CD47 / PD-L1 targeting protein complexes and methods of use thereof

Abstract

The present disclosure relates to protein complexes targeting CD47, PD-L1 and / or TGF [beta] and methods of use thereof. In one aspect, the protein complex comprises: a CD47 binding domain having all or a portion of the SIRP [alpha] extracellular region; a PD-L1 binding structural domain, wherein the PD-L1 binding structural domain is provided with a VHH (Vitamin Horse Hierarchy) which is bound with PD-L1; and optionally a TGF [beta] binding domain, said TGF [beta] binding domain having all or a portion of the extracellular region of TGFBRII.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63 / 470,003, filed on May 31, 2023, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically as an XML file named "52246-0014WO1.XML". The size of the XML file, created on May 15, 2024, is 45,056 bytes. The material in the XML file is hereby incorporated by reference in its entirety. Technical Field

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

[0006] Signal regulatory protein α (SIRPα) is a regulatory membrane glycoprotein in the SIRP family. The SIRPα is mainly expressed in myeloid cells, and additional expression can be detected in stem cells or neurons. SIRPα acts as an inhibitory receptor that interacts with the widely expressed transmembrane protein CD47. This interaction negatively regulates the effector functions of innate immune cells, such as host cell phagocytosis. SIRPα spreads laterally on the macrophage membrane and aggregates at the phagocytic synapse to bind to CD47, thereby inhibiting the cytoskeleton-intensive phagocytosis process performed by macrophages. CD47 provides a "don't eat" signal by binding to the N-terminus of signal regulatory protein α (SIRPα). CD47 has been found to be overexpressed in many different tumor cells. Targeting CD47 and / or SIRPα can be used for cancer immunotherapy. However, given that CD47 is also expressed on red blood cells (RBC) and platelets, inhibiting the CD47 / SIRPα interaction may lead to phagocytosis of RBC and platelets.

[0007] Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is considered to be a co-inhibitory factor in the immune response. The PD-L1 can be combined with programmed cell death protein 1 (PD-1) to reduce the proliferation of PD-1 positive cells, inhibit the PD-1 positive cell cytokine secretion and induce apoptosis. PD-L1 also plays an important role in various malignancies, where it can weaken the host's immune response to tumor cells. Therefore, the PD-1 / PD-L1 axis is responsible for cancer immune escape and has a huge impact on cancer therapy. It is necessary to develop therapies targeting the CD47 / SIRPα pathway and / or the PD-1 / PD-L1 pathway. Summary of the invention

[0008] The present disclosure relates to protein complexes targeting CD47 and PD-L1 and methods of use thereof. In some cases, the protein complex can also target TGFβ.

[0009] On the one hand, the present disclosure relates to a protein complex comprising: (a) Fc; (b) CD47 binding domain; and (c) PD-L1 (programmed death ligand 1) binding domain, in some embodiments, the PD-L1 binding domain is a VHH (heavy chain single variable domain) that binds to PD-L1. In some embodiments, the protein complex described herein further includes a TGFβ (transforming growth factor β) binding domain. 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 α (SIRPα). In some embodiments, the CD47 binding domain is or includes a SIRPα extracellular domain. In some embodiments, the SIRPα extracellular domain includes a sequence that is at least 80% identical to SEQ ID NO: 32, and optionally in some embodiments, the SIRPα extracellular domain includes one or more amino acid mutations at positions corresponding to H24, I31, E54, G55, H56 and / or E70 of SEQ ID NO: 32. In some embodiments, the SIRPα extracellular domain includes one or more of the following: (a) the amino acid corresponding to H24 of SEQ ID NO: 32 is T; (b) the amino acid corresponding to I31 of SEQ ID NO: 32 is Y; (c) the amino acid corresponding to E54 of SEQ ID NO: 32 is R; d) the amino acid corresponding to G55 of SEQ ID NO: 32 is Q; (e) the amino acid corresponding to H56 of SEQ ID NO: 32 is T; and (f) the amino acid corresponding to E70 of SEQ ID NO: 32 is F. In some embodiments, the CD47 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. In some embodiments, the VHH can bind to cells expressing PD-L1 (e.g., cancer cells) and / or block the interaction between PD-L1 and programmed cell death protein 1 (PD-1).In some embodiments, the VHH comprises complementarity determining regions (CDR) 1, 2, and 3; in some embodiments, the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence; in some embodiments, the selected VHH CDR1, 2, and 3 amino acid sequences are one of the following: (1) the selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 26, 27, and 28, respectively; and (2) the selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 29, 30, and 31, respectively. In some embodiments, the VHH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8. In some embodiments, the TGFβ binding domain is capable of capturing TGFβ, thereby increasing immune response and / or improving tumor microenvironment. In some embodiments, the TGFβ binding domain is or comprises a TGFBRII extracellular domain. In some embodiments, the TGFBRII extracellular domain comprises a sequence that is at least 80% identical to SEQ ID NO: 33, and optionally in some embodiments, the TGFBRII extracellular domain comprises one or more amino acid mutations at positions corresponding to S49 and / or D118 of SEQ ID NO: 33. In some embodiments, the TGFβ binding domain comprises one or more of the following: (a) the amino acid corresponding to S49 of SEQID NO: 33 is E; and (b) the amino acid corresponding to D118 of SEQ ID NO: 33 is E. In some embodiments, the TGFβ 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:9.

[0010] In some embodiments, the CD47 binding domain is connected to the N-terminus of the CH2 domain in the Fc, optionally through a hinge region. In some embodiments, the PD-L1 binding domain is connected to the N-terminus of the CD47 binding domain, optionally through a linker peptide. In some embodiments, the PD-L1 binding domain is connected to the N-terminus of the CH2 domain in the Fc, optionally through a hinge region. In some embodiments, the CD47 binding domain is connected to the N-terminus of the PD-L1 binding domain, optionally through a linker peptide. In some embodiments, the Fc is human IgG1 Fc. In some embodiments, the Fc is human IgG4 Fc. In some embodiments, the hinge region is a human IgG4 hinge region, and the human IgG4 hinge region optionally has an S228P mutation according to EU numbering. In some embodiments, the TGFβ binding domain is connected to the C-terminus of the CH3 domain in the Fc, optionally through a linker peptide.

[0011] In one aspect, the present disclosure relates to a protein complex comprising: (a) a first polypeptide, the first polypeptide comprising, from N-terminus to C-terminus: a first CD47 binding domain, an optional first linker peptide, a first VHH that binds to PD-L1, an optional first hinge region, and a first Fc region; and (b) a second polypeptide, the second polypeptide comprising, from N-terminus to C-terminus: a second CD47 binding domain, an optional second linker peptide, a second VHH that binds to PD-L1, an optional second hinge region, and a second Fc region. In some embodiments, the first VHH and / or the second VHH comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO: 8. 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: 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: 14. In some embodiments, the first Fc region and / or the second Fc region comprises a sequence at least 80% identical to SEQ ID NO: 15. In some embodiments, the first hinge region and / or the second hinge region comprises a sequence 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 at least 80% identical to SEQ ID NO: 17. In some embodiments, the first linker peptide and / or the second linker peptide comprises a sequence at least 80% identical to SEQ ID NO: 12. In some embodiments, the first polypeptide and / or the second polypeptide comprises a sequence at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 4 or 5. In some embodiments, the first polypeptide further comprises a first TGFβ binding domain, and the second polypeptide further comprises a second TGFβ binding domain. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain comprises a sequence at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 9. In some embodiments, the first TGFβ binding domain is linked to the C-terminus of the first Fc region, optionally through a third linker peptide, and in some embodiments, the second TGFβ binding domain is linked to the C-terminus of the second Fc region, optionally through a fourth linker peptide. In some embodiments, the third linker peptide and / or the fourth linker peptide comprises a sequence at least 80% identical to SEQ ID NO: 13. In some embodiments, the first polypeptide and / or the second polypeptide comprises a sequence at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 1 or 2.

[0012] In one aspect, the present disclosure relates to a protein complex comprising: (a) a first polypeptide, the first polypeptide comprising, from N-terminus to C-terminus: a first VHH that binds to PD-L1, an optional first linker peptide, a first CD47 binding domain, an optional first hinge region, and a first Fc region; and (b) a second polypeptide, the second polypeptide comprising, from N-terminus to C-terminus: a second VHH that binds to PD-L1, an optional second linker peptide, a second CD47 binding domain, an optional second hinge region, and a second Fc region. In some embodiments, the first VHH and / or the second VHH comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO: 8. 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: 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 at least 80% identical to SEQ ID NO: 17. In some embodiments, the first linker peptide and / or the second linker peptide comprises a sequence at least 80% identical to SEQ ID NO: 12. In some embodiments, the first polypeptide and / or the second polypeptide comprises a sequence at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide further comprises a first TGFβ binding domain, and the second polypeptide further comprises a second TGFβ binding domain. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain comprises a sequence at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 9. In some embodiments, the first TGFβ binding domain is linked to the C-terminus of the first Fc region, optionally via a third linker peptide, and in some embodiments, the second TGFβ binding domain is linked to the C-terminus of the second Fc region, optionally via a fourth linker peptide. In some embodiments, the third linker peptide and / or the fourth linker peptide comprises a sequence at least 80% identical to SEQ ID NO: 13. 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.

[0013] In one aspect, the disclosure relates to a nucleic acid comprising a polynucleotide encoding a protein complex described herein. In some embodiments, the nucleic acid is DNA (eg, cDNA) or RNA (eg, mRNA).

[0014] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0015] In one aspect, the disclosure relates to a cell comprising a vector described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure relates to a cell comprising one or more nucleic acids in the nucleic acids described herein.

[0016] In one aspect, the present 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) collecting the protein complex produced by the cell.

[0017] In one aspect, the present disclosure relates to a protein conjugate comprising a protein complex as described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent.

[0018] On the one hand, the present disclosure relates to a method for treating a subject. In some embodiments, the subject has cancer cells expressing 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 cancer, stomach cancer, bile duct cancer, head and neck cancer, blood cancer or a combination thereof.

[0019] 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 composition comprising the protein complex or a protein conjugate described herein.

[0020] In one aspect, the present disclosure relates to a method of killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising the protein complex or a protein conjugate described herein.

[0021] In one aspect, the present disclosure relates to a pharmaceutical composition comprising the protein complex and a pharmaceutically acceptable carrier.

[0022] As used herein, the term "protein complex" or "protein construct" refers to a complex having one or more polypeptides. In some embodiments, the protein complex has two or more polypeptides, wherein the polypeptides can associate with each other to form dimers or multimers.

[0023] As used herein, the term "CD47 binding domain" refers to a protein domain that can bind 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 includes or consists of a SIRPα extracellular domain. The SIRPα may be wild-type SIRPα, human SIRPα, a polypeptide derived from wild-type SIRPα (e.g., with a mutation) or a portion thereof (e.g., an extracellular region of SIRPα or an 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 includes or consists of substantially the entire extracellular region of SIRPα or a variant thereof. In some embodiments, the SIRPα extracellular domain includes or consists of an IgV domain of the SIRPα or a variant thereof. In some embodiments, the IgV domain has one or more mutations. In some embodiments, the SIRPα extracellular domain has one or more mutations (eg, mutations at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32).

[0024] As used herein, the term "PD-L1 binding domain" refers to a protein domain that can bind to PD-L1. In some embodiments, the PD-L1 binding domain can be an anti-PD-L1 antibody, an antigen-binding fragment thereof (e.g., scFv or VHH) or a PD-L1 binding protein or a portion thereof. In some embodiments, the PD-L1 binding domain is a VHH that binds to PD-L1 (e.g., anti-PD-L1 VHH (2H4)). In some embodiments, the PD-L1 binding domain is any VHH described herein. In some embodiments, the PD-L1 binding domain includes or consists of a PD-1 extracellular domain. PD-1 can 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., an extracellular region 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 includes or consists of substantially the entire extracellular region of PD-1 or a variant thereof. In some embodiments, the PD-1 extracellular domain comprises a portion of the extracellular region of PD-1 or a variant thereof. In some embodiments, the PD-1 extracellular domain has one or more mutations.

[0025] As used herein, the term "TGFβ binding domain" refers to a protein domain that can bind to TGFβ. In some embodiments, the TGFβ binding domain can be an anti-TGFβ antibody, an antigen binding fragment thereof (e.g., scFv or VHH) or a TGFβ binding protein or a portion thereof. In some embodiments, the TGFβ binding domain includes or consists of a TGFBRII extracellular domain. The TGFBRII can be wild-type TGFBRII, human TGFBRII, a polypeptide (e.g., with a mutation) derived from wild-type TGFBRII or a portion thereof (e.g., an extracellular region of TGFBRII). In some embodiments, the polypeptide derived from wild-type TGFBRII may have one or more mutations. In some embodiments, the TGFBRII extracellular domain includes or consists of substantially the entire extracellular region of TGFBRII or a variant thereof. In some embodiments, the TGFBRII extracellular domain includes a portion of the extracellular region of TGFBRII or a variant thereof. In some embodiments, the TGFBRII extracellular domain has one or more mutations (eg, mutations at positions corresponding to S49 and / or D118 of SEQ ID NO: 33).

[0026] As used herein, the term "cancer" refers to a cell with autonomous growth capacity. Examples of such cells include cells with abnormal states or conditions characterized by rapid cell growth. The term is intended to include cancerous growths, such as tumors; carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasive stage. Malignant tumors of various organ systems are also included, such as the respiratory system, cardiovascular system, renal system, reproductive system, blood system, nervous system, liver system, gastrointestinal system, and endocrine system; and adenocarcinomas, which include malignant tumors, such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, and small intestinal cancers. "Naturally occurring" cancers include any cancer that is not experimentally induced by transplanting cancer cells into a subject, and include, for example, spontaneously occurring cancers, cancers caused by exposure of a patient to a carcinogen, cancers caused by insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancers caused by infection, such as viral infection. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which includes malignant tumors composed of cancerous tissue and sarcomatous tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or a cancer in which the tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor derived from the mesenchyme. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative cells / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic disorders may be caused by myeloid, lymphoid or erythroid or their precursor cells. Hematological cancers are cancers that begin in hematopoietic tissues such as bone marrow or immune system cells. Examples of hematological cancers include, for example, leukemias, lymphomas, and multiple myeloma.

