of the extracellular domain of PD-1, e.g., amino acids 26-170 of the human PD-1 protein (NCBI Accession No.: NP_005009.2, SEQ ID NO: 11), or SEQ ID NO: 5. In some embodiments, the first and / or second PD-L1 binding domains are identical. In some embodiments, the first and / or second PD-L1 binding domains are different. In some embodiments, the first and / or second PD-L1 binding domains comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5.

[0081] In some embodiments, the first, second, third, and / or fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs:8-10. In some embodiments, the first, second, third, and / or fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO:18) or GSGGSG (SEQ ID NO:19).

[0082] In some embodiments, the first and / or second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2.

[0083] HCB301-1 In one aspect, the disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide comprises, preferably from N-terminus to C-terminus, a first CD47 binding domain, optionally a first linker peptide, a first PD-L1 binding domain, optionally a fifth linker peptide, a third PD-L1 binding domain, optionally a first hinge region, a first Fc region, optionally a second linker peptide, and optionally a first TGFβ binding domain. The second polypeptide comprises, preferably from N-terminus to C-terminus, a second CD47 binding domain, optionally a third linker peptide, a second PD-L1 binding domain, optionally a sixth linker peptide, a fourth PD-L1 binding domain, optionally a second hinge region, a second Fc region, optionally a fourth linker peptide, and optionally a second TGFβ binding domain. A schematic structure of an exemplary protein complex having the HCB301-2 format is shown in FIG. 1D.

[0084] In some embodiments, the first, second, third, and / or fourth PD-L1 binding domains comprise all or a portion of the extracellular domain of PD-1, e.g., amino acids 26-170 of the human PD-1 protein (NCBI Accession No.: NP_005009.2, SEQ ID NO: 11), or SEQ ID NO: 5. In some embodiments, the first, second, third, and / or fourth PD-L1 binding domains are identical. In some embodiments, the first, second, third, and / or fourth PD-L1 binding domains are different. In some embodiments, the first, second, third, and / or fourth PD-L1 binding domains comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5.

[0085] In some embodiments, the first, second, third, fourth, fifth, and / or sixth linker peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 8-10. In some embodiments, the first, second, third, fourth, fifth, and / or sixth linker peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO: 18) or GSGGSG (SEQ ID NO: 19).

[0086] In some embodiments, the first and / or second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4.

[0087] In any of the protein complexes described herein, the first and / or second CD47 binding domain can comprise all or a portion of the extracellular domain of SIRPα, e.g., amino acids 31-148 of the human SIRPα protein (NCBI Accession No.: AAH26692.1, SEQ ID NO: 12) or SEQ ID NO: 6. In some embodiments, the first and second CD47 binding domains are identical. In some embodiments, the first and / or second CD47 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the first and / or second CD47 binding domain comprises an IgV domain of SIRPα (e.g., human SIRPα). In some embodiments, the SIRPα IgV domain comprises one or more mutations. In some embodiments, the first and second CD47 binding domains are different.

[0088] In any of the protein complexes described herein, the first and / or second TGFβ binding domain can comprise all or a portion of the extracellular domain of TGFBR2, e.g., amino acids 24-159 of the human TGFBR2 protein (NCBI Accession No.: NP_003233.4, SEQ ID NO: 13) or SEQ ID NO: 7. In some embodiments, the first and / or second TGFβ binding domains are identical. In some embodiments, the first and / or second TGFβ binding domains are different. In some embodiments, the first and / or second TGFβ binding domains comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0089] In any of the protein complexes described herein, the first and / or second hinge region may comprise all or a portion of an immunoglobulin hinge region, e.g., a human IgG4 hinge region (SEQ ID NO: 16). In some embodiments, the first and / or second hinge region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16. In some embodiments, the first and second hinge regions are identical. In some embodiments, the first and second hinge regions are different. In some embodiments, the first and / or second hinge region comprises a proline at position 228 according to the EU numbering.

[0090] In any of the protein complexes described herein, the first and / or second Fc regions may be identical and may form an Fc homodimer. In some embodiments, the first and / or second Fc regions comprise all or a portion of an immunoglobulin Fc region, e.g., a human IgG4 Fc region (SEQ ID NO: 17). In any of the protein complexes described herein, the first and / or second Fc regions may be different. In some embodiments, the first and / or second Fc regions may form an Fc heterodimer by introducing one or more mutations. For example, the first and / or second Fc regions may comprise one or more knob-into-hole (KIH) mutations. In some embodiments, the first and / or second Fc regions may form an Fc heterodimer using other techniques known in the art. Detailed heterodimeric Fc technology can be found, for example, in Ha, et al. “Immunoglobulin Fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins.” Frontiers In Immunology 7 (2016): 394, which is incorporated by reference in its entirety.

[0091] In one aspect, the disclosure relates to a protein complex comprising a CD47 binding domain (e.g., any of the CD47 binding domains described herein) and a PD-L1 binding domain (e.g., any of the PD-L1 binding domains described herein). In some embodiments, the protein complex further comprises a TGFβ binding domain (e.g., any of the TGFβ binding domains described herein). In some embodiments, the CD47 binding domain is or comprises a SIRPα extracellular domain (e.g., any of the SIRPα extracellular domains described herein). In some embodiments, the PD-L1 binding domain is or comprises a PD-1 extracellular domain (e.g., any of the PD-1 extracellular domains described herein). In some embodiments, the TGFβ binding domain is or comprises a TGFBR2 extracellular domain (e.g., any of the TGFBR2 extracellular domains described herein).

[0092] Characterization of protein complexes In some embodiments, the protein complex may comprise any of the CD47-binding domains, PD-L1-binding domains, and / or TGFβ-binding domains described herein.The present disclosure also provides nucleic acids comprising polynucleotides encoding the polypeptides described herein.

[0093] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments, at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. For example, comparison of sequences and determination of percent identity between two sequences can be accomplished using the Blossum62 scoring matrix, which has a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0094] The protein complexes described herein may comprise an Fc of an antibody. These antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class, or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE1, IgE2). In some embodiments, the Fc region is derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc region is an IgG4 Fc region (e.g., a human IgG4 Fc region).

[0095] In some embodiments, the protein complexes described herein are linked to the Fc region via an antibody hinge region (e.g., IgG, IgE hinge region). In addition, the Fc region can be modified to provide desired effector functions or serum half-life.

[0096] The protein complexes described herein can block the binding between CD47 expressed on immune cells and endogenous SIRPα. In some embodiments, by binding to CD47, the protein complexes described herein can inhibit the binding of CD47 (e.g., expressed on tumor cells) to endogenous SIRPα expressed on immune cells (e.g., bone marrow cells, macrophages, and dendritic cells), thereby blocking the CD47 / SIRPα pathway, upregulating the immune response, and promoting phagocytosis.

[0097] The protein complexes described herein can block the binding between PD-L1 expressed on immune cells and endogenous PD-1. In some embodiments, by binding to PD-L1, the protein complexes described herein can inhibit the binding of PD-L1 (e.g., expressed on tumor cells) to endogenous PD-1 expressed on immune cells (e.g., T cells), thereby blocking the PD-1 / PD-L1 pathway and upregulating the immune response, activating T cell proliferation and cytokine release.

[0098] In some embodiments, the protein complexes described herein may increase an immune response, activity or number of immune cells (e.g., myeloid cells, macrophages, dendritic cells, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0099] In some embodiments, the protein complexes described herein are -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or equal to 0.00001s -1It may bind to CD47 (e.g., human CD47, monkey CD47, or mouse CD47), PD-L1 (e.g., human PD-L1, monkey PD-L1, or mouse PD-L1), or TGFβ (e.g., human TGFβ, monkey TGFβ, or mouse TGFβ) with a koff of less than 0.01 s. In some embodiments, the koff is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Greater than or equal to 0.000001s -1 In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or 1×10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms. The affinity can be estimated from the quotient of the kinetic rate constants (KD=koff / kon). In some embodiments, the KD is less than 1×10 -6 Less than M, 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M or 1×10 -10 In some embodiments, the KD is less than 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 More than M or 1×10 -12It's super M.

[0100] Common techniques for measuring affinity include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the protein complexes described herein may bind to monkey CD47 and / or mouse CD47. In some embodiments, the protein complexes described herein are unable to bind to monkey CD47 and / or mouse CD47. In some embodiments, the protein complexes described herein may bind to monkey PD-L1 and / or mouse PD-L1. In some embodiments, the protein complexes described herein are unable to bind to monkey PD-L1 and / or mouse PD-L1. In some embodiments, the protein complexes described herein may bind to monkey TGFβ and / or mouse TGFβ. In some embodiments, the protein complexes described herein are unable to bind to monkey TGFβ and / or mouse TGFβ.

[0101] In some embodiments, thermal stability is determined. Protein complexes described herein may have a Tm of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, the Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C.

[0102] In some embodiments, the protein complexes described herein have a tumor growth inhibition percentage (TGI%) of more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the protein complexes described herein have a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be determined, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after initiation of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after initiation of treatment. As used herein, tumor growth inhibition percentage (TGI%) is defined according to the following formula: TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100 where Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day zero. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day zero.

[0103] In some embodiments, the tumor inhibitory effect of the protein complexes described herein is comparable to that of an anti-CD47 reference antibody, e.g., Hu5F9-G4, or an anti-SIRPα antibody, e.g., CC-95251. Hu5F9-G4 is described, e.g., in Sikic et al. “First-in-human, first-in-class phase I trial of the anti-CD47 antibody Hu5F9-G4 in patients with advanced cancers.” Journal of Clinical Oncology 37.12(2019):946, which is incorporated herein by reference in its entirety. In some embodiments, the tumor inhibitory effect of the protein complexes described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold greater than that of an anti-CD47 reference antibody, e.g., Hu5F9-G4, or an anti-SIRPα antibody, e.g., CC-95251. In some embodiments, the tumor inhibitory effect of the protein complexes described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold greater than SIRPα_G4. Details of SIRPα_G4 (or hSIRPα-G4Fc-wt(Trillium), TTI-622) can be found, for example, in US Patent Application Publication No. 2015 / 0329616(A1), which is incorporated herein by reference in its entirety. The amino acid sequence of SIRPα_G4 is shown in SEQ ID NO:14.

[0104] In some embodiments, the tumor inhibitory effect of the protein complex described herein is comparable to that of an anti-PD-L1 reference antibody, e.g., MPDL3280A (atezolizumab), or an anti-PD-1 antibody, e.g., pembrolizumab. MPDL3280A is described, e.g., in Powles, T. et al. "MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer." Nature 515.7528 (2014):558-562, which is incorporated herein by reference in its entirety. In some embodiments, the tumor inhibitory effect of the protein complex described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold greater than that of an anti-PD-L1 reference antibody, e.g., MPDL3280A, or an anti-PD-1 antibody, e.g., pembrolizumab. In some embodiments, the tumor inhibitory effect of the protein complex described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold greater than that of PD1_G4. PD1_G4 is used as a control protein for HCB301 fusion protein (HCB301 protein). The amino acid sequence of PD1_G4 is shown in SEQ ID NO: 15.

[0105] In some embodiments, the tumor inhibitory effect of the protein complexes described herein is comparable to that of a TGFβ trap protein, such as an IgG4-TGFβ trap, or an anti-PD-L1 antibody x TGFβ trap M7824. M7824 is described, for example, in Gatti-Mays, ME, et al. "M7824: a promising new strategy to combat cancer immune evasion." Oncoscience 5.11-12 (2018): 269, which is incorporated by reference in its entirety. In some embodiments, the tumor inhibitory effect of the protein complexes described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold greater than that of an IgG4-TGFβ trap or M7824. In some embodiments, the tumor-inhibiting effect of the protein complexes described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, or 5-fold greater than that of IgG4-TGFβ Trap. IgG4-TGFβ Trap is used as a control protein for HCB301 fusion protein (HCB301 protein). The amino acid sequence of IgG4-TGFβ Trap is shown in SEQ ID NO:20.

[0106] In some embodiments, the protein complexes described herein have a functional Fc. In some embodiments, the Fc is from human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the effector function of the functional Fc is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc is phagocytosis. In some embodiments, the effector function of the functional Fc is ADCC and phagocytosis. In some embodiments, the protein constructs described herein have an Fc region without effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering), or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering).

[0107] Several other modifications to the Fc region can be made. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric fusion protein thus generated may optionally have increased half-life in vitro and / or in vivo.

[0108] In some embodiments, the IgG4 has a S228P mutation (EU numbering), which prevents IgG4 Fab-arm exchange in vivo and in vitro.

[0109] In some embodiments, the Fc region has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an Fc region composition can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (position 314 in the Eu numbering of Fc region residues, or Kabat numbering), although due to minor sequence variations in the Fc region sequence, Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i.e., positions 294-300. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region can be further engineered to replace asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0110] In some embodiments, the major HPLC-SEC peak represents at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the protein complexes described herein after purification by Protein A-based affinity chromatography and / or size-exclusion chromatography.

[0111] In some embodiments, a protein complex described herein may bind to human CD47-expressing tumor cells (e.g., human CD47 tf CHO-S cells, or FaDu cells) with an affinity that is at least 10%, at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, or at least 120% greater than an anti-CD47 reference antibody (e.g., Hu5F9-G4) or SIRPα_G4.

[0112] In some embodiments, a protein complex described herein may bind to human PD-L1-expressing tumor cells (e.g., transfected CHO-S cells expressing human PD-L1) with an affinity that is at least 10%, at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, or at least 120% greater than PD1_G4.

[0113] In some embodiments, the protein complexes described herein may bind to human TGFβ (e.g., TGFβ1, TGFβ2, or TGFβ3) with an affinity that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, or at least 120% greater than that of IgG4-TGFβ Trap. In some embodiments, the EC50 value of the protein complexes binding to human TGFβ (e.g., TGFβ1, TGFβ2, or TGFβ3) is less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 1-fold, less than 2-fold, less than 3-fold, less than 4-fold, less than 5-fold, or less than 10-fold greater than that of IgG4-TGFβ Trap.

[0114] In some embodiments, a protein complex described herein may bind to RBC cells or platelets (e.g., from a human donor) with less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, or less than 1% of the affinity compared to an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0115] In some embodiments, the protein complexes described herein do not induce hemagglutination, hi some embodiments, the protein complexes described herein may induce hemagglutination at a minimum concentration of 500-fold, 2000-fold, 5000-fold, 20000-fold, or 50000-fold greater than that of an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0116] In some embodiments, the protein complexes described herein may block the interaction between CD47 (e.g., human CD47 or a fragment thereof) and SIRPα (e.g., human SIRPα or a fragment thereof). In some embodiments, the protein complexes described herein may block the interaction between human CD47-expressing cells (e.g., CD47 tf CHO-S cells or FaDu cells) and human SIRPα. In some embodiments, the blocking ability of the protein complexes described herein is at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% compared to an anti-CD47 reference antibody (e.g., Hu5F9-G4) or SIRPa_G4.