[0027] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification, and describe the animal, human or non-human being provided for treatment according to the method of the present invention. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adults or adolescents (e.g., people 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, porcine animals (e.g., pigs, miniature pigs), horses, dogs, cats, cattle and other livestock animals, farm animals and zoo animals are included.

[0028] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length of at least two amino acids.

[0029] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to a polymer of nucleotides of any length of at least two nucleotides, and include but are not limited to DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only 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 the event of a conflict, the present specification (including definitions) shall prevail.

[0031] 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

[0032] 1A-1F respectively show the schematic structure of the HCB301_v5 format including SPT_G4_v5, SPT_G1_v5, PST_G1_v5, SP_G4_v5, SP_G1_v5 and PS_G1_v5.

[0033] Figure 2 Shown are the results of whole cell binding of HCB301_v5 protein to transfected CHO-S cells expressing CD47.

[0034] Figure 3 Shown are the results of whole cell binding of HCB301_v5 protein to transfected CHO-S cells expressing PD-L1.

[0035] Figure 4A The results of human TGFβ1 binding of HCB301_v5 protein determined by ELISA are shown. G4_TGFβRII_mt4 was used as a positive control.

[0036] Figure 4B The results of human TGFβ2 binding of HCB301_v5 protein determined by ELISA are shown. G4_TGFβRII_mt4 was used as a positive control.

[0037] Figure 4C The results of human TGFβ3 binding of HCB301_v5 protein determined by ELISA are shown. G4_TGFβRII_mt4 was used as a positive control.

[0038] Figure 5A-5BThe whole-cell binding results of HCB301_v5 protein to FaDu cells and NCI-H460 cells expressing CD47 are shown respectively.

[0039] 6A-6K show the results of HCB301_v5 protein binding to DLD1 cells expressing PD-L1 and whole cells of DLD1 cells, respectively.

[0040] Figure 7 The HCB301_v5 protein-induced RBC hemagglutination activity is shown.

[0041] Fig. 8A Platelet binding results for HCB301_v5 protein are shown.

[0042] Figure 8B The results of macrophage-mediated phagocytosis of platelets induced by HCB301_v5 protein are shown.

[0043] Fig. 9A RBC binding results of HCB301_v5 protein SPT_G4_v5 are shown. Hu5F9-G4 was used as a positive control. Anti-PD-L1 VHH (2H4) was used as a negative control.

[0044] Fig. 9B Results of macrophage-mediated phagocytosis of RBCs induced by HCB301_v5 protein SPT_G4_v5 are shown. Hu5F9-G4 was used as a positive control. H1G4 was used as a negative control.

[0045] Fig.10 The results of SIRPα / CD47 blocking by HCB301_v5 protein on CD47-expressing transfected CHO-S cells are shown. "Biotin SIRP Fc" indicates biotinylated SIRPα_G4.

[0046] Fig.11 Shown are the results of macrophage-mediated phagocytosis of OVA-GFP DLD1 cells expressing PD-L1.

[0047] Fig.12 The results of PD-1 / PD-L1 blocking of HCB301_v5 protein and transfected CHO-S cells expressing PD-L1 are shown. "Biotin PD1 Fc" indicates biotinylated PD1_G4.

[0048] Fig.13A Shown is the effect of HCB301_v5 protein on TGFβ1-mediated smad2 reporter gene activity.

[0049] Fig. 13BShown is the effect of HCB301_v5 protein on TGFβ2-mediated smad2 reporter gene activity.

[0050] Fig. 13C Shown is the effect of HCB301_v5 protein on TGFβ3-mediated smad2 reporter gene activity.

[0051] Fig.14 Shown is HCB301_v5 protein-induced IL-2 secretion in the MLR assay in the presence of TGFβ1 as determined by ELISA.

[0052] Fig.15A The in vivo antitumor efficacy of SPT_G4_v5 was shown by plotting the average tumor volume at multiple time points during 19 days after tumor cell inoculation.

[0053] Fig. 15B The in vivo antitumor efficacy of SPT_G4_v5 was shown by displaying the individual tumor volumes on day 19 after tumor cell inoculation.

[0054] Fig.16 The Kabat and Chothia CDR sequences of anti-PD-L1 VHH (2H4) are shown.

[0055] Fig.17 Sequences discussed in this disclosure are listed. DETAILED DESCRIPTION

[0056] 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 complex can effectively bind to cancer cells expressing CD47 and block the interaction between endogenous SIRPα and CD47, thereby inducing an innate immune response (e.g., phagocytosis of cancer cells by macrophages). On the other hand, the protein complex shows minimal binding to RBC cells or platelets, thereby inhibiting the clearance of host cells, as observed by the anti-CD47 antibody magrolimab. In addition, the results show that the protein complex can selectively bind to cancer cells expressing PD-L1 and block the interaction between endogenous PD-1 and PD-L1. Further, the protein complex can also include TGFβtrap, thereby inhibiting the immunosuppressive response induced by TGFβ, increasing T cell proliferation and cytokine secretion. In a mouse xenograft model, the protein complex (e.g., SPT_G4_v5) also exhibited better anti-tumor activity than commercial anti-PD-L1 or anti-PD-1 antibodies (e.g., Atezolizumab and Pembrolizumab).

[0057] Thus, the protein complexes described herein can be used for cancer therapy, wherein tumor immunogenicity and antigen presentation are enhanced by increasing phagocytosis by macrophages (e.g., inhibition of phagocytosis mediated by inactivation of CD47); and T cell activation is enhanced by inhibiting PD-1 / PD-L1 and TGFβ signaling pathways.

[0058] SIRPα extracellular domain

[0059] Signal regulatory protein alpha (SIRPα, SIRPa, Sirpa or CD172A) is a transmembrane protein. The signal regulatory protein alpha has an extracellular region including three Ig-like domains and a cytoplasmic region containing an immunoreceptor tyrosine-based inhibitory motif that mediates the binding of protein tyrosine phosphatases SHP1 and SHP2. Tyrosine phosphorylation of SIRPα is regulated by various growth factors and cytokines as well as integrin-mediated cell adhesion to extracellular matrix proteins. SIRPα is particularly abundant in myeloid cells such as macrophages and dendritic cells, but is only expressed at low levels in T cells, B cells, NK cells, and NKT cells.

[0060] The extracellular region of SIRPα can interact with its ligand CD47. The interaction of SIRPα on macrophages with CD47 on erythrocytes prevents macrophages from phagocytizing Ig opsonized erythrocytes in vitro and in vivo. The connection of SIRPα on phagocytes to CD47 expressed on adjacent cells causes phosphorylation of the tyrosine-based inhibitory motif of the SIRPα cytoplasmic immune receptor, leading to the recruitment of SHP-1 and SHP-2 phosphatases. One of the resulting downstream effects is to prevent myosin-IIA from accumulating at the phagocytic synapse and thus inhibiting phagocytosis. Therefore, the CD47-SIRPα interaction is used to send a "don't eat me" signal in the form of a negative immune checkpoint to ensure that healthy autologous cells are not inappropriately phagocytosed. However, overexpression of CD47 has also been found in almost all types of tumors, some of which include acute myeloid leukemia, non-Hodgkin's lymphoma, bladder cancer, and breast cancer. This negative regulation of macrophages can be minimized by blocking the binding of CD47 to SIRPα. Therefore, agents that block the CD47 / SIRPα interaction can both promote antibody-dependent cellular phagocytosis (ADCP) and, in some cases, trigger antibody-dependent cellular cytotoxicity (ADCC), and thus may be useful in treating various cancers.

[0061] Blocking the CD47 / SIRPα interaction can promote phagocytosis, which can be used to treat various cancers. It triggers the recognition and elimination of cancer cells by innate immunity. Agents targeting CD47 or SIRPα can be used to treat various tumors and cancers, for example, 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.

[0062] In addition, SIRPα acts to inhibit the in vivo clearance of CD47-expressing host cells by macrophages, including red blood cells and platelets. The CD47-SIRPα interaction has also been shown to be crucial for transplantation on hematopoietic stem cells. Blocking the CD47 / SIRPα interaction may lead to the accidental killing of normal red blood cells, which may cause anemia and induce inflammation. Therefore, it is very important to regulate the interaction of SIRPα targeting agents with CD47, for example, to have a limited or controlled effect on red blood cells.

[0063] 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); 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; the documents are incorporated herein by reference in their entirety.

[0064] 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. SIRPs all contain an N-terminal extracellular region, a single transmembrane domain, and a C-terminal intracellular region.

[0065] The extracellular region of human SIRPα (UniProt identifier: P78324) has an IgV domain, an Ig-like C1-type 1 domain, and an Ig-like C1-type 2 domain. They correspond to amino acids 32-137, amino acids 148-247, and amino acids 254-348 of human SIRPα protein (NP_542970.1). Amino acids 1-30 are signal peptides. Human SIRPα also has a long intracellular domain that includes two putative immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Activation of SIRPα ITIM delivers inhibitory signals that negatively regulate cellular responses.

[0066] In some embodiments, the protein complex includes one or more CD47 binding domains. In some embodiments, the CD47 binding domain includes or consists of a SIRPα extracellular domain. As used herein, "SIRPα extracellular domain" refers to all or part of the extracellular region of SIRPα or its variant, wherein a part of the extracellular region can bind to CD47. The SIRPα extracellular domain may have one or more protein domains that can be folded independently and form a self-stabilizing structure. In some embodiments, the SIRPα extracellular domain includes 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 includes or consists of an IgV domain. In some embodiments, the SIRPα extracellular domain includes or consists of an IgV domain and an Ig-like C1-1 type domain. In some embodiments, the SIRPα extracellular domain includes or consists of an IgV domain, an Ig-like C1-1 type domain, and an Ig-like C1-2 type domain.

[0067] In some embodiments, the 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 human SIRPα protein (NCBI accession number: AAH26692.1; SEQ ID NO: 24). In some embodiments, the CD47 binding domain or 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 SEQ ID NO: 32, and further comprises one or more (e.g., 1, 2, 3, 4, 5 or 6) amino acid mutations at positions corresponding to H24, I31, E54, G55, H56 and / or E70 of SEQ ID NO: 32. In some embodiments, the CD47 binding domain or SIRPα extracellular domain described herein comprises one or more of the following (1, 2, 3, 4, 5, or 6): (a) the amino acid corresponding to H24 of SEQ ID NO: 32 is T; (b) the amino acid corresponding to I31 of SEQ ID NO: 32 is Y; (c) the amino acid corresponding to E54 of SEQ ID NO: 32 is R; (d) the amino acid corresponding to G55 of SEQ ID NO: 32 is Q; (e) the amino acid corresponding to H56 of SEQ ID NO: 32 is T; and (f) the amino acid corresponding to E70 of SEQ ID NO: 32 is F. In some embodiments, the CD47 binding domain or 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 SEQ ID NO: 7.

[0068] In some embodiments, the CD47 binding domain or SIRPα extracellular domain described herein comprises the IgV domain of human SIRPα protein (wild type or mutant). In some embodiments, the CD47 binding domain or SIRPα extracellular domain described herein comprises the IgV domain of mouse SIRPα protein (wild type or mutant).

[0069] Anti-PD-L1 VHH

[0070] Monoclonal antibodies and recombinant antibodies are important tools in medicine and biotechnology. Like all mammals, Camelidae (e.g., llamas) can produce conventional antibodies consisting of two heavy chains and two light chains that are bound together in a Y shape (e.g., IgG1) with a disulfide bond. However, Camelidae also produces two unique IgG subclasses: IgG2 and IgG3, also referred to as heavy chain IgG. These antibodies are composed of only two heavy chains that lack the CH1 region but still carry the antigen-binding domain known as VHH (or nanobody) at its N-terminus. Conventional Ig requires the association of the variable regions from both heavy and light chains to allow for the high diversity of antigen-antibody interactions. Although the separated heavy and light chains still show this ability, when compared with paired heavy and light chains, they show very low affinity. The unique feature of heavy chain IgG is that its monomeric antigen-binding region is capable of binding antigens with specificity, affinity, and diversity in particular, which is comparable to conventional antibodies that do not need to be paired with another region. This feature is mainly due to some major variations in the amino acid sequence of the variable regions of the two heavy chains, which induce profound conformational changes compared to conventional Ig. The major substitutions in the variable regions prevent the light chain from binding to the heavy chain, but also prevent the unbound heavy chain from being retrieved by the immunoglobulin binding protein.

[0071] The single variable domain (named as VHH, sdAb or nanobody) of these antibodies is the minimum antigen binding domain produced by the adaptive immune system. It has been found that the third complementarity determining region (CDR3) of the variable region of these antibodies is twice as long as conventional antibodies. This increases the interaction surface with the antigen and the diversity of antigen-antibody interactions, which compensates for the lack of light chain. In the case of long complementarity determining region 3 (CDR3), VHH can extend into the protein cleft that conventional antibodies cannot reach, including the site of interest in function, such as the active site of an enzyme or the receptor binding gorge on the viral surface. In addition, other cysteine ​​residues make the structure more stable, thus increasing the intensity of interaction.

[0072] Compared with conventional antibodies carrying the variable domains (VH and VL) of conventional antibodies, VHH provides many other advantages, including higher stability, solubility, expression yield and refolding ability and better in vivo tissue penetration. In addition, compared with the VH domain of conventional antibodies, VHH does not show an inherent tendency to bind to the light chain. This is conducive to inducing heavy chain antibodies in the presence of functional light chain loci. Further, since VHH does not bind to the VL domain, it is much easier to reformat VHH into a bispecific antibody construct than to reformat it into a construct containing a conventional VH-VL pair or a single domain based on the VH domain.

[0073] The PD-1 / PD-L1 pathway controls the induction and maintenance of immune tolerance in the tumor microenvironment. The activity of PD-1 and its ligands PD-L1 or PD-L2 is responsible for T cell activation, proliferation, and cytotoxic secretion in cancer to degrade antitumor immune responses.

[0074] PD-1, also known as CD279, is a 55-kDa transmembrane protein containing 288 amino acids with an extracellular N-terminal domain (IgV-like), a membrane permeation domain, and a cytoplasmic tail with two tyrosine bases at the N-terminus and C-terminus, respectively.

[0075] PD-1 is an inhibitor of both adaptive and innate immune responses and is expressed on activated T cells, natural killer (NK) and B lymphocytes, macrophages, dendritic cells (DCs), and monocytes.