[0117] In some embodiments, a protein complex described herein may block the interaction between PD-L1 (e.g., human PD-L1 or a fragment thereof) and PD-1 (e.g., human PD-1 or a fragment thereof). In some embodiments, a protein complex described herein may block the interaction between human PD-L1-expressing cells (e.g., PD-L1 tf CHO-S cells) and human PD-1. In some embodiments, the blocking ability of a protein complex described herein is at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% compared to an anti-PD-L1 reference antibody (e.g., MPDL3280A) or PD1_G4.

[0118] In some embodiments, the protein complexes described herein may induce phagocytosis of CD47-expressing tumor cells (e.g., Jurkat cells) by mouse macrophages (e.g., Raw264.7 cells). In some embodiments, the EC50 value of the protein complexes described herein for inducing phagocytosis of CD47-expressing tumor cells is less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some embodiments, the EC50 value of the protein complexes described herein for inducing phagocytosis of CD47-expressing tumor cells is comparable to the EC50 value of an anti-CD47 reference antibody (e.g., Hu5F9-G4) or SIRPα_G4. In some embodiments, the protein complexes described herein are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as capable of inducing phagocytosis of CD47-expressing tumor cells by mouse macrophages as compared to the ability of Hu5F9-G4 or SIRPα_G4. In some embodiments, the protein complexes described herein are less capable (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%) of inducing phagocytosis of RBC cells or platelets by mouse macrophages (e.g., Raw264.7 cells) than an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0119] In some embodiments, the protein complexes described herein may induce phagocytosis of CD47-expressing tumor cells (e.g., Jurkat cells) by human macrophages (e.g., MDM cells). In some embodiments, the protein complexes described herein are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% capable of inducing phagocytosis of CD47-expressing tumor cells by human macrophages compared to the ability of an anti-CD47 reference antibody (e.g., Hu5F9-G4) or SIRPα_G4. In some embodiments, the protein complexes described herein are less capable (less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%) of inducing phagocytosis of RBC cells or platelets by human macrophages (e.g., MDM cells) than an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0120] Endogenous expression of CD47 on various cell types, including red blood cells, creates a formidable "antigen sink" that may limit the efficacy of CD47-targeted therapy. Thus, the weak ability of the protein complexes described herein to induce phagocytosis of RBC cells and / or platelets may increase the in vivo efficacy of the protein complexes. In addition, the protein complexes may be administered at lower dose levels and / or less frequent dosing schedules with efficacy similar to that of anti-CD47 reference antibodies (e.g., Hu5F9-G4).

[0121] In some embodiments, the protein complexes described herein may inhibit TGFβ-induced downstream pathways, such as the smad2 reporter pathway. In some embodiments, the protein complexes may inhibit TGFβ1-mediated smad2 reporter activity by less than 150%, less than 140%, less than 130%, less than 120%, less than 110%, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% compared to IgG4-TGFβ trap, anti-TGFβ, or M7824.

[0122] In some embodiments, the protein complexes described herein may enhance T cell responses (e.g., in an MLR assay). The principle of the mixed lymphocyte reaction (MLR) is that T cells from one donor proliferate in the presence of APCs from a different donor. This is triggered by the recognition of HLA mismatches between two unrelated donors, eliciting an immune response from the T cells. MLR is often used as a means to induce general stimulation / activation of T cells in culture. In some embodiments, the protein complexes may increase T cell proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% over control molecules or combinations thereof used herein. In some embodiments, the protein complexes described herein may increase cytokine (e.g., IFN-γ and / or IL-2) production by at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, or 10000-fold over a control molecule or combination thereof used herein.

[0123] In some embodiments, the protein complexes described herein do not induce a cytokine storm in humans. In some embodiments, the protein complexes described herein are not superagonists. Details of cytokine storm and superagonists can be found, for example, in Shimabukuro-Vornhagen, A. et al. "Cytokine release syndrome." Journal for ImmunoTherapy of Cancer 6.1 (2018): 1-14, which is incorporated herein by reference in its entirety.

[0124] In some embodiments, the protein complexes described herein can inhibit tumor growth.

[0125] Methods for making protein complexes Variants of the protein complexes described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the polypeptide or a portion thereof, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence.

[0126] Screening may be performed to increase the binding affinity of the CD47-binding domain and the PD-L1-binding domain. Any combination of deletions, insertions, and / or combinations may be performed to arrive at a variant with increased binding affinity to the target. The amino acid changes introduced into the variant may also alter or introduce new post-translational modifications to the polypeptide, such as altering the number (e.g., increasing or decreasing) of glycosylation sites, altering the type of glycosylation site (e.g., altering the amino acid sequence so that different sugars are attached by enzymes present in the cell), or introducing new glycosylation sites.

[0127] The CD47-binding domain and / or the PD-L1-binding domain may be derived from any species of animal, including mammals. Non-limiting examples of binding domain variants include sequences derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits).

[0128] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cell contains the polynucleotide and / or the vector comprising the polynucleotide), and the production of the recombinant polypeptides or fragments thereof by recombinant techniques.

[0129] As used herein, a "vector" is any construct that can deliver one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" can deliver and express one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, the polynucleotide of interest is arranged to be expressed in the vector by being operably linked to regulatory elements such as promoters, enhancers, and / or polyA tails, either within the vector or in the genome of the host cell, at or near or adjacent to the integration site of the polynucleotide of interest, such that the polynucleotide of interest is translated in the host cell into which the expression vector is introduced.

[0130] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0131] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may use a replication-defective virus. Techniques for incorporating DNA into such expression systems are well known to those of skill in the art. The DNA may also be "naked." Uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0132] For expression, the DNA inserts comprising the polypeptide-encoding polynucleotides disclosed herein may be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs, to name a few. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct may further contain sites for transcription initiation, termination, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcripts expressed by the construct may include a termination codon (UAA, UGA, or UAG) appropriately positioned at the beginning of translation to initiate and at the end of the polypeptide to be translated.

[0133] As indicated, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are known in the art.

[0134] Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0135] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral LTRs such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0136] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used.

[0137] Introduction of the construct into the host cell may be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0138] Transcription of DNA encoding the polypeptides of the present disclosure by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located on the late side of the replication origin at base pairs 100-270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0139] For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

[0140] Polypeptides can be expressed in modified forms such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain additional heterologous functional regions as well as secretion signals. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and persistence within the host cell, during purification, or during subsequent handling and storage. Peptide moieties can also be added to the polypeptide to facilitate purification. Such regions can be removed before final preparation of the polypeptide. The addition of peptide moieties to polypeptides to cause secretion or excretion, improve stability, and facilitate purification, among others, is a routine technique well known in the art.

[0141] Treatment methods The protein constructs or polypeptides of the present disclosure may be used for a variety of therapeutic purposes.

[0142] In one aspect, the present disclosure provides a method for treating cancer in a subject, reducing the rate of tumor volume growth over time in a subject, reducing the risk of developing metastasis, or reducing the risk of developing additional metastases in a subject. In some embodiments, the treatment may stop, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment may result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0143] In one aspect, the disclosure features a method comprising administering a therapeutically effective amount of a protein construct or polypeptide disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, cancer), such as breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancies. In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple negative breast cancer (TNBC), or colorectal carcinoma. In some embodiments, the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

[0144] In some embodiments, the compositions and methods disclosed herein may be used to treat patients at risk for cancer. Cancer patients may be identified in a variety of ways known in the art.

[0145] As used herein, "effective amount" refers to an amount or dosage sufficient to bring about a beneficial or desired result, including halting, slowing, retarding, or inhibiting the progression of a disease, e.g., cancer. The effective amount varies depending on, for example, the age and weight of the subject to whom the protein construct or polypeptide, the vector comprising the polynucleotide encoding the protein construct or polypeptide, and / or the composition thereof is administered, the severity of symptoms, and the route of administration, and thus administration can be determined on an individual basis.

[0146] An effective amount may be administered in one or more doses. For example, an effective amount of a protein construct or polypeptide is an amount sufficient to improve, stop, stabilize, reverse, inhibit, slow down, and / or delay the progression of cancer in a patient, or to improve, stop, stabilize, reverse, slow down, and / or delay the proliferation of cells in vitro (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)). As understood in the art, an effective amount may vary depending on other factors, such as the medical history of the patient and the type (and / or dosage) of protein construct or polypeptide used, among others.

[0147] Effective amounts and schedules for administering a protein construct or polypeptide, a polynucleotide encoding the protein construct or polypeptide, and / or a composition disclosed herein may be determined empirically, and making such determinations is within the skill of the art. One of ordinary skill in the art will understand that the dosage required to be administered will vary depending, for example, on the mammal receiving the protein construct or polypeptide, polynucleotide, and / or composition disclosed herein, the route of administration, the particular type of polynucleotide and / or composition disclosed herein used, and other drugs administered to the mammal.

[0148] A typical daily dosage of an effective amount of protein construct and / or polypeptide is 0.1 mg / kg to 100 mg / kg (mg per kg patient weight). In some embodiments, the dosage may be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage may be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, In some embodiments, the dosage is about 1-10 mg / kg, about 1-5 mg / kg, or about 2-5 mg / kg.

[0149] In any of the methods described herein, the protein construct or polypeptide may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

[0150] In some embodiments, the one or more additional therapeutic agents may be administered to the subject prior to or after administration of the protein construct or polypeptide. In some embodiments, the one or more additional therapeutic agents are administered to the subject such that there is an overlap in the period of biological activity of the one or more additional therapeutic agents and the protein construct or polypeptide in the subject.

[0151] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agents may include one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, inhibitors of MEK, inhibitors of ERK, inhibitors of K-Ras, inhibitors of c-Met, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), inhibitors of Akt, inhibitors of mTOR, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).

[0152] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated Hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0153] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, cyclosporine ... The therapeutic agent may include one or more therapeutic agents selected from the group consisting of zantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomid, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0154] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapy, a CXCL9 targeted therapy, a CXCL10 targeted therapy, a CCL5 targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0155] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0156] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-SIRPα antibody, an anti-CD47 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody. In some embodiments, the additional therapeutic agent is an anti-CD20 antibody (e.g., rituximab) or an anti-EGF receptor antibody (e.g., cetuximab).

[0157] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions containing the protein constructs or polypeptides described herein. Pharmaceutical compositions may be formulated in any manner known in the art.

[0158] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, and isotonic agents such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions may also be used as pharma- ceutically acceptable carriers. Preparations of these compositions may be formulated and enclosed in ampoules, disposable syringes, or multiple dose vials. Where necessary (e.g., injectable formulations, etc.), proper fluidity can be maintained by the use of a coating, for example, lecithin or a surfactant. Absorption of the agent can be prolonged by the inclusion of an agent which delays absorption, for example, aluminum monostearate and gelatin. Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems which can include biodegradable, biocompatible polymers, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

[0159] Compositions containing the protein constructs or polypeptides described herein may be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage).

[0160] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., dosage for single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. For injection, the agent may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the protein construct or polypeptide may be in lyophilized form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0161] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, one can determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population), and the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indexes are preferred. If an agent exhibits undesirable side effects, care must be taken to minimize potential damage (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0162] Exemplary doses include milligram or microgram amounts of any of the protein constructs or polypeptides described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, about 1 μg / kg to about 50 μg / kg, about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg). While these doses cover a wide range, one of skill in the art will understand that therapeutic substances can vary in their potency and that effective amounts can be determined by methods known in the art. Typically, a relatively low dose is administered initially, and the attending medical or veterinary professional (for therapeutic applications) or researcher (if still working in the development phase) may then gradually increase the dose until an appropriate response is obtained. In addition, it will be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the particular compound used, the subject's age, weight, general health, sex, and diet, time of administration, route of administration, rate of excretion, and half-life in vivo.

[0163] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.The disclosure also provides methods of producing the protein constructs or polypeptides for the various uses described herein. EXAMPLES

[0164] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0165] Example 1. Design of an Fc-Based Designer Biologic (FBDB™) with a PD1 x SIRPα x TGFβ Trap Format The distinct triple targeting formats of FBDB™ include: (1) targeting therapeutic agents to PD-L1; +They have been designed and developed to be directed against tumor cells, (2) combine the power of innate immunity (e.g., by targeting the SIRPα / CD47 pathway) and adaptive immunity (e.g., by targeting the PD-1 / PD-L1 pathway), and (3) reverse TGFβ-induced immune suppression.

[0166] Each format includes at least three different types of immune modules directly or indirectly connected to the Fc region of IgG (e.g., human IgG4): (1) one or more SIRPα extracellular domains, which can stimulate antigen presentation by inducing phagocytosis, (2) one or more PD-1 extracellular domains, which can block the PD-1 / PD-L1 pathway to enhance T cell function and target SIRPα×PD-1×TGFβ trap molecules to PD-L1-expressing tumors, and (3) one or more TGFβ trap molecules (e.g., the extracellular domain of TGF-beta receptor type 2 (TGFBR2)), which can capture immunosuppressive TGFβ and improve the tumor microenvironment to enhance the immune response. For example, the SIRPα ectodomain can release a "don't eat me" brake on macrophages by blocking the interaction between CD47 on tumor cells and SIRPα on macrophages, the PD-1 ectodomain can release an inhibitory brake on effector T cells by blocking the interaction between PD-1 and PD-L1, and TGFβ trap molecules can inhibit TGFβ function in the tumor microenvironment (TME).

[0167] Four triple target formats of FBDB™ were designed, as shown in Figures 1A-1D. HCB301-4 (or PST_v2, schematic structure shown in Figure 1A) contains two identical polypeptide chains, each having the amino acid sequence shown in SEQ ID NO:1. Specifically, each polypeptide chain comprises, from the N-terminus to the C-terminus, a PD-1 extracellular domain (SEQ ID NO: 5, having a sequence identical to amino acids 26-170 of the human PD-1 protein (NCBI Accession No.: NP_005009.2, SEQ ID NO: 11)), a SIRPα extracellular domain (SEQ ID NO: 6, having a sequence identical to amino acids 31-148 of the human SIRPα protein (NCBI Accession No.: AAH26692.1, SEQ ID NO: 12)), a human IgG4 hinge region (with an S228P mutation according to EU numbering), a human IgG4 Fc, and a TGFBR2 extracellular domain (SEQ ID NO: 7, NCBI Accession No.: NP_003233.4, SEQ ID NO: 13) having a sequence identical to amino acids 24-159 of the TGFBR2 extracellular domain). The PD-1 extracellular domain and the SIRPα extracellular domain are connected via a (GSG)6 linker peptide (SEQ ID NO: 8). The TGFBR2 extracellular domain is connected to the C-terminus of human IgG4 Fc via a (G4S)4G linker peptide (SEQ ID NO:9).

[0168] HCB301-3 (or SPT_v2, schematic structure shown in FIG. 1B) comprises two identical polypeptide chains, each having the amino acid sequence shown in SEQ ID NO:2. Specifically, each polypeptide chain comprises, from N-terminus to C-terminus, a SIRPα extracellular domain (SEQ ID NO:6), a PD-1 extracellular domain (SEQ ID NO:5), a human IgG4 hinge region (with an S228P mutation according to EU numbering), a human IgG4 Fc, and a TGFBR2 extracellular domain (SEQ ID NO:7). The SIRPα extracellular domain and the PD-1 extracellular domain are connected via a (GSG)6 linker peptide (SEQ ID NO:8). The TGFBR2 extracellular domain is connected to the C-terminus of the human IgG4 Fc via a (G4S)4G linker peptide (SEQ ID NO:9).