[0076] PD-1 ligand (PD-L1 or PD-L1; also known as CD279 and B7-H1), belongs to the B7 family and is a 33-kDa type 1 transmembrane glycoprotein that contains 290 amino acids and has Ig and IgC domains in its extracellular region.

[0077] PD-L1 is normally expressed by macrophages, some activated T and B cells, DCs, and some epithelial cells, especially under inflammatory conditions. In addition, PD-L1 is expressed by tumor cells as an “adaptive immune mechanism” to evade antitumor responses. PD-L1 is associated with an immune environment rich in CD8 T cells, the production of Th1 cytokines and chemokines, as well as interferons and specific gene expression characteristics. It has been demonstrated that IFN-γ leads to PD-L1 upregulation in ovarian cancer cells, which is responsible for disease progression, while IFN-γ receptor 1 inhibition can reduce PD-L1 expression in acute myeloid leukemia mouse models through the MEK / extracellular signal-regulated kinase (ERK) and MYD88 / TRAF6 pathways. IFN-γ induces protein kinase D isoform 2 (PKD2), which is important for the regulation of PD-L1. Inhibition of PKD2 activity suppresses PD-L1 expression and promotes a strong antitumor immune response. NK cells secrete IFN-γ through the Janus kinase (JAK) 1, JAK2, and signal transducer and activator of transcription (STAT) 1 pathways, increasing the expression of PD-L1 on the surface of tumor cells. Studies on melanoma cells have shown that IFN-γ secreted by T cells through the JAK1 / JAK2-STAT1 / STAT2 / STAT3-IRF1 pathway can regulate the expression of PD-L1. T cells and NK cells appear to secrete IFN-γ, thereby inducing PD-L1 expression on the surface of target cells (including tumor cells).

[0078] PD-L1 acts as a tumor-promoting factor in cancer cells by binding to its receptor and activating proliferation and survival signaling pathways. This finding further suggests that PD-L1 is associated with subsequent tumor progression. In addition, PD-L1 has been shown to exert non-immune proliferative effects on a variety of tumor cell types. For example, PD-L1 induces epithelial-mesenchymal transition (EMT) and stem cell-like phenotypes in renal cancer cells, indicating that the presence of PD-L1 intrinsic pathways promotes the progression of renal cancer.

[0079] Detailed reviews of PD-L1 and its functions can be found in Han, Yanyan, Dandan Liu, and Lianhong Li. "PD-1 / PD-L1 pathway: current researches in cancer". American journal of cancer research 10.3 (2020): 727; Liu, Jinhua et al. "PD-1 / PD-L1 checkpoint inhibitors in tumor immunotherapy". Frontiers in Pharmacology 12 (2021); each of which is incorporated herein by reference in its entirety.

[0080] In some embodiments, the protein complexes described herein include one or more PD-L1 binding domains. In some embodiments, the PD-L1 binding domain is a VHH that binds to PD-L1.

[0081] The present disclosure provides VHHs that bind to PD-L1, e.g., anti-PD-L1 VHH (2H4) ("2H4"), chimeric antibodies thereof, and human or humanized antibodies thereof. According to the Kabat definition, the CDR sequences of 2H4 and 2H4-derived antibodies (e.g., humanized antibodies) include VHH CDR1, 2, and 3 as shown in SEQ ID NOs: 26, 27, and 28, respectively. VHH CDRs may also be defined by the Chothia system. Under the Chothia definition, the CDR sequences of 2H4 and 2H4-derived antibodies (e.g., humanized antibodies) include VHH CDR1, 2, and 3 as shown in SEQ ID NOs: 29, 30, and 31, respectively. The amino acid sequence of anti-PD-L1 VHH (2H4) is shown in SEQ ID NO: 7. Detailed information on anti-PD-L1 VHH (2H4) can be found in, for example, PCT Application No. PCT / CN2022 / 143510, which is incorporated herein by reference in its entirety.

[0082] In some embodiments, the amino acid sequences of the VHHs described herein are provided. In some embodiments, the VHHs are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8. In some embodiments, the VHHs described herein may contain one, two or three CDRs selected from the group consisting of SEQ ID NOs: 26-28 and SEQ ID NOs: 29-31.

[0083] In some embodiments, the VHHs described herein may have complementarity determining regions (CDR) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH CDR3 amino acid sequence. Fig.16 Selected VHH CDR1, 2, 3 amino acid sequences (CDRs under Kabat definition and Chothia definition) are shown in .

[0084] In some embodiments, the VHHs described herein may contain one, two, or three of the following: SEQ ID NO: 26 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 28 with zero, one, or two amino acid insertions, deletions, or substitutions. In some embodiments, the VHHs described herein may contain one, two, or three of the following: SEQ ID NO: 29 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 30 with zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 31 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0085] Insertions, deletions and substitutions can be within a CDR sequence, or at one or both ends of a CDR sequence. In some embodiments, CDRs are determined based on the Kabat definition scheme. In some embodiments, CDRs are determined based on the Chothia definition scheme. In some embodiments, CDRs are determined based on a combination of the Kabat definition scheme and the Chothia definition scheme. In some embodiments, CDRs are determined based on the IMGT definition. In some embodiments, CDRs are determined based on the contact definition.

[0086] TGFBRII extracellular domain

[0087] TGF-β receptor type 2 (TGFBRII) is a ligand binding receptor for all members of the TGF-β family and is expressed in almost all cell types comprising fibroblasts. Ligand-induced cell responses are mediated by typical Smad-dependent or atypical Smad-independent signal transduction pathways, such as c-Jun N-terminal kinase, Akt, Src, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase pathways. The ligand bound to TGFBRII causes receptor dimerization and autophosphorylation, and then binds to TGF-β receptor type 1 (TGFBR1) or type 3 (TGFBR3). The newly formed heterotetrameric complex then recruits and phosphorylates regulatory SMAD (SMAD2 or SMAD3), which is combined with the co-SMAD molecule SMAD4 in the phosphorylated state. Regulatory SMAD / co-SMAD complex translocations to the nucleus, where it acts as a transcription factor regulating target gene expression.

[0088] Detailed descriptions of the use of TGFβ, TGFBRII, and TGFβtrap for treating cancer are described in, for example, 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-βpathway". Journal of Hematology & Oncology 14.1 (2021): 1-20; and Lind, H. et al. "Dual targeting of TGF-βandPD-L1 via a bifunctional anti-PD-L1 / TGF-βRII agent: preclinical and clinical progress status". "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.

[0089] According to the UniProt identifier P37173, the extracellular region of human TGFBRII corresponds to amino acids 23-166 of NP_003233.4 (SEQ ID NO: 25), the transmembrane region of human TGFBRII corresponds to amino acids 167-187 of SEQ ID NO: 25, and the cytoplasmic region of human TGFBRII corresponds to amino acids 188-567 of SEQ ID NO: 25. The signal peptide corresponds to amino acids 1-22 of SEQ ID NO: 25.

[0090] In some embodiments, the protein complex includes one or more TGFβ binding domains. In some embodiments, the TGFβ binding domain includes or consists of a TGFBRII extracellular domain. As used herein, "TGFBRII extracellular domain" refers to all or part of the extracellular region of TGFBRII or its variants, wherein a part of the extracellular region can bind to TGFβ. The TGFBRII extracellular domain may have one or more protein domains that can be independently folded and form a self-stabilizing structure. In some embodiments, the TGFBRII extracellular domain does not include a signal peptide.

[0091] In some embodiments, the TGFBRII 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 human TGFBRII protein (NCBI accession number: NP_003233.4; SEQ ID NO: 25). In some embodiments, the TGFβ binding domain or TGFBRII 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: 33. In some embodiments, the TGFBRII extracellular domain also comprises one or more (e.g., 1 or 2) amino acid mutations at positions corresponding to S49 and / or D118 of SEQ ID NO: 33. In some embodiments, the TGFβ binding domain or TGFBRII extracellular domain described herein comprises one or more (1 or 2) of the following: (a) the amino acid corresponding to S49 of SEQ ID NO: 33 is E; and (b) the amino acid corresponding to D118 of SEQ ID NO: 33 is E. In some embodiments, the TGFβ binding domain or TGFBRII 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: 9.

[0092] In some embodiments, the TGFβ binding domain or TGFBRII extracellular domain described herein comprises all or a portion of a human TGFBRII extracellular domain (wild type or mutant). In some embodiments, the TGFβ binding domain or TGFBRII extracellular domain described herein comprises all or a portion of an extracellular domain of a mouse TGFBRII protein (wild type or mutant).

[0093] Protein complex targeting CD47, PD-L1 and / or TGFβ

[0094] 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, thereby increasing the immune response. In some embodiments, these protein complexes can induce phagocytosis.

[0095] 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, thereby increasing the immune response. In some embodiments, these protein complexes can induce T cell activation, proliferation and / or cytokine release.

[0096] On the one hand, the present disclosure provides a protein complex or protein construct, which includes or consists of 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 crystallizable fragment 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 domain (e.g., CH2 and CH3) in the heavy chain peptide forming the Fc region. In some embodiments, the protein complex or protein construct includes 1, 2, 3, 4, 5 or 6 CD47 binding domains. In some embodiments, the protein complex or protein construct includes 1, 2, 3, 4, 5 or 6 PD-L1 binding domains. In some embodiments, the protein complex or protein construct includes 1, 2, 3, 4, 5 or 6 TGFβ binding domains.

[0097] In some embodiments, the protein complex or protein construct comprises or consists of an Fc, a first domain that specifically binds to cluster of differentiation 47 (CD47), and a second domain that specifically binds to programmed death-ligand 1 (PD-L1).

[0098] In some embodiments, the first 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 first domain includes all or part of the extracellular region of SIRPα. In some embodiments, SIRPα is a human SIRPα extracellular domain having one or more mutations (e.g., any SIRPα mutation in the SIRPα mutations described herein). In some embodiments, the first 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.

[0099] In some embodiments, the second domain can 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 a VHH that binds to PD-L1. 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: 8.

[0100] In some embodiments, the Fc is a human IgG1 Fc. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the first domain is connected to the N-terminus of the CH2 domain in the Fc, optionally through the hinge region. In some embodiments, the second domain is connected to the N-terminus of the CH2 domain in the Fc, optionally through the hinge region. In some embodiments, the hinge region is a human IgG4 hinge region, and the human IgG4 hinge region optionally has an S228P mutation according to EU numbering.

[0101] In some embodiments, the protein complex or protein construct further includes a third domain that specifically binds to transforming growth factor β (TGFβ). In some embodiments, the third domain is connected to the C-terminus of the CH3 domain in the Fc, optionally through a linker peptide. In some embodiments, the third domain includes all or part of the extracellular region of TGFBRII. In some embodiments, TGFBRII is a human TGFBRII extracellular domain with one or more mutations (e.g., any TGFBRII mutation in the TGFBRII mutation described herein). In some embodiments, the first 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:9.

[0102] 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.

[0103] 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 as described herein.

[0104] Some examples of protein complexes are shown in Figures 1A-1F. The examples are described in detail below.

[0105] SPT_G4_v5

[0106] In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises 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 preferably comprises 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. The schematic structure of an exemplary protein complex with SPT_G4_v5 format is shown in FIG. 1A.

[0107] In any of the protein complexes described herein, the first CD47 binding domain and / or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any of the SIRPα extracellular domains described herein). In some embodiments, the SIRPα extracellular domain comprises one or more mutations (e.g., at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are identical. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain 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. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain comprises the IgV domain of SIRPα (e.g., human SIRPα) with one or more mutations (at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different.

[0108] In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain comprises a VHH that binds to PD-L1, e.g., an anti-PD-L1 VHH (2H4). In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain 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: 8.

[0109] In some embodiments, the first hinge region and / or the second hinge region may comprise all or a portion of a hinge region of an immunoglobulin, e.g., a human IgG4 hinge region (SEQ ID NO: 14). In some embodiments, the first hinge region and / or the 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: 14. In some embodiments, the first hinge region and the second hinge region are the same. In some embodiments, the first hinge region and the second hinge region are different. In some embodiments, the first hinge region and / or the second hinge region comprise a proline at position 228 according to EU numbering.

[0110] In some embodiments, the first Fc region and / or the second Fc region are identical and can form Fc homodimers. In some embodiments, the first Fc region and / or the second Fc region comprise all or a portion of an Fc region of an immunoglobulin, e.g., a human IgG4 Fc region (SEQ ID NO: 15). In some embodiments, the first Fc region and / or the second Fc region 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: 15.

[0111] In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain may comprise a TGFBRII extracellular domain (e.g., any TGFBRII extracellular domain described herein). In some embodiments, the TGFBRII extracellular domain comprises one or more mutations (e.g., at a position corresponding to S49 and / or D118 of SEQ ID NO: 33). In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain are identical. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain are different. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain 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: 9.

[0112] In some embodiments, the first and / or third joint peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 12. In some embodiments, the second and / or fourth joint peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 13. In some embodiments, the first, second, third and / or fourth joint peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 13. In some embodiments, the first, second, third and / or fourth joint 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).

[0113] In some embodiments, the first polypeptide and / or the 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.

[0114] SPT_G1_v5

[0115] In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises 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 preferably comprises 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. The schematic structure of an exemplary protein complex with SPT_G1_v5 format is shown in FIG. 1B .

[0116] In any of the protein complexes described herein, the first CD47 binding domain and / or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any of the SIRPα extracellular domains described herein). In some embodiments, the SIRPα extracellular domain comprises one or more mutations (e.g., at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are identical. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain 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. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain comprises the IgV domain of SIRPα (e.g., human SIRPα) with one or more mutations (at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different.

[0117] In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain comprises a VHH that binds to PD-L1, e.g., an anti-PD-L1 VHH (2H4). In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain 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: 8.

[0118] In some embodiments, the first hinge region and / or the second hinge region may comprise all or a portion of a hinge region of an immunoglobulin, e.g., a human IgG1 hinge region (SEQ ID NO: 16). In some embodiments, the first hinge region and / or the 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 hinge region and the second hinge region are the same. In some embodiments, the first hinge region and the second hinge region are different.