[0169] HCB301-2 (or P2ST_v2, schematic structure shown in FIG. 1C) comprises two identical polypeptide chains, each having the amino acid sequence shown in SEQ ID NO:3. Specifically, each polypeptide chain comprises, from N-terminus to C-terminus, two identical PD-1 extracellular domains (SEQ ID NO:5), SIRPα extracellular domain (SEQ ID NO:6), human IgG4 hinge region (with S228P mutation according to EU numbering), human IgG4 Fc, and TGFBR2 extracellular domain (SEQ ID NO:7), which are linked via a (G4S)3 linker (SEQ ID NO:10). The two PD-1 extracellular domains are connected to the N-terminus of the SIRPα extracellular domain via a (GSG)6 linker peptide (SEQ ID NO:8). The TGFBR2 extracellular domain is connected to the C-terminus of the human IgG4 Fc via a (G4S)4G linker peptide (SEQ ID NO:9).

[0170] HCB301-1 (or SP2T_v2, schematic structure shown in FIG. 1D) comprises two identical polypeptide chains, each having the amino acid sequence shown in SEQ ID NO: 4. Specifically, each polypeptide chain comprises, from N-terminus to C-terminus, a SIRPα extracellular domain (SEQ ID NO: 6), two identical PD-1 extracellular domains (SEQ ID NO: 5) linked via a (G4S)3 linker (SEQ ID NO: 10), a human IgG4 hinge region (with a S228P mutation according to EU numbering), a human IgG4 Fc, and a TGFBR2 extracellular domain (SEQ ID NO: 7). The SIRPα extracellular domain is connected to one of the two PD-1 extracellular domains via a (GSG)6 linker peptide (SEQ ID NO: 8). The TGFBR2 extracellular domain is connected to the C-terminus of the human IgG4 Fc via a (G4S)4G linker peptide (SEQ ID NO: 9).

[0171] The expressed proteins were purified by protein A column followed by HPLC-SEC (high performance liquid chromatography coupled with size exclusion chromatography, Agilent) to determine the percentage of high molecular weight peak (HMW%), the percentage of the major peak (major%), and the percentage of low molecular weight peak (LMW%).

[0172] Specifically, HCB301 protein was expressed in CHO-S cells. The culture supernatant was collected and subjected to Protein A purification. The pH of the culture supernatant was adjusted to 8.0 by adding concentrated equilibration buffer (250 mM Tris, 1500 mM NaCl, pH 8.0). Next, the Protein A column was equilibrated with 10× column volume of equilibration buffer (25 mM Tris, 150 mM NaCl, pH 8.0), and then the culture supernatant was loaded onto the equilibrated Protein A column. The column was then washed with 6× column volume of wash buffer (100 mM citric acid, 500 mM NaCl). The protein sample was eluted by 6× column volume of elution buffer (100 mM acetate, 200 mM NaCl, pH 3.0) and the pH was adjusted to 6.5-7 by a buffer containing 1 M Hepes, pH 8.0. As shown in the table below, the expression levels (titers) of HCB301-3 and HCB301-4 were comparable and higher than HCB301-1 or HCB302-2. After Protein A purification, the percentage of the major peak (major%) of HCB301 protein ranged from 87.88% to 94.38%, the percentage of the high molecular weight peak (HMW%) of HCB301 protein ranged from 1.35% to 5.43%, and the percentage of the low molecular weight peak (LMW%) of HCB301 protein ranged from 1.27% to 10.78%. The results indicate that all four HCB301 proteins (HCB301-1, HCB301-2, HCB301-3, and HCB301-4) can be expressed and harvested with high purity.

[0173] [Table 1]

[0174] In addition, the amino acid sequences of HCB301-1, HCB301-2, HCB301-3, and HCB301-4 were analyzed using an immune depletion tool (a database and analysis resource for immune epitopes, Dhanda et al. "Development of a strategy and computational application to select candidate protein analogues with reduced HLA binding and immunogenicity." Immunology 153.1 (2018): 118-132) to identify immunogenic regions. No immunogenicity was identified.

[0175] Example 2. Determination of overall cell binding capacity for CD47 tf CHO-S cells To assess the overall cell-binding ability of HCB301 protein to CD47 expressed on the cell surface, transfected CHO-S cells expressing human CD47 (CD47 tf CHO-S) were used as target cells. 5×10 4The cells were incubated with HCB301 protein serially diluted at the indicated concentrations (6.4 pM, 32 pM, 160 pM, 0.8 nM, 4 nM, 20 nM, 100 nM, and 500 nM) in FACS buffer (phosphate-buffered saline (PBS) supplemented with 4% fetal bovine serum (FBS)) for 30 min at 4°C. After incubation, the cells were washed twice with FACS buffer and then incubated with R-Phycoerythrin-AffiniPure Goat Anti-Human IgG (Jackson ImmunoResearch, Catalog No.: 109-115-098) for 30 min at 4°C. The samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc., CA, USA). SIRPα_G4 was used as a positive control. SIRPα_G4 contains two identical polypeptide chains, each having the amino acid sequence shown in SEQ ID NO: 14. PD1_G4 was used as a negative control. PD1_G4 contains two identical polypeptide chains, each having the amino acid sequence shown in SEQ ID NO: 15.

[0176] As shown in Figure 2, all four HCB301 proteins can bind to CD47 tf CHO-S cells. Specifically, the positive control SIRPα_G4 exhibited the highest CD47 binding ability, followed by HCB301-3 and HCB301-1. HCB301-4 and HCB301-2 can also bind to CD47-expressing cells. In contrast, no binding signal was detected for the negative control PD1_G4.

[0177] Example 3. Determination of overall cell binding capacity to PDL1 tf CHO-S cells To determine the binding ability of HCB301 protein to PD-L1, transfected CHO-S cells expressing human PD-L1 (PDL1 tf CHO-S) were used as target cells. 4The cells were incubated with HCB301 protein serially diluted at the indicated concentrations (6.4 pM, 32 pM, 160 pM, 0.8 nM, 4 nM, 20 nM, and 100 nM) in FACS buffer (PBS supplemented with 4% FBS) for 30 min at 4 °C. After incubation, the cells were washed twice with FACS buffer and then incubated with R-Phycoerythrin-AffiniPure Goat Anti-Human IgG (Jackson ImmunoResearch, Catalog No.: 109-115-098) for 30 min at 4 °C. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc., CA, USA). PD1_G4 was used as a positive control. Hu5F9-G4 (anti-CD47 reference antibody) and SIRPα_G4 were used as negative controls.

[0178] As shown in Figure 3, all four HCB301 proteins can bind to PDL1 tf CHO-S cells. Specifically, HCB301-1 and HCB301-2 exhibited higher binding ability than the positive control PD1_G4, which exhibited higher binding ability than HCB301-4 and HCB301-3. In contrast, no binding signal was detected for the negative controls Hu5F9-G4 and SIRPα_G4.

[0179] Example 4. Determination of overall cell binding capacity to CD47-expressing tumor cells To assess the overall cell-binding ability of HCB301 protein to CD47 expressed on the tumor cell surface, hypopharyngeal carcinoma FaDu cells expressing endogenous CD47 were used as target cells. 4FaDu cells were incubated with HCB301 protein serially diluted at the indicated concentrations (6.4 pM, 32 pM, 160 pM, 0.8 nM, 4 nM, 20 nM, and 100 nM) in FACS buffer (PBS supplemented with 4% FBS) for 30 min at 4 °C. After incubation, cells were washed twice with FACS buffer and then incubated with R-Phycoerythrin-AffiniPure Goat Anti-Human IgG (Jackson ImmunoResearch, Catalog No.: 109-115-098) for 30 min at 4 °C. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc., CA, USA). Hu5F9-G4 and SIRPα_G4 were used as positive controls. PD1_G4 was used as a negative control.