[0119] In some embodiments, the first Fc region and / or the second Fc region are identical and can form Fc homodimers. In some embodiments, the first Fc region and / or the second Fc region comprise all or a portion of an Fc region of an immunoglobulin, e.g., a human IgG1 Fc region (SEQ ID NO: 17). In some embodiments, the first Fc region and / or the second Fc region 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: 17.

[0120] In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain may comprise a TGFBRII extracellular domain (e.g., any TGFBRII extracellular domain described herein). In some embodiments, the TGFBRII extracellular domain comprises one or more mutations (e.g., at a position corresponding to S49 and / or D118 of SEQ ID NO: 33). In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain are identical. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain are different. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain 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: 9.

[0121] In some embodiments, the first and / or third joint peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 12. In some embodiments, the second and / or fourth joint peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 13. In some embodiments, the first, second, third and / or fourth joint peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 13. In some embodiments, the first, second, third and / or fourth joint 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).

[0122] In some embodiments, the first polypeptide and / or the 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.

[0123] PST_G1_v5

[0124] In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises 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 preferably comprises 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. The schematic structure of an exemplary protein complex with a PST_G1_v5 format is shown in FIG. 1C.

[0125] In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain comprises a VHH that binds to PD-L1, e.g., an anti-PD-L1 VHH (2H4). In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain 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: 8.

[0126] In any of the protein complexes described herein, the first CD47 binding domain and / or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any of the SIRPα extracellular domains described herein). In some embodiments, the SIRPα extracellular domain comprises one or more mutations (e.g., at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are identical. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain 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. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain comprises the IgV domain of SIRPα (e.g., human SIRPα) with one or more mutations (at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different.

[0127] In some embodiments, the first hinge region and / or the second hinge region may comprise all or a portion of a hinge region of an immunoglobulin, e.g., a human IgG1 hinge region (SEQ ID NO: 16). In some embodiments, the first hinge region and / or the 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 hinge region and the second hinge region are the same. In some embodiments, the first hinge region and the second hinge region are different.

[0128] In some embodiments, the first Fc region and / or the second Fc region are identical and can form Fc homodimers. In some embodiments, the first Fc region and / or the second Fc region comprise all or a portion of an Fc region of an immunoglobulin, e.g., a human IgG1 Fc region (SEQ ID NO: 17). In some embodiments, the first Fc region and / or the second Fc region 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: 17.

[0129] In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain may comprise a TGFBRII extracellular domain (e.g., any TGFBRII extracellular domain described herein). In some embodiments, the TGFBRII extracellular domain comprises one or more mutations (e.g., at a position corresponding to S49 and / or D118 of SEQ ID NO: 33). In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain are identical. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain are different. In some embodiments, the first TGFβ binding domain and / or the second TGFβ binding domain 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: 9.

[0130] In some embodiments, the first and / or third joint peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 12. In some embodiments, the second and / or fourth joint peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 13. In some embodiments, the first, second, third and / or fourth joint peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 13. In some embodiments, the first, second, third and / or fourth joint 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).

[0131] In some embodiments, the first polypeptide and / or the 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.

[0132] SP_G4_v5

[0133] In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises, 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, and a first Fc region. The second polypeptide preferably comprises, from N-terminus to C-terminus: a second CD47 binding domain, an optional second linker peptide, a second PD-L1 binding domain, an optional second hinge region, and a second Fc region. The schematic structure of an exemplary protein complex having the SP_G4_v5 format is shown in FIG. 1D.

[0134] In any of the protein complexes described herein, the first CD47 binding domain and / or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any of the SIRPα extracellular domains described herein). In some embodiments, the SIRPα extracellular domain comprises one or more mutations (e.g., at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are identical. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain 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. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain comprises the IgV domain of SIRPα (e.g., human SIRPα) with one or more mutations (at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different.

[0135] In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain comprises a VHH that binds to PD-L1, e.g., an anti-PD-L1 VHH (2H4). In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain 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: 8.

[0136] In some embodiments, the first hinge region and / or the second hinge region may comprise all or a portion of a hinge region of an immunoglobulin, e.g., a human IgG4 hinge region (SEQ ID NO: 14). In some embodiments, the first hinge region and / or the 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: 14. In some embodiments, the first hinge region and the second hinge region are the same. In some embodiments, the first hinge region and the second hinge region are different. In some embodiments, the first hinge region and / or the second hinge region comprise a proline at position 228 according to EU numbering.

[0137] In some embodiments, the first Fc region and / or the second Fc region are identical and can form Fc homodimers. In some embodiments, the first Fc region and / or the second Fc region comprise all or a portion of an Fc region of an immunoglobulin, e.g., a human IgG4 Fc region (SEQ ID NO: 15). In some embodiments, the first Fc region and / or the second Fc region 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: 15.

[0138] In some embodiments, the first and / or second linker peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12. In some embodiments, the first and / or second 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).

[0139] In some embodiments, the first polypeptide and / or the 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.

[0140] SP_G1_v5

[0141] In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises, 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, and a first Fc region. The second polypeptide preferably comprises, from N-terminus to C-terminus: a second CD47 binding domain, an optional second linker peptide, a second PD-L1 binding domain, an optional second hinge region, and a second Fc region. The schematic structure of an exemplary protein complex having the SP_G1_v5 format is shown in FIG. 1E.

[0142] In any of the protein complexes described herein, the first CD47 binding domain and / or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any of the SIRPα extracellular domains described herein). In some embodiments, the SIRPα extracellular domain comprises one or more mutations (e.g., at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are identical. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain 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. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain comprises the IgV domain of SIRPα (e.g., human SIRPα) with one or more mutations (at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different.

[0143] In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain comprises a VHH that binds to PD-L1, e.g., an anti-PD-L1 VHH (2H4). In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain 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: 8.

[0144] In some embodiments, the first hinge region and / or the second hinge region may comprise all or a portion of a hinge region of an immunoglobulin, e.g., a human IgG1 hinge region (SEQ ID NO: 16). In some embodiments, the first hinge region and / or the 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 hinge region and the second hinge region are identical.

[0145] In some embodiments, the first Fc region and / or the second Fc region are identical and can form Fc homodimers. In some embodiments, the first Fc region and / or the second Fc region comprise all or a portion of an Fc region of an immunoglobulin, e.g., a human IgG1 Fc region (SEQ ID NO: 17). In some embodiments, the first Fc region and / or the second Fc region 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: 17.

[0146] In some embodiments, the first and / or second linker peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12. In some embodiments, the first and / or second 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).

[0147] In some embodiments, the first polypeptide and / or the 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:5.

[0148] PS_G1_v5

[0149] In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises, 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, and a first Fc region. The second polypeptide preferably comprises, from N-terminus to C-terminus: a second PD-L1 binding domain, an optional second linker peptide, a second CD47 binding domain, an optional second hinge region, and a second Fc region. The schematic structure of an exemplary protein complex having a PS_G1_v5 format is shown in FIG. 1F.

[0150] In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain comprises a VHH that binds to PD-L1, e.g., an anti-PD-L1 VHH (2H4). In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and / or the second PD-L1 binding domain 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: 8.

[0151] In any of the protein complexes described herein, the first CD47 binding domain and / or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any of the SIRPα extracellular domains described herein). In some embodiments, the SIRPα extracellular domain comprises one or more mutations (e.g., at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are identical. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain 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. In some embodiments, the first CD47 binding domain and / or the second CD47 binding domain comprises the IgV domain of SIRPα (e.g., human SIRPα) with one or more mutations (at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO: 32). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different.

[0152] In some embodiments, the first hinge region and / or the second hinge region may comprise all or a portion of a hinge region of an immunoglobulin, e.g., a human IgG1 hinge region (SEQ ID NO: 16). In some embodiments, the first hinge region and / or the 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 hinge region and the second hinge region are identical.

[0153] In some embodiments, the first Fc region and / or the second Fc region are identical and can form Fc homodimers. In some embodiments, the first Fc region and / or the second Fc region comprise all or a portion of an Fc region of an immunoglobulin, e.g., a human IgG1 Fc region (SEQ ID NO: 17). In some embodiments, the first Fc region and / or the second Fc region 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: 17.

[0154] In some embodiments, the first and / or second linker peptides described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12. In some embodiments, the first and / or second 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).

[0155] In some embodiments, the first polypeptide and / or the 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:6.

[0156] In any protein complex in the protein complex described herein, the first hinge and / or the second hinge region may be different. In any protein complex in the protein complex described herein, the first Fc region and / or the second Fc region may be different. In some embodiments, the first Fc region and / or the second Fc region may form an Fc heterodimer by introducing one or more mutations. For example, the first Fc region and / or the second Fc region may include one or more knob-hole (KIH) mutations. In some embodiments, the first Fc region and / or the second Fc region may form an Fc heterodimer using other techniques known in the art. The detailed information of heterodimer Fc technology can be found in, for example, 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 herein by reference in its entirety.

[0157] On the one hand, the present disclosure relates to a protein complex comprising a CD47 binding domain (e.g., any CD47 binding domain described herein) and a PD-L1 binding domain (e.g., any PD-L1 binding domain described herein). In some embodiments, the protein complex further comprises a TGFβ binding domain (e.g., any TGFβ binding domain described herein). In some embodiments, the CD47 binding domain is or includes a SIRPα extracellular domain (e.g., any SIRPα extracellular domain described herein). In some embodiments, the PD-L1 binding domain is or includes a VHH that binds to PD-1 (e.g., any VHH described herein). In some embodiments, the TGFβ binding domain is or includes a TGFBRII extracellular domain (e.g., any TGFBRII extracellular domain described herein).

[0158] Properties of protein complexes

[0159] In some embodiments, the protein complex may include any CD47 binding domain, PD-L1 binding domain, and / or TGFβ binding domain as described herein. The present disclosure also provides nucleic acids comprising polynucleotides encoding the polypeptides described herein.

[0160] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (e.g., a gap can be introduced in one or both of the first amino acid and the second amino acid or nucleic acid sequence for optimal comparison, and for the purpose of comparison, non-homologous sequences can be ignored). The length of the reference sequence aligned for the purpose of comparison 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 the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at the position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, and the function needs to be introduced to perform the optimal comparison of the two sequences. For example, the comparison of the sequences and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0161] The protein complexes described herein may comprise the 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).

[0162] In some embodiments, the protein complexes described herein are connected 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.

[0163] The protein complex described herein can block the binding between CD47 and endogenous SIRPα expressed on immune cells. In some embodiments, by binding to CD47, the protein complex described herein can inhibit the binding of CD47 (e.g., expressed on tumor cells) to endogenous SIRPα expressed on immune cells (e.g., myeloid cells, macrophages, and dendritic cells), thereby blocking the CD47 / SIRPα pathway, thereby upregulating the immune response and promoting phagocytosis.

[0164] The protein complexes described herein can block the binding between PD-L1 and endogenous PD-1 expressed on immune cells. 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, upregulating immune responses, activating T cell proliferation and cytokine release.

[0165] In some embodiments, the protein complexes described herein can increase the 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.

[0166] In some embodiments, the protein complexes described herein can 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) in less than 0.1 s. -1 , less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 In some embodiments, the kinetic association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms. Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 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 greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M or greater than 1×10 -12 M.

[0167] 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 may not 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 may not 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 may not bind to monkey TGFβ and / or mouse TGFβ.

[0168] In some embodiments, thermal stability is determined. The Tm of the protein complexes described herein may be greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, Tm is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C.

[0169] In some embodiments, the protein complexes described herein have a tumor growth inhibition percentage (TGI%) greater 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 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 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days after the start of treatment, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months after the start of treatment. As used herein, tumor growth inhibition percentage (TGI%) is calculated using the following formula:

[0170] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100

[0171] Ti is the average tumor volume of the treatment group on day i. T0 is the average tumor volume of the treatment group on day 0. Vi is the average tumor volume of the control group on day i. V0 is the average tumor volume of the control group on day 0.

[0172] In some embodiments, the tumor inhibitory effect of the protein complex 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 in, for example, 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 complex described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold or 5-fold greater than the tumor inhibitory effect 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 complex described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold or 5-fold greater than the tumor inhibitory effect of HCB101. The amino acid sequence of HCB101 is shown in SEQ ID NO: 20.

[0173] 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., atezolizumab (MPDL3280A)) or an anti-PD-1 antibody (e.g., pembrolizumab). MPDL3280A is described, for example, 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 of the tumor inhibitory effect of an anti-PD-L1 reference antibody, such as MPDL3280A or an anti-PD-1 antibody, such as 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 of the tumor inhibitory effect of an anti-PD-L1 VHH (2H4). c is used as a control protein for HCB301_v5 fusion protein. The amino acid sequence of anti-PD-L1 VHH (2H4) is shown in SEQ ID NO: 8.

[0174] In some embodiments, the tumor suppressive effect of the protein complex described herein is comparable to that of a TGFβtrap protein, such as G4_TGFβRII_mt4 or an anti-PD-L1 antibody × TGFβtrap M7824. M7824 is described in, for example, Gatti-Mays, ME et al. "M7824: a promising new strategy to combat cancer immune evasion". Oncoscience 5.11-12 (2018): 269, which is incorporated herein by reference in its entirety. In some embodiments, the tumor suppressive 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 that of the tumor suppressive effect of G4_TGFβRII_mt4 or M7824. In some embodiments, the tumor suppressive 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 G4_TGFβRII_mt4. G4_TGFβRII_mt4 is used as a control protein for the HCB301_v5 fusion protein. The amino acid sequence of G4_TGFβRII_mt4 is shown in SEQ ID NO: 21.

[0175] In some embodiments, the protein complex described herein has a functional Fc. In some embodiments, Fc is from human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the effector function of functional Fc is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of functional Fc is phagocytosis. In some embodiments, the effector function of functional Fc is ADCC and phagocytosis. In some embodiments, the protein construct as described herein has an Fc region without effector function. In some embodiments, Fc is human IgG4 Fc. In some embodiments, Fc does not have a functional Fc region. For example, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

[0176] Some other modifications can be made to the Fc region. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The resulting homodimeric fusion protein can have any increased half-life in vitro and / or in vivo.

[0177] In some embodiments, the IgG4 has an S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0178] In some embodiments, the Fc region is provided as a carbohydrate structure lacking fucose connected to the Fc region (directly or indirectly). For example, the amount of fucose in such Fc region compositions may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn 297 as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue at approximately position 297 (Eu numbering of Fc region residues; or position 314 in Kabat numbering) in the Fc region; however, due to minor sequence variations in the Fc region sequence, Asn297 may also be located at approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0179] In some embodiments, the major peak by HPLC-SEC after purification by Protein A-based affinity chromatography and / or size exclusion chromatography accounts for at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of the protein complex described herein.

[0180] In some embodiments, the protein complexes described herein can bind to tumor cells expressing human CD47 (e.g., human CD47 tf CHO-S cells, FaDu cells, or NCI-H460 cells) with 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 affinity than an anti-CD47 reference antibody (e.g., Hu5F9-G4) or HCB101.

[0181] In some embodiments, the protein complexes described herein can bind to tumor cells expressing human PD-L1 (e.g., transfected CHO-S cells expressing human PD-L1) with 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 affinity than anti-PD-L1 VHH (2H4).

[0182] In some embodiments, the protein complexes described herein can selectively bind to transfected cells expressing PD-L1 (e.g., transfected DLD1 cells expressing PD-L1; or PD-L1 tf DLD1 cells), e.g., in a cell mixture of transfected DLD1 cells expressing PD-L1 and untransfected DLD1 cells. In some embodiments, the selectivity of the protein complex is comparable to that of atezolizumab and / or anti-PD-L1 VHH (2H4).

[0183] In some embodiments, the protein complexes described herein can bind to human TGFβ (e.g., TGFβ1, TGFβ2, or TGFβ3) with an affinity of 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% compared to G4_TGFβRII_mt4. In some embodiments, the EC50 value of the protein complex that binds to human TGFβ (e.g., TGFβ1, TGFβ2, or TGFβ3) is less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold that of the EC50 value of G4_TGFβRII_mt4.

[0184] In some embodiments, the protein complexes described herein can bind to RBC cells or platelets (e.g., from human donors) with an affinity of 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% as compared to an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0185] In some embodiments, the protein complexes described herein do not induce hemagglutination. In some embodiments, the protein complexes described herein can induce hemagglutination at a minimum concentration greater than 500-fold, 2000-fold, 5000-fold, 20000-fold, or 50000-fold compared to the minimum concentration of an anti-CD47 reference antibody (e.g., Hu5F9-G4).

[0186] In some embodiments, the protein complex described herein can 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 complex described herein can block the interaction between cells expressing human CD47 (e.g., CD47 tf CHO-S cells or FaDu cells) and human SIRPα. In some embodiments, the blocking ability of the 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-CD47 reference antibody (e.g., Hu5F9-G4) or HCB101.

[0187] In some embodiments, the protein complexes described herein can 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, the protein complexes described herein can block the interaction between cells expressing human PD-L1 (e.g., PD-L1 tf CHO-S cells) and human PD-1. 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-PD-L1 reference antibody (e.g., atezolizumab) or an anti-PD-L1 VHH (2H4).

[0188] In some embodiments, the protein complexes described herein can induce phagocytosis of tumor cells by mouse macrophages (e.g., Raw264.7 cells). In some embodiments, the protein complexes described herein induce phagocytosis of tumor cells by mouse microphages by 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% compared to Hu5F9-G4 or HCB101. In some embodiments, the protein complexes described herein have a weaker (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%) ability to induce phagocytosis of RBC cells or platelets (e.g., Raw264.7 cells) by mouse macrophages compared to anti-CD47 reference antibodies (e.g., Hu5F9-G4) or HCB101.

[0189] In some embodiments, the protein complexes described herein can induce phagocytosis of tumor cells expressing PD-L1 (e.g., PD-L1 tf DLD1 cells) by human macrophages (e.g., MDM cells). In some embodiments, the protein complexes described herein induce phagocytosis of tumor cells expressing PD-L1 by human macrophages compared to anti-CD47 reference antibodies (e.g., Hu5F9-G4) or HCB101 by at least 10%, at least 20%, 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%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200%. In some embodiments, the protein complexes described herein have a weaker (less than 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%) ability to induce phagocytosis of RBC cells or platelets by human macrophages (e.g., MDM cells) compared to an anti-CD47 reference antibody (e.g., Hu5F9-G4) or HCB101.

[0190] In some embodiments, the protein complexes described herein can induce phagocytosis of tumor cells expressing CD47 by mouse macrophages (e.g., Raw264.7 cells). In some embodiments, the protein complexes described herein can induce phagocytosis of tumor cells expressing CD47 by human macrophages (e.g., MDM cells).

[0191] Endogenous expression of CD47 on a variety of cell types including red blood cells produces a strong "antigenic silencing" that may limit the efficacy of therapies targeting CD47. Therefore, the weaker 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, compared to an anti-CD47 reference antibody (e.g., Hu5F9-G4), the protein complexes can be administered at lower dose levels and / or less frequent dose regimens with similar efficacy.

[0192] In some embodiments, the protein complexes described herein can inhibit TGFβ-induced downstream pathways, for example, smad2 reporter gene pathways. In some embodiments, the protein complexes can inhibit TGFβ1 and / or TGFβ3-mediated smad2 reporter gene activity to 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 G4_TGFβRII_mt4.

[0193] In some embodiments, the protein complex described herein can enhance T cell response (for example, in MLR determination). The principle of mixed lymphocyte reaction (MLR) is that T cells from one donor will proliferate in the presence of APCs from different donors. This is due to the recognition of HLA mismatches between two unrelated donors, thereby stimulating the immune response of T cells. MLR is generally used as a means of inducing universal stimulation / activation of T cells in culture. In some embodiments, compared with the control molecule used herein or its combination, the protein complex can increase T cell proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, compared with the control molecule used herein or its combination, the protein complex described herein can increase cytokine (for example, IFN-γ and / or IL-2) production by at least 1 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1000 times, 2000 times or 10000 times.

[0194] In some embodiments, the protein complexes described herein do not induce cytokine storms in humans. In some embodiments, the protein complexes described herein are not superagonists. Detailed information on cytokine storms and superagonists can be found in, for example, Shimabukuro-Vornhagen, A. et al. "Cytokine release syndrome". Journal of Cancer Immunotherapy 6.1 (2018): 1-14, which is incorporated herein by reference in its entirety.

[0195] In some embodiments, the protein complex described herein can inhibit tumor growth. In some embodiments, compared with vehicle control, the protein complex described herein (e.g., SPT_G4_v5) can significantly inhibit tumor growth. In some embodiments, in a mouse xenograft model, compared with HCB101, anti-PD-L1 VHH (2H4), G4_TGFβRII_mt4, atezolizumab and / or pembrolizumab, the protein complex described herein (e.g., SPT_G4_v5) can inhibit tumor growth, wherein the TGI value is at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or at least 10 times. In some embodiments, tumor cells (eg, pharyngeal cancer cells) are subcutaneously inoculated with NPG TMIn mice, a xenograft model was generated. TM Mice (NOD.Cg-PrkdcscidIl2rgtm1 Vst / Vst mice) were obtained from Beijing Vitalstar Biotechnology Co., Ltd. and are NOD / SCID mice with knockout interleukin-2 gamma chain receptor. In some embodiments, the tumor volume of mice is analyzed at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days after inoculation. In some embodiments, the TGI value of mice treated with the protein complex described herein (e.g., SPT_G4_v5) is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90%.

[0196] Method for preparing protein complex

[0197] 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.

[0198] Screening can be performed to increase the binding affinity of the CD47 binding domain, the PD-L1 binding domain, and / or the TGFβ binding domain. Any combination of deletions, insertions, and / or combinations can be performed to obtain variants with increased binding affinity for the target. Amino acid changes introduced into the variant can also change the polypeptide or introduce new post-translational modifications into the polypeptide, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in the cell) or introducing new glycosylation sites.

[0199] The CD47 binding domain, PD-L1 binding domain and / or TGFβ binding domain can be derived from any animal species, including mammals. Non-limiting examples of binding domain variants include sequences derived from humans, primates (e.g., monkeys and apes), cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits).

[0200] 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., so that the host cells contain the polynucleotides and / or vectors comprising the polynucleotides), and production of the recombinant polypeptides or fragments thereof by recombinant techniques.

[0201] As used herein, "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. "Expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector has been introduced. Therefore, in an expression vector, the polynucleotide of interest is positioned to be expressed in the vector by: being operably connected to regulatory elements such as promoters, enhancers and / or poly-A tails in the vector or in the genome of the host cell located at or near the integration site of the polynucleotide of interest or flanking the integration site, so that the polynucleotide of interest is translated in the host cell introduced with the expression vector.

[0202] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., using a recombinant virus). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with a cationic condensing agent.

[0203] In some embodiments, polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of non-pathogenic (defective), replication-capable viruses, or replication-defective viruses may be used. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. DNA may also be "naked." The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into cells.

[0204] For expression, a DNA insert comprising a polynucleotide encoding a polypeptide disclosed herein can be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as the bacteriophage λ PL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled person. 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 transcript expressed by the construct may include a translation starting at the beginning and a stop codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0205] As indicated, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase resistance or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include but are not limited to bacterial cells, such as Escherichia coli, Streptomyces (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium) cells; fungal cells, such as yeast cells; insect cells, such as fruit fly S2 and noctuid Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma and HK 293 cells; and plant cells. Suitable culture media and conditions for host cells described herein are known in the art.

[0206] Non-limiting vectors for use in bacteria include: pQE70, pQE60, and pQE-9, which are available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, which are available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, which are available from Pharmacia. Non-limiting eukaryotic vectors include: pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, which are available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL, which are available from Pharmacia. Other suitable vectors will be apparent to the skilled artisan.

[0207] Non-limiting bacterial promoters suitable for use include E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters, and trp promoter. Suitable eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, promoters of retroviral LTRs, such as the promoter of Rous sarcoma virus (RSV), and metallothionein promoters, such as mouse metallothionein-I promoter.

[0208] In the yeast Saccharomyces cerevisiae, a number of vectors are available containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.

[0209] The introduction of constructs into host cells may be effected 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.

[0210] Transcription of the DNA encoding the polypeptide of the present disclosure by higher eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, usually about 10 to 300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located on the rear side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the rear side of the replication origin, and adenovirus enhancers.

[0211] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signal may be endogenous to the polypeptide, or it may also be a heterologous signal.

[0212] The polypeptide can be expressed in a modified form, such as a fusion protein (e.g., GST-fusion) or with a histidine tag, and can contain not only a secretion signal, but also additional heterologous functional regions. For example, additional amino acids, particularly regions of charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or during subsequent processing and storage. Similarly, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. Adding peptide moieties to polypeptides to cause secretion or excretion, improve stability and facilitate purification (among others) is a well-known and conventional technique in the art.

[0213] Treatment

[0214] The protein constructs or polypeptides of the present disclosure can be used for various therapeutic purposes.

[0215] In one aspect, the present disclosure provides a method for treating cancer in a subject, a method for reducing the rate at which the volume of a tumor in a subject increases over time, a method for reducing the risk of metastasis, or a method for reducing the risk of additional metastasis in a subject. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can reduce the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0216] In one aspect, the disclosure features methods comprising administering a therapeutically effective amount of a protein construct or polypeptide disclosed herein to a subject in need thereof (e.g., a subject suffering from cancer or identified or diagnosed as suffering from cancer), wherein the cancer is, for example, breast cancer (e.g., triple-negative breast cancer), carcinoid, 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, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancies. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject suffers from 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 cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, a hematological malignancy, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or an advanced solid tumor.

[0217] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for developing cancer. Patients with cancer can be identified using various methods known in the art.

[0218] As used herein, "effective amount" means an amount or dosage sufficient to produce a beneficial or desired result, including stopping, slowing, delaying or inhibiting the progression of a disease, e.g., cancer. The effective amount will vary depending on, for example, the age and weight of the subject to whom the protein construct or polypeptide, vector comprising a polynucleotide encoding the protein construct or polypeptide, and / or a composition thereof is to be administered, the severity of the symptoms, and the route of administration, and thus administration can be determined on an individual basis.

[0219] An effective amount can be administered in one or more administrations. For example, an effective amount of a protein construct or polypeptide is an amount sufficient to improve, prevent, stabilize, reverse, inhibit, slow down and / or delay the progression of a patient's cancer in vitro, or an amount sufficient to improve, stop, stabilize, reverse, slow down and / or delay the proliferation of cells (e.g., biopsy cells, any cancer cell or cell line (e.g., cancer cell line) described herein) in vitro. As is known in the art, an effective amount can vary, particularly depending on the patient's medical history and other factors, such as the type (and / or dosage) of the protein construct or polypeptide used.

[0220] The effective amount and schedule for administering the protein constructs or polypeptides, polynucleotides encoding protein constructs or polypeptides, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. It will be appreciated by those skilled in the art that the dosage that must be administered will vary depending on, for example, the mammal to which the protein constructs or polypeptides, polynucleotides and / or compositions disclosed herein will be administered, the route of administration, the specific type of polynucleotides and / or compositions disclosed herein used, and other drugs administered to the mammal.

[0221] The typical daily dosage of an effective amount of a protein construct and / or polypeptide is 0.1 mg / kg to 100 mg / kg (mg / kg patient body 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, or 1 mg / kg. In some embodiments, the dosage is about 1 mg / kg to 10 mg / kg, about 1 mg / kg to 5 mg / kg, or about 2 mg / kg to 5 mg / kg.

[0222] In any of the methods described herein, the protein construct or polypeptide can be administered to the 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).

[0223] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of a protein construct or polypeptide. In some embodiments, the one or more additional therapeutic agents are administered to a subject such that there is an overlap in the biologically active time period of the one or more additional therapeutic agents and the protein construct or polypeptide in the subject.

[0224] In certain embodiments, one or more additional therapeutic agents may be administered to a subject. 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), inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or inhibitors of isocitrate dehydrogenase 2 (IDH2). In certain embodiments, additional therapeutic agents are inhibitors of indoleamine 2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).

[0225] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, activated hedgehog signaling pathway inhibitors, and agents that selectively degrade estrogen receptors.

[0226] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, ib), Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, Hsp90 inhibitors, Ad-GM-CSF, Temozolomide, IL-2, IFNa, Vinblastine, Thalomid, dacarbazine, Cyclophosphamide, Lenalidomide, Azacytidine, Lenalidomide, Bortezomid, Amrubicin, Carfilzomib, Pralatrexate, and Enzastaurin.