[0180] As shown in Figure 4, all four HCB301 proteins can bind to FaDu cells. Specifically, HCB301-1 showed the highest binding ability, followed by HCB301-3, HCB301-4, and HCB301-2. In contrast, no binding signal was detected for the negative control PD1_G4.

[0181] Example 5. Determination of overall cell binding capacity to RBCs To determine the binding capacity of HCB301 protein to red blood cells (RBCs), 1 × 10 5Human RBCs were incubated with HCB301 protein serially diluted at the indicated concentrations (6.4 pM, 32 pM, 160 pM, 0.8 nM, 4 nM, 20 nM, 100 nM, and 500 nM) in FACS buffer (PBS supplemented with 4% FBS) for 30 min at 4 °C. After incubation, RBCs were washed twice with FACS buffer and then incubated with R-Phycoerythrin-AffiniPure Goat Anti-Human IgG (Jackson ImmunoResearch, Catalog No.: 109-115-098) for 30 min at 4 °C. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc., CA, USA). Hu5F9-G4 was used as a positive control. PD1_G4 was used as a negative control.

[0182] As shown in Figures 5A-5B, the positive control Hu5F9-G4 exhibited strong binding ability to RBCs. In contrast, all four HCB301 proteins showed weak binding to RBCs. Specifically, HCB301-3 showed higher RBC binding ability than HCB301-1, and the binding ability of both HCB301-3 and HCB301-1 was higher than that of SIRPα_G4. HCB301-4 and HCB301-2 showed lower RBC binding ability, which was comparable to PD1_G4.

[0183] Example 6. Determination of overall cell binding capacity for platelets To determine the binding ability of HCB301 protein to platelets, 5 × 10 5Human platelets were incubated with HCB301 protein serially diluted at the indicated concentrations (6.4 pM, 32 pM, 160 pM, 0.8 nM, 4 nM, 20 nM, 100 nM, and 500 nM) in FACS buffer (PBS supplemented with 4% FBS) for 30 min at 4 °C. After incubation, platelets were washed twice with FACS buffer and then incubated with R-Phycoerythrin-AffiniPure Goat Anti-Human IgG (Jackson ImmunoResearch, Catalog No.: 109-115-098) for 30 min at 4 °C. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc., CA, USA). Hu5F9-G4 was used as a positive control. PD1_G4 and SIRPα_G4 were used as negative controls.

[0184] As shown in Figures 6A-6B, the positive control Hu5F9-G4 exhibited strong binding ability to platelets. In contrast, all HCB301 proteins showed weak binding to platelets. Specifically, HCB301-3 showed higher platelet binding ability than HCB301-2 and HCB301-1. HCB301-4 showed lower RBC binding ability, which was comparable to SIRPα_G4.

[0185] Example 7. Determination of binding ability to hTGFβ1, hTGFβ2, and hTGFβ3 To determine the binding ability of HCB301 protein to human TGFβ, a binding titration ELISA assay was performed using HCB301 protein at the indicated concentrations (25.4 pM, 76.2 pM, 228.6 pM, 685.8 pM, 2.1 nM, 6.2 nM, 18.5 nM, 55.6 nM, 166.7 nM, or 500 nM). IgG4-TGFβ trap was used as a positive control. SIRPα_G4 was used as a negative control. 96-well EIA microplates were coated with 1 μg / ml human TGFβ (TGFβ1, TGFβ2, or TGFβ3) overnight at 4°C. After blocking with 1×PBS containing 5% skim milk, diluted HCB301 protein was added and incubated for 1 h at room temperature (RT). Unbound proteins were removed by washing the wells three times with 1x PBST (1x PBS containing 0.1% Tween 20). HRP-conjugated secondary antibody (1:5000) was added to the wells for 1 h at room temperature. After incubation, excess secondary antibody was removed by washing the wells three times with 1x PBST. Finally, 3,3',5,5'-tetramethylbenzidine (TMB) was added for color development. The reaction was stopped and HRP activity was measured using a spectrophotometer at 450 nm.

[0186] As shown in Figures 7A-7C, the EC50 values ​​were also determined according to the binding curves. The results show that HCB301 protein can bind to hTGFβ1 and hTGFβ3, but exhibits weak binding ability to hTGFβ2. Specifically, the EC50 values ​​of HCB301-1, HCB301-3, and HCB301-4 binding to hTGFβ3 were lower than their respective EC50 values ​​binding to hTGFβ1.

[0187] Example 8. Selective binding to PD-L1 expressing cells The selective binding ability of HCB301 protein to PD-L1 expressing cells was determined as follows: OE19 cells were labeled with CellTrace™ CFSE (Thermo, Catalog No.: C34554) and transfected OE19 cells expressing PD-L1 (PD-L1 tf OE19) were labeled with Celltrace™ Violet (Thermo, Catalog No.: C34557) according to the instructions provided by the manufacturer. 2×10 4 CellTrace™ CFSE-labeled OE19 cells (OE19-CFSE) at 10 cells / well and 2×10 4 CellTrace™ Violet-labeled transfected OE19 cells expressing PD-L1 tf OE19 cells (OE19-Violet) at 100 cells / well were incubated with HCB301 protein serially diluted at the indicated concentrations (6.4 pM, 32 pM, 160 pM, 0.8 nM, 4 nM, 20 nM, 100 nM, and 500 nM) in FACS buffer (PBS supplemented with 4% FBS) for 30 min at 4°C. After incubation, cells were washed twice with FACS buffer and then incubated with R-Phycoerythrin-AffiniPure Goat Anti-Human IgG (Jackson ImmunoResearch, Catalog No.: 109-115-098) for 30 min at 4°C. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc., CA, USA). Cells were then gated according to the PE signal, and the percentages of different cell types were calculated by Kaluza analysis software (Beckman Coulter Inc.). PD1_G4 was used as a positive control, and SIRPα_G4 was used as a negative control.

[0188] As shown in Figure 8A, at 32 pM, all HCB301 proteins showed selective binding to PD-L1-expressing OE19-Violet cells versus OE19-CFSE cells. Figures 8B-8G show the binding curves of SIRPα_G4, PD1_G4, and four HCB301 proteins (HCB301-1, HCB301-2, HCB301-3, and HCB301-4), respectively, to the two cell types.

[0189] Example 9. Hemagglutination (HA) activity To determine the HA activity induced by HCB301 protein, a 10% RBC solution was prepared from whole blood of a healthy donor. RBCs were washed twice with 0.9% NaCl buffer and then diluted to 10% by volume in 0.9% NaCl buffer. HCB301 protein was serially diluted (3-fold) to final concentrations of 8.4 pM, 25.4 pM, 76.2 pM, 228.6 pM, 685.8 pM, 2.1 nM, 6.2 nM, 18.5 nM, 55.6 nM, 166.7 nM, or 500 nM. The diluted protein was incubated overnight at room temperature with 12 μl of 10% RBC solution in a round-bottom 96-well plate. The aggregated RBCs evenly coated the wells, whereas the non-aggregated cells formed distinct red dots at the bottom of the wells. Hu5F9-G4 was used as a positive control PC. SIRPα_G4, PD1_G4, and IgG4-TGFβ trap were used as negative controls.