[0227] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapeutic targeting CX3CL1, therapeutic targeting CXCL9, therapeutic targeting CXCL10, therapeutic targeting CCL5, LFA-1 agonists, ICAM1 agonists and selectin agonists.

[0228] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0229] 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).

[0230] Pharmaceutical compositions and routes of administration

[0231] Also provided herein are pharmaceutical compositions containing the protein constructs or polypeptides described herein.The pharmaceutical compositions can be formulated in any manner known in the art.

[0232] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvent, an antibacterial or antifungal agent, such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate or phosphate; and an isotonic agent, such as a sugar (e.g., dextrose), a polyol (e.g., mannitol or sorbitol) or a salt (e.g., sodium chloride) or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. The preparation of the composition may be formulated and packaged in an ampoule, a disposable syringe or a multiple dose vial. Where necessary (e.g., in injectable formulations), appropriate fluidity can be maintained, for example, by using a coating such as lecithin or a surfactant. Absorption of the agent can be extended by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems that can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0233] Compositions containing the protein constructs or polypeptides described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing predetermined quantities of active compound for ease of administration and uniformity of dosage).

[0234] The pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic, and is manufactured under good manufacturing practice (GMP) conditions. The pharmaceutical composition can be provided in a unit dosage form (i.e., a single dose). The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution can contain a formulation agent, such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, the protein construct or polypeptide can be in a lyophilized form for use in conjunction with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use.

[0235] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell culture or experimental animals (e.g., monkeys). For example, the LD50 (the dose that is lethal to 50% of the population) and the ED50 (the dose that is therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indexes are preferred. In the case where the agent exhibits undesirable side effects, care should be taken to minimize potential damage (i.e., reduce adverse side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0236] Exemplary dosages include milligram or microgram amounts of any of the protein constructs or polypeptides described herein per kilogram of body weight of the subject (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). Although these dosages cover a wide range, it will be appreciated by one of ordinary skill in the art that the efficacy of the therapeutic agent can vary, and that an effective amount can be determined by methods known in the art. Typically, a relatively low dose is initially administered, and the attending health care professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the research and development phase) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it should be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, the age, weight, general health, sex and diet of the subject, time of administration, route of administration, excretion rate and half-life.

[0237] The pharmaceutical composition may be contained in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods of making protein constructs or polypeptides for the various uses described herein.

[0238] Examples

[0239] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0240] Example 1. Design of Fc-based Designer Biologics using the anti-PD-L1×SIRPα×TGFβtrap format (FBDB TM )

[0241] Developed and optimized various FBDB TM The triple targeting format is designed to achieve specific functional effects as follows: (1) Directing therapeutic agents to PD-L1 + tumor cells; (2) enhance the synergy between innate immunity (e.g., targeting the SIRPα / CD47 pathway) and adaptive immunity (e.g., targeting the PD-1 / PD-L1 pathway); (3) reverse TGFβ-induced immunosuppression.

[0242] Each format contains at least three different types of immune modules, which are directly or indirectly connected to the Fc region of IgG (e.g., human IgG4 or human IgG1). The three immune modules are: (1) one or more SIRPα extracellular domains, which can stimulate antigen presentation by inducing phagocytosis; (2) one or more anti-PD-L1 extracellular domains, which can block the PD-1 / PD-L1 pathway to enhance T cell function and guide SIRPα×PD-1×TGFβ-trap molecules to tumors expressing PD-L1; and (3) one or more TGFβ-trap molecules (e.g., the extracellular domain of TGF-β receptor type 2 (TGFBRII)), which can capture immunosuppressive TGFβ and improve the tumor microenvironment to enhance the immune response. For example, the SIRPα extracellular domain disrupts the interaction between CD47 expressed on tumor cells and SIRPα present on macrophages, thereby releasing inhibitory signals and enabling macrophages to overcome the “don’t eat me” mechanism; the anti-PD-L1 extracellular domain disrupts the interaction between PD-1 expressed on T cells and PD-L1 present on tumor cells, thereby releasing the inhibitory brake on effector T cells and promoting T cell function; and the TGFβ-trap molecule can counteract TGFβ-induced immunosuppression, thereby improving the tumor microenvironment (TME) to enhance the immune response.

[0243] Three types of FBDB are designed TM The triple targeting format, such as Figures 1A-1C As shown in FIG. SPT_G4_v5 (schematic structure shown in FIG. 1A ) comprises two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 1. Specifically, each polypeptide chain comprises, from N-terminus to C-terminus, a SIRPα extracellular domain SIRPα_mt10 (SEQ ID NO: 7), an anti-PD-L1 VHH (2H4) (SEQ ID NO: 8), a hinge region containing a human IgG4 Fc region (S228P) (SEQ ID NO: 10), and a mutant TGFBRII extracellular domain TGFβRⅡ_mt4 (SEQ ID NO: 9). SIRPα_mt10 is connected to the N-terminus of anti-PD-L1 VHH (2H4) via a (GSG)6 linker peptide (SEQ ID NO: 12). TGFβRⅡ_mt4 is connected to the C-terminus of human IgG4 Fc via a (G4S)4G linker peptide (SEQ ID NO: 13).

[0244] SPT_G1_v5 (schematic structure shown in FIG. 1B ) comprises two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 2. Specifically, each polypeptide chain comprises, from N-terminus to C-terminus, SIRPα extracellular domain SIRPα_mt10 (SEQ ID NO: 7), anti-PD-L1 VHH (2H4) (SEQ ID NO: 8), a hinge region containing human IgG1 Fc region (SEQ ID NO: 11), and TGFBRII extracellular domain TGFβRⅡ_mt4 (SEQ ID NO: 9). SIRPα_mt10 and anti-PD-L1 VHH (2H4) are connected by a (GSG) 6 linker peptide (SEQ ID NO: 12). TGFβRⅡ_mt4 is connected to the C-terminus of human IgG1 Fc by a (G4S) 4G linker peptide (SEQ ID NO: 13).

[0245] PST_G1_v5 (schematic structure shown in FIG. 1C ) comprises two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 3. Specifically, each polypeptide chain comprises anti-PD-L1 VHH (2H4) (SEQ ID NO: 8), SIRPα extracellular domain SIRPα_mt10 (SEQ ID NO: 7), a hinge region containing human IgG1 Fc region (SEQ ID NO: 11), and TGFBRII extracellular domain TGFβRⅡ_mt4 (SEQ ID NO: 9) from N-terminus to C-terminus. Anti-PD-L1 VHH (2H4) is connected to the N-terminus of SIRPα_mt10 via a (GSG)6 linker peptide (SEQ ID NO: 12). TGFβRⅡ_mt4 is connected to the C-terminus of human IgG1 Fc via a (G4S)4G linker peptide (SEQ ID NO: 13).

[0246] Three types of FBDB are designed TM Dual targeting formats such as Figures 1D-1F As shown in . SP_G4_v5 (schematic structure is shown in FIG. 1D ) comprises two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 4. Specifically, each polypeptide chain comprises a SIRPα extracellular domain SIRPα_mt10 (SEQ ID NO: 7), an anti-PD-L1 VHH (2H4) (SEQ ID NO: 8), and a hinge region containing a human IgG4 Fc region (S228P) (SEQ ID NO: 10) from the N-terminus to the C-terminus. SIRPα_mt10 is connected to the N-terminus of the anti-PD-L1 VHH (2H4) via a (GSG)6 linker peptide (SEQ ID NO: 12).

[0247] SP_G1_v5 (schematic structure shown in FIG. 1E ) comprises two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 5. Specifically, each polypeptide chain comprises the SIRPα extracellular domain SIRPα_mt10 (SEQ ID NO: 7), anti-PD-L1 VHH (2H4) (SEQ ID NO: 8), and a hinge region containing a human IgG1 Fc region (SEQ ID NO: 11) from the N-terminus to the C-terminus. SIRPα_mt10 and anti-PD-L1 VHH (2H4) are connected by a (GSG)6 linker peptide (SEQ ID NO: 12).

[0248] PS_G1_v5 (schematic structure shown in FIG. 1F ) comprises two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 6. Specifically, each polypeptide chain comprises anti-PD-L1 VHH (2H4) (SEQ ID NO: 8), SIRPα extracellular domain SIRPα_mt10 (SEQ ID NO: 7) and a hinge region containing a human IgG1 Fc region (SEQ ID NO: 11) from the N-terminus to the C-terminus. Anti-PD-L1 VHH (2H4) is connected to the N-terminus of SIRPα_mt10 via a (GSG)6 linker peptide (SEQ ID NO: 12).

[0249] The expressed proteins were passed through a protein A column and then purified by HPLC-SEC (high performance liquid chromatography coupled with size exclusion chromatography; Agilent). Specifically, six HCB301_v5 proteins were expressed in CHO-S cells. The culture supernatant was collected and subjected to protein A purification. Next, the protein A column was equilibrated with 10× column volumes of equilibration buffer (25 mM Tris, 150 mM NaCl, pH 8.0), and the culture supernatant was then loaded onto the equilibrated protein A column. The column was then washed with 6× column volumes of equilibration buffer (25 mM Tris, 150 mM NaCl, pH 8.0). The protein sample was eluted with 6× column volumes of elution buffer (100 mM acetate, 20 mM NaCl, pH 3.0) and washed with 1 M HEPES, pH 7.0. The pH was adjusted to 6.5-7 with a buffer of 8.0. The results showed that all six HCB301_v5 proteins (SPT_G4_v5, SPT_G1_v5, PST_G1_v5, SP_G4_v5, SP_G1_v5, and PS_G1_v5) could be expressed and collected with high purity.

[0250] In addition, the amino acid sequences of SEQ ID NO: 1-6 were analyzed using the Deimmunization Tool (Immune Epitope Database and Analysis Resource; 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.

[0251] Example 2. Determination of whole cell binding capacity to CD47-transfected (tf) CHO-S cells

[0252] In order to determine the whole-cell binding ability of HCB301_v5 protein to CD47 expressed on the cell surface, tf CHO-S cells expressing human CD47 (CD47 tf CHO-S) were used as target cells. 5×10 4 Individual cells were incubated with HCB301_v5 proteins serially diluted at specified concentrations (0.2pM, 1.91pM, 15.3pM, 122.1pM, 1.0nM, 7.8nM, 62.5nM and 500nM) in FACS buffer (phosphate buffered saline (PBS) supplemented with 4% fetal bovine serum (FBS)) at 4°C for 30 minutes. After incubation, the cells were washed twice with FACS buffer and then incubated with R-phycoerythrin-AffiniPure goat anti-human IgG (Jackson ImmunoResearch, catalog number: 109-115-098) at 4°C for 30 minutes. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc., CA, USA). Hu5F9-G4 was used as a positive control. Anti-PD-L1 VHH (2H4) was used as a negative control.

[0253] like Figure 2 As shown in , all six HCB301_v5 proteins can bind to CD47 tf CHO-S cells. EC50 values ​​were determined based on the binding curves. Specifically, SPT_G1_v5 only showed comparable binding efficacy at high concentrations, while at low concentrations, it showed reduced effectiveness. In contrast, for binding to CD47 tf CHO-S cells, the other five HCB301_v5 proteins showed comparable effectiveness to the positive control Hu5F9-G4 (anti-47 reference antibody) over the entire concentration range. In contrast, no binding signal was detected for the negative control anti-PD-L1 VHH (2H4).

[0254] Example 3. Determination of whole cell binding capacity to PD-L1 tf CHO-S cells

[0255] In order to determine the whole-cell binding ability of HCB301_v5 protein to PD-L1 expressed on the cell surface, tf CHO-S cells expressing human PD-L1 (PD-L1 tf CHO-S) were used as target cells. 4The cells were incubated with HCB301_v5 proteins serially diluted at specified concentrations (0.2pM, 1.91pM, 15.3pM, 122.1pM, 1.0nM, 7.8nM, 62.5nM, and 500nM) in FACS buffer (phosphate-buffered saline (PBS) supplemented with 4% fetal bovine serum (FBS)) at 4°C for 30 minutes. After incubation, the cells were washed twice with FACS buffer and then incubated with R-phycoerythrin-AffiniPure goat anti-human IgG (Jackson ImmunoResearch Institute Laboratory, catalog number: 109-115-098) at 4°C for 30 minutes. The samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, California, USA). Anti-PD-L1VHH (2H4) was used as a positive control. Hu5F9-G4 was used as a negative control.

[0256] like Figure 3 As shown in , all six HCB301_v5 proteins can bind to PD-L1 tf CHO-S cells. EC50 values ​​were also determined based on the binding curves. Similarly, for binding to CD47 tf CHO-S cells, SPT_G1_v5 only showed comparable binding efficacy at high concentrations, while the other five HCB301_v5 proteins showed comparable effectiveness to the positive control anti-PD-L1 VHH (2H4) over the entire concentration range. In contrast, no binding signal was detected for the negative control Hu5F9-G4 (anti-47 reference antibody).

[0257] Example 4. Determination of the binding capacity of HCB301_v5 to plate-bound human TGFβ protein

[0258] To determine the binding ability of HCB301_v5 protein to human TGFβ, a binding titration ELISA assay was performed using HCB301_v5 protein at a specified concentration (6.4pM, 32.0pM, 160.0pM, 0.8nM, 4.0nM, 20nM, 100nM or 500nM). G4_TGFβRII_mt4 (SEQ ID NO: 21) was used as a positive control. HCB101 (SEQ ID NO: 20) was used as a negative control. At 4°C, 96-well EIA microplates were coated with 0.5μg / ml human TGFβ (TGFβ1, TGFβ2 or TGFβ3) overnight. After blocking with 1×PBS containing 5% skim milk, diluted HCB301_v5 protein was added and incubated at 24°C for 1 hour. Unbound proteins were removed by washing the wells three times with 1×PBST (1×PBS containing 0.1% Tween 20). HRP-conjugated secondary antibodies (1:5000) were added to the wells at 24°C for 1 hour. After incubation, excess secondary antibodies were removed by washing the wells three times with 1×PBST. Finally, 3,3',5,5'-tetramethylbenzidine (TMB) was added for color development. The reaction was stopped and HRP activity was measured at 450nm using a spectrophotometer.

[0259] like Figures 4A-4C As shown in Figure 2, three triple targeting formats of HCB301_v5 protein, SPT_G4_v5, SPT_G1_v5, and PST_G1_v5, showed strong affinity with hTGFβ1 ( Figure 4A ) and hTGFβ3( Figure 4C ) but hardly binds to hTGFβ2 ( Figure 4B ). In contrast, three dual targeting formats of HCB301_v5 protein without the TGFβRⅡ_mt4 extracellular domain, SP_G4_v5, SP_G1_v5, PS_G1_v5, and the negative control did not bind to hTGFβ1, hTGFβ2, or hTGFβ3.

[0260] Example 5. Determination of whole cell binding capacity to CD47 expressing tumor cells

[0261] To determine the whole-cell binding ability of HCB301_v5 protein to CD47 and PD-L1 expressed on the surface of tumor cells, hypopharyngeal carcinoma FaDu cells expressing endogenous CD47 and non-small cell lung cancer NCI-H460 were used as target cells. 4Tumor cells were incubated with HCB301_v5 proteins serially diluted at specified concentrations (0.2pM, 1.91pM, 15.3pM, 122.1pM, 1.0nM, 7.8nM, 62.5nM and 500nM) in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. After incubation, the cells were washed twice with FACS buffer and then incubated with R-phycoerythrin-AffiniPure goat anti-human IgG (Jackson ImmunoResearch Institute Laboratories, catalog number: 109-115-098) at 4°C for 30 minutes. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, California, USA). HCB101 was used as a positive control. G4_TGFβRII_mt4 was used as a negative control.

[0262] like Figure 5A-5B As shown in Figure 2, the three triple targeting formats of HCB301_v5 protein, SPT_G4_v5, SPT_G1_v5, and PST_G1_v5, can bind to FaDu cells as effectively as the positive control HCB101 ( Figure 5A ) and NCI-H460 cells ( Figure 5B ) In contrast, no binding signal was detected for the negative control G4_TGFβRII_mt4.

[0263] Example 6. Selective binding to cells expressing PD-L1

[0264] The ability of HCB301_v5 protein to selectively bind to cells expressing PD-L1 was determined as follows. DLD1 cells were cultured with CellTrace TM CFSE (Thermo, catalog number: C34554) was used to label the PD-L1-expressing tf DLD1 cells (PD-L1 tf DLD1) and the cells were analyzed by Celltrace TM (Thermo Fisher Scientific, catalog number: C34557). 5×10 4 CellTrace for cells / well TM CFSE-labeled DLD1 cells and 5 × 10 4 CellTrace for cells / well TMViolet-labeled PD-L1 tf DLD1 cells were incubated with HCB301_v5 proteins serially diluted at specified concentrations (0.2pM, 1.91pM, 15.3pM, 122.1pM, 1.0nM, 7.8nM, 62.5nM and 500nM) in FACS buffer (PBS supplemented with 4% FBS) at 4 ° C for 30 minutes. After incubation, the cells were washed twice with FACS buffer and then incubated with R-phycoerythrin-AffiniPure goat anti-human IgG (Jackson ImmunoResearch Institute Laboratory, catalog number: 109-115-098) at 4 ° C for 30 minutes. Samples were analyzed using CytoFLEX flow cytometer (Beckman Coulter, California, USA). Afterwards, cells were gated according to PE signals to calculate the percentage of different cell types by Kaluza analysis software (Beckman Coulter). Hu5F9-G4, HCB101, Atezolizumab, and anti-PD-L1 VHH (2H4) were used as positive controls and G4_TGFβRII_mt4 was used as a negative control.

[0265] As shown in Figures 6F-6K, all six HCB301_v5 proteins preferentially bound to PD-L1 tf DLD1 cells in a mixture of PD-L1 tf DLD1 cells and DLD1 cells (ratio 1:1). This selective binding to PD-L1 tf DLD1 cells was also evident in the two positive controls, atezolizumab and anti-PD-L1 VHH (2H4) ( Figure 6C-6D ). In contrast, the other two positive controls, Hu5F9-G4 and HCB101 ( Figure 6A-6B ) showed similar binding effectiveness between PD-L1 tf DLD1 cells and DLD1 cells. No binding signal was detected in the negative control G4_TGFβRII_mt4 ( Fig. 6E ).

[0266] Example 7. Determination of hemagglutination (HA) activity

[0267] To determine the HA activity induced by HCB301_v5, RBCs were prepared from whole blood of healthy donors by washing twice with 0.9% sodium chloride buffer and then resuspending the RBCs in 0.9% sodium chloride buffer. HCB301_v5 protein was serially diluted (3-fold) to a final concentration of 500 nM, 166.7 nM, 55.6 nM, 18.5 nM, 6.2 nM, 2.1 nM, 685.8 pM, 228.6 pM, 76.2 pM, 25.4 pM or 8.4 pM and incubated in a round-bottom 96-well plate with 8×10 6The solution of RBCs was incubated overnight at room temperature (RT). After incubation, the agglutinated RBCs uniformly coated the wells, while the non-agglutinated cells formed obvious red spots at the bottom of the wells. Hu5F9-G4 was used as a positive control. h1G4 (heavy chain: SEQ ID NO: 36; light chain: SEQ ID NO: 37) was used as a negative control. The sequence of G1_TGFβRII_mt4 is shown in SEQ ID NO: 22.

[0268] After overnight incubation, images of the plates were captured the next day, e.g. Figure 7 The images show that only Hu5F9-G4 induced HA activity at high concentrations, while the six HCB301_v5 proteins did not induce HA activity in the entire concentration range mentioned above.

[0269] Example 8. Determination of whole cell binding capacity to platelets and subsequent induction of phagocytosis of platelets by mouse macrophages

[0270] In order to determine the binding ability of HCB301_v5 protein to platelets, 5×10 5 Individual platelets were incubated with HCB301_v5 proteins serially diluted at designated concentrations (0.07pM, 0.4pM, 2.6pM, 15.4pM, 92.5pM, 0.6nM, 3.3nM, and 20.0nM) in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. After incubation, platelets were washed twice with FACS buffer and then incubated with R-phycoerythrin-AffiniPure goat anti-human IgG (Jackson ImmunoResearch Laboratories, catalog number: 109-115-098) at 4°C for 30 minutes. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, California, USA). Hu5F9-G4 was used as a positive control. h1G4 was used as a negative control.

[0271] To determine the macrophage-mediated phagocytosis induced by HCB301_v5 protein on platelets, a phagocytosis assay was performed as follows. Platelets were treated with 5 nM CellTrace TM CFSE (Thermo Fisher Scientific, catalog number: C34554) was labeled at 37°C for 10 minutes and then washed with complete DMEM medium. 5×10 5 CFSE-labeled platelet cells (target cells) at 10 cells / well were incubated with serially diluted HCB301_v5 proteins at designated concentrations (0.5 pM, 5.0 pM, 50.0 pM, 0.5 nM, 5.0 nM, 50.0 nM, and 500.0 nM) in a low binding 96-well U-bottom plate at 37°C for 30 minutes. 4Raw264.7 mouse microphages were added to each well, and the plates were incubated at 37°C for 2 hours. Raw264.7 cells were stained with PE-cyanine 7 conjugated F4 / 80 antibody (eBioscience, catalog number: 25-4801-82). The phagocytic ability of HCB301_v5 protein was evaluated by calculating the percentage of CFSE+F4 / 80+ (indicating that macrophages engulfed CFSE-labeled Jurkat cells) from macrophages by CytoFlex flow cytometer (Beckman Coulter). Hu5F9-G4 was used as a positive control. h1G4 was used as a negative control.

[0272] like Fig. 8A As shown in , all six HCB301_v5 proteins, like the positive control Hu5F9-G4, showed strong binding ability to platelets. Figure 8B It shows that SPT_G4_v5 is less likely to trigger phagocytosis of platelets by mouse macrophage Raw264.7 at lower concentrations. In contrast, the other five HCB301_v5 proteins and the positive control Hu5F9-G4 induced significant phagocytosis when bound to platelets.

[0273] Example 9. Determination of whole cell binding capacity to RBCs and subsequent induction of phagocytosis of RBCs by human macrophages

[0274] In order to determine the binding ability of SPT_G4_v5 protein to RBC, 5×10 5 Individual RBCs were incubated with SPT_G4_v5 proteins serially diluted at specified concentrations (0.3pM, 1.3pM, 6.4pM, 32.0pM, 160.0pM, 0.8nM, 4.0nM, 20nM, 100.0nM, and 500.0nM) in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. After incubation, RBCs were washed twice with FACS buffer and then incubated with R-phycoerythrin-AffiniPure goat anti-human IgG (Jackson ImmunoResearch Institute Laboratories, catalog number: 109-115-098) at 4°C for 30 minutes. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, California, USA). Hu5F9-G4 was used as a positive control. Anti-PD-L1 VHH (2H4) was used as a negative control.

[0275] To determine whether SPT_G4_v5 protein induces macrophage-mediated phagocytosis of RBCs by human MDM cells, a phagocytosis assay was performed as follows. RBCs were stained with 5 nM CellTrace TMCFSE (Thermo Fisher Scientific, catalog number: C34554) was labeled at 37°C for 10 minutes and washed with complete RPMI-1640 medium. 5 CFSE-labeled RBCs (target cells) at 10 cells / well were incubated with SPT_G4_v5 proteins serially diluted at the indicated concentrations (2.0 pM, 20.0 pM, 0.2 nM, 2.0 nM, and 20.0 nM) in a low binding 96-well U-bottom plate at 37°C for 30 min. 4 Individual MDM cells were added to each well, and the plate was incubated at 37°C for 2 hours. Human MDM cells were stained with PE-cyanine 7-conjugated CD14 antibody (e Biosciences, catalog number: 25-0149-42). The phagocytic ability of SPT_G4_v5 protein was evaluated by calculating the percentage of CFSE+CD14+ (indicating that macrophages engulfed CFSE-labeled RBCs) from macrophages by CytoFlex flow cytometer (Beckman Coulter). Hu5F9-G4 was used as a positive control. h1G4 was used as a negative control.

[0276] like Figures 9A-9B As shown in , the positive control Hu5F9-G4 exhibited strong binding ability to human RBC ( Fig. 9A ), and this leads to phagocytosis of human RBCs by human MDM cells ( Fig. 9B ). In contrast, SPT_G4_v5 showed low binding affinity to human RBCs, and this binding did not result in phagocytosis.

[0277] Example 10. Determination of the blocking effect of the interaction between SIRPα and CD47

[0278] To determine the SIRPα ligand blocking ability of HCB301_v5 protein, a flow cytometry-based assay was performed using CD47 tf CHO-S cells as target cells. 4CD47 tf CHO-S cells of 1 cell / well were incubated with HCB301_v5 protein serially diluted at designated concentrations (0.2pM, 1.9pM, 15.2pM, 122.1pM, 976.6pM, 7.8nM, 62.5nM and 500nM) and biotinylated SIRPα_G4 ("biotin SIRP Fc"; SEQ ID NO: 23) at a fixed concentration in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. After washing, streptavidin-PE (e Biosciences, catalog number: 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). Hu5F9-G4 and HCB101 were used as positive controls. Anti-PD-L1 VHH (2H4) was used as a negative control.

[0279] like Fig.10 As shown in , all six HCB301_v5 proteins as well as the positive controls Hu5F9-G4 and HCB101 exhibited comparable blocking effects on the interaction between SIRPα and CD47. In contrast, the interaction between SIRPα and CD47 was not interfered with by the negative control anti-PD-L1 VHH (2H4).

[0280] Example 11. Induction of phagocytosis of PD-L1 TF tumor cells by human MDM cells

[0281] To determine whether HCB301_v5 protein induces macrophage-mediated phagocytosis of PD-L1 tf cancer cells by human MDM cells, a phagocytosis assay was performed as follows. PD-L1 tf OVA-GFP DLD1 cells were treated with 5 nM CellTrace TM CFSE (Thermo Fisher Scientific, catalog number: C34554) was labeled at 37°C for 10 minutes and then washed with complete RPMI-1640 medium. 4 CFSE-labeled PD-L1 tf OVA-GFP DLD1 cells (target cells) were incubated with serially diluted HCB301_v5 proteins at the indicated concentrations (5 pM, 50.0 pM, 0.5 nM, 5.0 nM, 50.0 nM, and 500.0 nM) in a low binding 96-well U-bottom plate at 37°C for 30 min. 4Individual MDM cells were added to each well, and the plate was incubated at 37°C for 2 hours. Human MDM cells were stained with PE-cyanine 7-conjugated CD14 antibodies (e Biosciences, catalog number: 25-0149-42). The CFSE+CD14+ from macrophages (indicating that macrophages engulfed CFSE-labeled PD-L1 tf OVA-GFP DLD1 cells) was calculated by CytoFlex flow cytometer (Beckman Coulter) as a percentage of the total CD14 signal from macrophages to assess the phagocytic ability of HCB301_v5 protein. Hu5F9-G4 and HCB101 were used as positive controls. h1G4 was used as a negative control.

[0282] like Fig.11 As shown in , all six HCB301_v5 proteins triggered stronger phagocytic activity of human MDM cells against PD-L1 tf OVA-GFP DLD1 cells compared to the positive controls Hu5F9-G4 and HCB101. In contrast, the negative control h1G4 did not induce phagocytosis at the tested concentrations.

[0283] Example 12. Determination of the blocking effect of the interaction between PD-1 and PD-L1

[0284] The blocking effect of HCB301_v5 protein on the interaction between PD-1 and PD-L1 tf CHO-S cells was determined as follows. 3×10 4 PD-L1 tf CHO-S cells of cells / well were incubated with HCB301_v5 proteins serially diluted at designated concentrations (0.2pM, 1.9pM, 15.2pM, 122.1pM, 976.6pM, 7.8nM, 62.5nM and 500nM) and fixed concentrations of biotinylated PD1_G4 ("biotin PD1 Fc"; SEQ ID NO: 34) in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. After washing, streptavidin-PE (e Biosciences, catalog number: 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). Atezolizumab (anti-PD-L1 antibody) and anti-PD-L1 VHH (2H4) were used as positive controls. HCB101 was used as a negative control.

[0285] like Fig.12As shown in, all six HCB301_v5 proteins as well as the positive control atezolizumab (anti-PD-L1 antibody) and anti-PD-L1 VHH (2H4) showed similar blocking effects on the interaction between PD-1 and PD-L1. In contrast, the interaction between PD-1 and PD-L1 was not interfered by the negative control HCB101.