[0190] The next day, images of the plates were taken, as shown in Figure 9. The images show that only Hu5F9-G4 induced HA activity at high concentrations, while none of the other tested molecules, including the four HCB301 proteins, induced HA activity within the concentration ranges shown above.

[0191] Example 10. Determining the effect of blocking SIRPα / CD47 interaction To determine the SIRPα ligand blocking ability of HCB301 protein, a flow cytometry-based assay was performed using CD47 tf CHO-S cells as target cells. 4CD47 tf CHO-S cells at 10 cells / well were incubated with HCB301 protein serially diluted at the indicated concentrations (0.2 pM, 1.9 pM, 15.2 pM, 122.1 pM, 876.6 pM, 7.8 nM, 62.5 nM, and 500 nM) in FACS buffer (PBS supplemented with 4% FBS) with a fixed concentration of biotinylated SIRPα_G4 for 30 min at 4°C. After washing, streptavidin-PE (eBioscience, Catalog No.: EBS12-4317-87) was added at 0.3 μg per well, and the PE signal from the cells was analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc.). Hu5F9 and SIRPa_G4 were used as positive controls. Anti-PD-1 antibody (with human IgG4 Fc) was used as an isotype control.

[0192] As shown in Figure 10, Hu5F9-G4 exhibited the strongest blocking effect, followed by SIRPα_G4 and the four HCB301 proteins. More specifically, the blocking effects of the four HCB301 proteins can be ranked from strong to weak as follows: HCB301-3, HCB301-1, HCB301-4, and HCB301-2.

[0193] Example 11. Determining the effect of blocking PD-1 / PD-L1 interaction The blocking effect of HCB301 protein on the interaction between PD-1 and PD-L1 tf CHO-S cells was determined as follows: 3×10 4PD-L1 tf CHO-S cells at 100 cells / well were incubated with HCB301 protein serially diluted at the indicated concentrations (0.2 pM, 1.9 pM, 15.2 pM, 122.1 pM, 876.6 pM, 7.8 nM, 62.5 nM, and 500 nM) in FACS buffer (PBS supplemented with 4% FBS) with a fixed concentration of biotinylated PD1_G4 for 30 min at 4 °C. After washing, streptavidin-PE (eBioscience, Catalog No.: EBS12-4317-87) was added at 0.3 μg per well, and the PE signal from the cells was analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc.). MPDL3280A (anti-PD-L1 antibody) and PD1_G4 were used as positive controls.

[0194] As shown in Figure 11, MPDL3280A exhibited the strongest blocking effect. HCB301-1 and HCB301-2 exhibited stronger blocking effects than that of PD1_G4. HCB301-4 exhibited a weaker blocking effect than PD1_G4, but a stronger blocking effect than HCB301-3. The results indicate that the HCB301 protein, which has two PD-1 extracellular domains on each polypeptide chain, can block PD-1 / PD-L1 interaction more efficiently than one having one PD-1 extracellular domain on each polypeptide chain.

[0195] Example 12. Inhibition of TGFβ-induced smad2 reporter activity The ability of HCB301 protein to inhibit TGFβ1-induced smad2 reporter activity was determined as follows. HCB301 protein was serially diluted (5-fold) to final concentrations of 6.4 pM, 32 pM, 160 pM, 0.8 nM, 4 nM, 20 nM, 100 nM, and 500 nM. M7824 (anti-PD-L1 x TGFβ trap), anti-TGFβ (anti-TGFβ antibody, BioXcell, Cat. No.: BE0057), and IgG4-TGFβ trap were used as positive controls. SIRPα_G4 was used as a negative control. 4 x 10 3Transfected HEK293T cells expressing smad2 reporters were incubated with diluted HCB301 protein along with 20ng / ml TGFβ1, TGFβ2, and TGFβ3, respectively. After 24 hours of incubation at 37°C in a 5% CO2 incubator, luminescence signals were detected by a Varioskan™ LUX multimode microplate reader (Thermo).

[0196] As shown in FIG. 12A, all four HCB301 proteins can inhibit TGFβ1-mediated smad2 reporter activity. Each HCB301 protein exhibited stronger inhibitory ability than M7824 and IgG4-TGFβ trap. Specifically, HCB301-3, HCB301-4, and HCB301-1 exhibited stronger inhibitory ability than HCB301-2. As shown in FIG. 12B-12C, HCB301-3, HCB301-4, and HCB301-1 can inhibit TGFβ3-mediated reporter activity, but cannot inhibit TGFβ2-mediated smad2 reporter activity. Notably, HCB301-3, HCB301-4, and HCB301-1 exhibited comparable TGFβ3 inhibitory ability compared to IgG4-TGFβ trap or M7824.

[0197] Example 13. Induction of macrophages to phagocytose CD47-expressing tumor cells To assess the macrophage-mediated phagocytosis induced by HCB301 protein against cancer cells, a phagocytosis assay was performed as follows: CD47-expressing Jurkat cells were labeled with 5 nM CellTrace™ CFSE (Thermo, Cat. No.: C34554) for 10 min at 37° C., and then washed with complete RPMI-1640 medium. 5 CFSE-labeled Jurkat cells (target cells) at 5 × 10 cells / well were incubated with serially diluted HCB301 protein at the indicated concentrations (10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM) in low-binding 96-well U-bottom plates for 30 min at 37 °C.4 Raw264.7 mouse macrophages were added to each well and the plate was incubated at 37°C for 2 hours. Raw264.7 cells were stained with PE-Cyanine 7 conjugated F4 / 80 antibody (eBioscience, Catalog No: 25-4801-82). The phagocytosis ability of HCB301 protein was evaluated by calculating the percentage of CFSE+F4 / 80+ (indicating macrophage phagocytosed CFSE-labeled Jurkat cells) from macrophages relative to the total F4 / 80 signal from macrophages by CytoFlex flow cytometer (Beckman Coulter Inc.). Hu5F9-G4 and SIRPα_G4 were used as positive controls. PD1_G4 was used as negative control. Anti-PD-1 antibody (with human IgG4 Fc) was used as isotype control.

[0198] As shown in Figure 13, Hu5F9-G4 exhibited the strongest ability to induce Raw264.7-mediated phagocytosis of CD47-expressing Jurkat cells, followed by SIRPα_G4. HCB301-3, HCB301-1, and HCB301-4 also induced macrophages to phagocytose Jurkat cells. In contrast, PD1_G4 and isotype control did not induce phagocytosis at the concentrations tested.

[0199] Example 14. Enhancement of T cell responses in MLR assays in the presence of TGFβ1 A mixed lymphocyte reaction (MLR) assay in the presence of TGFβ1 was performed to determine the enhancement of T cell responses by HCB301 protein. T cells were labeled with 5 nM CellTrace™ Violet (Thermo, Catalog No.: C34557) for 10 min at 37° C. and then washed twice with complete RPMI-1640 medium. 1×10 5 CellTrace™ Violet-labeled CD4+ T cells and 1×10 4of dendritic cells (DCs) were incubated with 2 nM, 20 nM, or 200 nM of HCB301 protein. Control molecules or combinations thereof, such as PDGFR-Fc, SIRPα_G4, PD1_G4, IgG4-TGFβ trap, M7824, SIRPα_G4+PD1_G4+IgG4-TGFβ trap, SIRPα_G4+PD1_G4, SIRPα_G4+IgG4-TGFβ trap, and PD1_G4+IgG4-TGFβ trap, were also used in the incubation. PDGFR-Fc served as a negative control. PDGFR-Fc contains two identical polypeptide chains, each with a PDGFR extracellular domain fused to Fc. TGFβ1-containing conditioned medium from NCl-H650 cells was then added and co-incubated for 5 days. After co-incubation, cells were harvested and cell proliferation was analyzed by a CytoFLEX-S flow cytometer (Beckman Coulter Inc.). Culture supernatants were also collected and IL-2 and IFN-γ secretion were measured using a human IL-2 ELISA MAX Deluxe kit (BioLegend, Catalog No.: 431805) and an IFNγ ELISA MAX Deluxe kit (BioLegend, Catalog No.: 430105), respectively.