[0286] Example 13. Inhibition of TGFβ-induced smad2 reporter gene activity

[0287] The ability of HCB301_v5 protein to inhibit smad2 reporter gene activity induced by TGFβ1, TGFβ2 or TGFβ3 was determined as follows. HCB301_v5 protein was serially diluted (5 times) to a final concentration of 6.4pM, 32.0pM, 160.0pM, 0.8nM, 4.0nM, 20.0nM, 100.0nM and 500.0nM. G4_TGFBRII_mt4 was used as a positive control, and anti-PD-L1 VHH (2H4) was used as a negative control. 4×10 3 HEK293T cells expressing the smad2 reporter gene were incubated with diluted HCB301_v5 protein and 20 ng / ml TGFβ1, TGFβ2 and TGFβ3. After incubation at 37°C in a 5% CO2 incubator for 24 hours, the cells were analyzed by Varioskan TM The luminescent signal was detected by LUX multi-mode microplate reader (Thermo Scientific).

[0288] like Figures 13A-13C As shown in Figure 3, three triple targeting formats of HCB301_v5 protein, SPT_G4_v5, SPT_G1_v5 and PST_G1_v5, as well as the positive control G4_TGFBRII_mt4, can inhibit TGFβ1-( Fig.13A ) or TGFβ3-( Fig. 13B )-mediated smad2 reporter gene activity. In contrast, TGFβ2-( Fig. 13B )-mediated smad2 reporter gene activity was not interfered with by any of the test proteins.

[0289] Example 14. Assessment of enhancement of T cell activation by MLR assay

[0290] Mixed lymphocyte reaction (MLR) assays were performed in the presence of TGFβ1 to determine the enhancement of T cell responses by HCB301_v5 protein. 5 CD4 + T cells and 1×10 4Dendritic cells (DC) were incubated with 0.1nM, 1.0nM, 10.0nM or 100.0nM HCB301_v5 proteins. Control molecules or their combinations were also used for incubation, for example, PDGFR-Fc, Atezolizumab, anti-PD-L1 VHH (2H4), HCB101 and G4_TGFBRII_mt4. PDGFR-Fc (SEQ ID NO: 35) was used as a negative control. Atezolizumab was used as a positive control. Conditioned medium containing TGFβ1 from NCl-H650 cells was then added and incubated for 5 days. After incubation, culture supernatants were collected and IL-2 secretion was determined using human IL-2ELISA MAXDeluxe kit (BioLegend, catalog number: 431805).

[0291] like Fig.14 As shown in, IL-2 secretion (indicating T cell activation) was determined by MLR assay using human IL-2 ELISA MAX Deluxe kit. The results showed that SPT_G4_v5 and SP_G4_v5 as well as the positive control atezolizumab can promote T cell activation in the presence of conditioned medium containing TGFβ1. In contrast, no increased IL-2 secretion was detected in the negative control PDGFR-Fc and the other four HCB301_v5 proteins.

[0292] Example 15. Determination of in vivo anti-tumor efficacy

[0293] FaDu cells (pharyngeal carcinoma) were used to determine the in vivo antitumor efficacy of SPT_G4_v5. 6 FaDu (pharyngeal cancer) and 1×10 6 PBMC blends were subcutaneously inoculated into immunodeficient NPG TM Mice. The same molar concentration of the test article was then administered to the mice by intraperitoneal injection on days 5, 8, 12, 15 and 19 after inoculation. An equal volume of 0.9% sodium chloride injection was administered to the control group of mice. The tumor volume of each group of mice was measured on days 5, 8, 12, 15 and 19 after inoculation. The average tumor volume of mice in each group on day 19 is shown in the table. Tumor growth inhibition (TGI) and the corresponding p value were also determined.

[0294] like Figures 15A-15B As shown in Figure , SPT_G4_v5 treatment suppressed the proliferation of NPG bearing FaDu (human pharyngeal carcinoma) / PBMC xenografts compared with HCB101, anti-PD-L1 VHH (2H4), G4_TGFβRII_mt4, atezolizumab, and pembrolizumab during a time course of 2.5 weeks after administration. TMTumor growth in mice. Specifically, mice treated with SPT_G4_v5 showed smaller tumors at day 19 after administration compared to mice treated with other antibodies or vehicles. TGI evaluation showed that SPT_G4_v5 had better anti-tumor activity by significantly inhibiting tumor growth 19 days after administration, as shown in the table below.

[0295] Table 1

[0296]

[0297] Other embodiments

[0298] It should be understood that although the invention has been described in conjunction with the specific description of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A protein complex comprising: (a) Fc; (b) CD47 binding domain; as well as (c) a PD-L1 (programmed death-ligand 1) binding domain, wherein the PD-L1 binding domain is a VHH (heavy chain single variable domain) that binds to PD-L1. 2 . The protein complex according to claim 1 , further comprising a TGFβ (transforming growth factor β) binding domain.

3. The protein complex according to claim 1 or 2, wherein the CD47 binding domain is capable of binding to cells expressing CD47 (e.g., cancer cells) and / or blocking the interaction between CD47 and signal regulatory protein α (SIRPα). 4 . The protein complex according to claim 1 , wherein the CD47 binding domain is or comprises a SIRPα extracellular domain.

5. The protein complex according to claim 4, wherein the SIRPα extracellular domain comprises a sequence that is at least 80% identical to SEQ ID NO:32, optionally wherein the SIRPα extracellular domain comprises one or more amino acid mutations at positions corresponding to H24, I31, E54, G55, H56 and / or E70 of SEQ ID NO:

32.

6. The protein complex according to claim 4 or 5, The SIRPα extracellular domain comprises one or more of the following: (a) the amino acid corresponding to H24 of SEQ ID NO:32 is T; (b) the amino acid corresponding to I31 of SEQ ID NO:32 is Y; (c) the amino acid corresponding to E54 of SEQ ID NO:32 is R; (d) the amino acid corresponding to G55 of SEQ ID NO:32 is Q; (e) the amino acid corresponding to H56 of SEQ ID NO:32 is T; and (f) the amino acid corresponding to E70 of SEQ ID NO:32 is F.

7. The protein complex of any one of claims 1 to 6, wherein 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:

7.

8. The protein complex according to any one of claims 1 to 7, wherein the VHH is capable of binding to cells expressing PD-L1 (e.g., cancer cells) and / or blocking the interaction between PD-L1 and programmed cell death protein 1 (PD-1).

9. The protein complex of any one of claims 1 to 8, wherein the VHH comprises complementarity determining regions (CDRs) 1, 2 and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence; The selected VHH CDR1, 2 and 3 amino acid sequences are one of the following: (1) the selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 26, 27 and 28, respectively; and (2) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 29, 30 and 31, respectively.

10. The protein complex of any one of claims 1 to 9, wherein the VHH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

8.

11. The protein complex according to any one of claims 2 to 10, wherein the TGFβ binding domain is capable of capturing TGFβ, thereby increasing the immune response and / or improving the tumor microenvironment.

12. The protein complex according to any one of claims 2 to 11, wherein the TGFβ binding domain is or comprises the TGFBRII extracellular domain.

13. The protein complex according to any one of claims 2 to 12, wherein the TGFBRII extracellular domain comprises a sequence at least 80% identical to SEQ ID NO: 33, optionally wherein the TGFBRII extracellular domain comprises one or more amino acid mutations at positions corresponding to S49 and / or D118 of SEQ ID NO:

33.

14. The protein complex according to any one of claims 2 to 13, Wherein the TGFβ binding domain comprises one or more of the following: (a) the amino acid corresponding to S49 of SEQ ID NO:33 is E; and (b) The amino acid corresponding to D118 of SEQ ID NO: 33 is E.

15. The protein complex of any one of claims 2 to 14, wherein the TGFβ 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:

9.

16. The protein complex according to any one of claims 1 to 15, wherein the CD47 binding domain is linked to the N-terminus of the CH2 domain in the Fc, optionally via a hinge region.

17. The protein complex according to claim 16, wherein the PD-L1 binding domain is linked to the N-terminus of the CD47 binding domain, optionally via a linker peptide.

18. The protein complex according to any one of claims 1 to 15, wherein the PD-L1 binding domain is linked to the N-terminus of the CH2 domain in the Fc, optionally through a hinge region.

19. The protein complex according to claim 18, wherein the CD47 binding domain is linked to the N-terminus of the PD-L1 binding domain, optionally via a linker peptide.

20. The protein complex according to any one of claims 1 to 19, wherein the Fc is human IgG1 Fc.

21. The protein complex according to any one of claims 1 to 19, wherein the Fc is human IgG4 Fc.

22. The protein complex according to any one of claims 16 to 19 and 21, wherein the hinge region is a human IgG4 hinge region, optionally with an S228P mutation according to EU numbering.

23. The protein complex according to any one of claims 2 to 22, wherein the TGFβ binding domain is linked to the C-terminus of the CH3 domain in the Fc, optionally via a linker peptide.

24. A protein complex comprising (a) a first polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus: a first CD47 binding domain, an optional first linker peptide, a first VHH that binds to PD-L1, an optional first hinge region, a first Fc region; as well as (b) a second polypeptide, which comprises, from N-terminus to C-terminus: a second CD47 binding domain, an optional second linker peptide, a second VHH binding to PD-L1, an optional second hinge region, and a second Fc region.

25. The protein complex of claim 24, wherein the first VHH and / or the second VHH comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO:

8.

26. The protein complex of claim 24 or 25, wherein 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:

7.

27. The protein complex according to any one of claims 24 to 26, wherein the first hinge region and / or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO:

14.

28. The protein complex according to any one of claims 24 to 27, wherein the first Fc region and / or the second Fc region comprises a sequence at least 80% identical to SEQ ID NO:

15.

29. The protein complex according to any one of claims 24 to 26, wherein the first hinge region and / or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO:

16.

30. The protein complex according to any one of claims 24 to 26 and 29, wherein the first Fc region and / or the second Fc region comprises a sequence at least 80% identical to SEQ ID NO:

17.

31. The protein complex according to any one of claims 24 to 30, wherein the first linker peptide and / or the second linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO:

12.

32. The protein complex of any one of claims 24 to 31, wherein 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 or 5.

33. The protein complex of any one of claims 24 to 32, wherein the first polypeptide further comprises a first TGFβ binding domain and the second polypeptide further comprises a second TGFβ binding domain.

34. The protein complex of claim 33, wherein 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:

9.

35. The protein complex according to claim 33 or 34, wherein the first TGFβ binding domain is linked to the C-terminus of the first Fc region, optionally via a third linker peptide, and wherein the second TGFβ binding domain is linked to the C-terminus of the second Fc region, optionally via a fourth linker peptide.

36. The protein complex according to claim 35, wherein the third linker peptide and / or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO:

13.

37. The protein complex of any one of claims 33 to 36, wherein 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 or 2.

38. A protein complex comprising (a) a first polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus: a first VHH that binds to PD-L1, an optional first linker peptide, a first CD47 binding domain, an optional first hinge region, a first Fc region; as well as (b) a second polypeptide, which comprises, from N-terminus to C-terminus: a second VHH that binds to PD-L1, an optional second linker peptide, a second CD47 binding domain, an optional second hinge region, and a second Fc region.

39. The protein complex of claim 38, wherein the first VHH and / or the second VHH comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO:

8.

40. The protein complex of claim 38 or 39, wherein 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:

7.

41. The protein complex of any one of claims 38 to 40, wherein the first hinge region and / or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO:

16.

42. The protein complex of any one of claims 38 to 41, wherein the first Fc region and / or the second Fc region comprises a sequence at least 80% identical to SEQ ID NO:

17.

43. The protein complex of any one of claims 38 to 42, wherein the first linker peptide and / or the second linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO:

12.

44. The protein complex of any one of claims 38 to 43, wherein 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:

6.

45. The protein complex of any one of claims 38 to 44, wherein the first polypeptide further comprises a first TGFβ binding domain and the second polypeptide further comprises a second TGFβ binding domain.

46. ​​The protein complex of claim 45, wherein 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:

9.

47. The protein complex of claim 45 or 46, wherein the first TGFβ binding domain is linked to the C-terminus of the first Fc region, optionally via a third linker peptide, and wherein the second TGFβ binding domain is linked to the C-terminus of the second Fc region, optionally via a fourth linker peptide.

48. The protein complex according to claim 47, wherein the third linker peptide and / or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO:

13.

49. The protein complex of any one of claims 45 to 48, wherein 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.

50. A nucleic acid comprising a polynucleotide encoding the protein complex according to any one of claims 1 to 49.

51. The nucleic acid of claim 50, wherein the nucleic acid is DNA (eg, cDNA) or RNA (eg, mRNA).

52. A vector comprising one or more nucleic acids according to claim 50 or 51.

53. A cell comprising the vector according to claim 52.

54. The cell of claim 53, wherein the cell is a CHO cell.

55. A cell comprising one or more nucleic acids according to claim 50 or 51.

56. A method for producing a protein complex, the method comprising (a) culturing the cell according to any one of claims 53 to 55 under conditions sufficient for the cell to produce the protein complex; and (b) collecting the protein complex produced by the cell.

57. A protein conjugate comprising the protein complex according to any one of claims 1 to 49, wherein the protein conjugate is covalently bound to a therapeutic agent.

58. The protein conjugate of claim 57, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

59. 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 according to any one of claims 1 to 49 or a protein conjugate according to claim 57 or 58.

60. The method of claim 59, wherein the subject has cancer cells that express CD47 and / or PD-L1.

61. The method of claim 59 or 60, 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 tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, stomach cancer, bile duct cancer, head and neck cancer, blood cancer, or a combination thereof.

62. A method for reducing tumor growth rate, the method comprising The tumor cells are contacted with an effective amount of a composition comprising a protein complex according to any one of claims 1 to 49 or a protein conjugate according to claim 57 or 58.

63. A method for killing tumor cells, the method comprising The tumor cells are contacted with an effective amount of a composition comprising a protein complex according to any one of claims 1 to 49 or a protein conjugate according to claim 57 or 58.

64. A pharmaceutical composition comprising the protein complex according to any one of claims 1 to 49 and a pharmaceutically acceptable carrier.

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