[0200] As shown in Figure 14A, cell proliferation in the MLR assay was measured using a CellTrace™ Violet Cell Proliferation Kit (Thermo, Catalog No.: C34557). Specifically, the percentage of weaker CellTrace™ Violet labeled cells (representing proliferating cells) relative to CD3+ / 7-ADD- cells (representing total T cells) was calculated. The results show that cells treated with HCB301-3, HCB301-4, and HCB301-1 did not significantly change cell proliferation compared to that of the control molecules or their combinations.

[0201] As shown in Figure 14B, IL-2 secretion in MLR assay was measured using human IL-2 ELISA MAX Deluxe kit. The results showed that M7824 exhibited the highest IL-2 secretion level, followed by HCB301-1, HCB301-3, and HCB301-4. Notably, cells treated with HCB301-4 had similar IL-2 secretion levels compared to those of IgG4-TGFβ Trap and SIRPα_G4+PD1_G4+IgG4-TGFβ Trap. Cells treated with SIRPα_G4+IgG4-TGFβ Trap or PD1_G4+IgG4-TGFβ Trap had lower IL-2 secretion levels.

[0202] As shown in Figure 14C, IFN-γ secretion in MLR assay was measured using Human IFNγ ELISA MAX Deluxe kit. The results showed that HCB301-1 exhibited the highest IFN-γ secretion level, followed by M7824, PD1_G4+IgG4-TGFβ Trap, HCB301-3, and HCB301-4. Cells treated with PD1_G4, SIRPα_G4, SIRPα_G4+PD1_G4, SIRPα_G4+PD1_G4+IgG4-TGFβ Trap, SIRPα_G4+PD1_G4, and SIRPα_G4+IgG4-TGFβ Trap had similar and low IFN-γ secretion levels.

[0203] In summary, Figures 15A-15B show the results of the in vitro assays discussed above. HCB301-3 was selected for subsequent experiments in part due to its relatively simple structure relative to HCB301-1 and HCB301-2, as well as its robust function as shown in the in vitro assays.

[0204] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. (a) Fc; and (b) a CD47 binding domain; and (c) a PD-L1 (programmed death ligand 1) binding domain; A protein complex comprising:

2. The protein complex of claim 1 , further comprising a TGFβ (transforming growth factor beta) binding domain.

3. the CD47 binding domain is or comprises the extracellular domain of SIRPα; or the CD47 binding domain is an anti-CD47 antibody or an antigen-binding fragment thereof; The protein complex of claim 1.

4. the PD-L1 binding domain is or comprises the PD-1 extracellular domain; or the PD-L1 binding domain is an anti-PD-L1 antibody or an antigen-binding fragment thereof; The protein complex of claim 1.

5. the TGFβ binding domain is or comprises the TGFBR2 extracellular domain, or the TGFβ-binding domain is an anti-TGFβ antibody or an antigen-binding fragment thereof; The protein complex of claim 2.

6. The protein complex of claim 1 , wherein the Fc is a human IgG4 Fc or an IgG1 Fc.

7. The protein complex of claim 2, wherein the PD-L1-binding domain is linked to the N-terminus of the CH2 domain of the Fc, the CD47-binding domain is linked to the N-terminus of the PD-L1-binding domain, and the TGFβ-binding domain is linked to the C-terminus of the CH3 domain of the Fc.

8. The protein complex of claim 2, comprising two or more CD47-binding domains, two or more PD-L1-binding domains, or two or more TGFβ-binding domains.

9. (a) a first polypeptide comprising, from N-terminus to C-terminus, a first PD-L1-binding domain, a first CD47-binding domain, a first Fc region, and a first TGFβ-binding domain; (b) a second polypeptide comprising, from N-terminus to C-terminus, a second PD-L1-binding domain, a second CD47-binding domain, a second Fc region, and a second TGFβ-binding domain; A protein complex comprising:

10. The protein complex of claim 9, wherein the first polypeptide further comprises a third PD-L1-binding domain and the second polypeptide further comprises a fourth PD-L1-binding domain, the third PD-L1-binding domain being linked to the N-terminus of the first PD-L1-binding domain and the fourth PD-L1-binding domain being linked to the N-terminus of the second PD-L1-binding domain.

11. (a) a first polypeptide comprising, from N-terminus to C-terminus, a first CD47-binding domain, a first PD-L1-binding domain, a first hinge region, a first Fc region, and a first TGFβ-binding domain; (b) a second polypeptide comprising, from N-terminus to C-terminus, a second CD47-binding domain, a second PD-L1-binding domain, a second hinge region, a second Fc region, and a second TGFβ-binding domain; A protein complex comprising:

12. The protein complex of claim 11, wherein the first polypeptide further comprises a third PD-L1-binding domain and the second polypeptide further comprises a fourth PD-L1-binding domain, the third PD-L1-binding domain being fused between the first PD-L1-binding domain and the first hinge region, and the fourth PD-L1-binding domain being fused between the second PD-L1-binding domain and the second hinge region.

13. A nucleic acid comprising a polynucleotide encoding the protein complex of any one of claims 1 to 12.

14. A vector comprising the nucleic acid of claim 13.

15. A cell comprising the nucleic acid of claim 13.

16. 1. A method for producing a protein complex, comprising: (a) culturing the cells of claim 15 under conditions sufficient for the cells to produce the protein complex; (b) collecting the protein complex produced by the cells; and The method comprising:

17. A protein conjugate comprising the protein complex of any one of claims 1 to 12 covalently bound to a therapeutic agent.

18. For use in a method of treating a subject having cancer, A pharmaceutical comprising the protein complex according to any one of claims 1 to 12, the method comprising administering the protein complex to the subject; The medicine.

19. For use in a method of treating a subject having cancer. A medicament comprising the protein conjugate of claim 17, the method comprising administering the protein conjugate to the subject; The medicine.

20. The pharmaceutical composition according to claim 18, wherein the subject has cancer cells that express CD47 or PD-L1.

21. The pharmaceutical described in claim 19, wherein the subject has cancer cells that express CD47 or PD-L1.

22. 19. The pharmaceutical composition of claim 18, wherein the cancer is breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, diffuse large B-cell lymphoma, mesothelioma, lung cancer, ovarian cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial carcinoma, stomach cancer, bile duct cancer, head and neck cancer, blood cancer, or a combination thereof.

23. The pharmaceutical composition of claim 19, wherein the cancer is breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, diffuse large B-cell lymphoma, mesothelioma, lung cancer, ovarian cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial carcinoma, stomach cancer, bile duct cancer, head and neck cancer, blood cancer, or a combination thereof.

24. The pharmaceutical described in claim 18, wherein the cancer is a solid tumor.

25. The pharmaceutical described in claim 19, wherein the cancer is a solid tumor.

26. A medicament comprising the protein complex of any one of claims 1 to 12 for use in a method for reducing tumor growth rate or killing tumor cells, the method comprising contacting a tumor cell with the protein complex; The medicine.

27. A pharmaceutical comprising the protein conjugate of claim 17 for use in a method for reducing tumor growth rate or killing tumor cells, comprising: the method comprising contacting a tumor cell with the protein conjugate; The medicine.

28. A pharmaceutical composition comprising the protein complex of any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition comprising the protein conjugate of claim 17 and a pharmaceutically acceptable carrier.

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