WO2024260419A1

WO2024260419A1 - Multi-targeting protein complex and methods of use thereof

Info

Publication number: WO2024260419A1
Application number: PCT/CN2024/100387
Authority: WO
Inventors: I-Yin CHEN; Chiu-Yueh SHIH; Yi-Shin LIAO; I-Chun Chen; Huan-Ching LIN; Chi-Ling Tseng; Zong Sean Juo
Original assignee: Fbd Biologics Ltd
Current assignee: Fbd Biologics Ltd
Priority date: 2023-06-22
Filing date: 2024-06-20
Publication date: 2024-12-26
Anticipated expiration: 2025-12-22
Also published as: CN121013868A; AU2024314312A1; TW202515923A

Abstract

Provided are protein complexes targeting CD47, PD-L1, and/or TIGIT, and methods of use thereof. In one aspect, the protein complexes include a CD47-binding domain having all or a portion of the SIRPα extracellular region; a PD-L1-binding domain having all or a portion of the PD-1 extracellular domain; and a TIGIT ligand-binding domain having all or a portion of the TIGIT extracellular region.

Description

MULTI-TARGETING PROTEIN COMPLEX AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This disclosure claims priority to and benefit of U.S. Provisional Patent Application Serial No. 63/509,693, filed June 22, 2023, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named “52246-0015WO1_SL_ST26. XML. ” The XML file, created on June 12, 2024, is 55, 931 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This disclosure relates to protein complexes targeting CD47, PD-L1, and a TIGIT ligand, and methods of use thereof.

BACKGROUND

PD-1/PD-L1 blockade monotherapy has emerged as a promising approach in cancer treatment by enhancing the anti-tumor immune response. However, the effectiveness of PD-1/PD-L1 blockade as a standalone therapy is often limited by various factors. For example, numerous types of cancer are resistant to anti-PD-1/PD-L1 therapies. Additionally, although the PD-1/PD-L1 axis plays a central role in regulating T cell functions, there are many other co-inhibitory receptor-ligand interactions, including TIGIT, that can restrain anti-tumor functions. TIGIT blockade therapy has demonstrated encouraging results as an immunotherapeutic strategy for the treatment of cancer, however, the anti-tumor efficacy of anti-TIGIT antibody alone is not good enough. There is a pressing demand for the development of immunotherapy combinations that effectively target co-inhibitory molecules expressed by T cells, aiming to enhance the anti-tumor efficacy.

SUMMARY

This disclosure relates to protein complexes targeting CD47, PD-L1, and a TIGIT ligand (e.g., PVR or Nectin-2) , and methods of use thereof.

In one aspect, the disclosure is related to a protein complex, comprising: (a) an Fc; (b) a TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains) ligand-binding domain; (c) a PD-L1 (programmed death-ligand 1) -binding domain; and (d) a CD47-binding domain.

In some embodiments, the TIGIT ligand-binding domain can bind to a cell (e.g., cancer cell) expressing a TIGIT ligand (e.g., PVR or Nectin-2) and/or block the interaction between TIGIT and the TIGIT ligand. In some embodiments, the TIGIT ligand-binding domain is or comprises a TIGIT extracellular domain. In some embodiments, the TIGIT extracellular 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: 1.

In some embodiments, the PD-L1-binding domain can bind to a cell (e.g., cancer cell) expressing PD-L1 and/or block the interaction between PD-1 (programmed cell death protein 1) and PD-L1. In some embodiments, the PD-L1-binding domain is or comprises a PD-1 extracellular domain. In some embodiments, the PD-1 extracellular 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: 2. In some embodiments, the PD-1 extracellular domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 36. In some embodiments, the amino acid that corresponds to S39 of SEQ ID NO: 36 is H. In some embodiments, the PD-1 extracellular domain further comprises a PD-L1 surface interaction sequence. In some embodiments, the PD-L1 surface interaction sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 37, 38, 39, or 40. In some embodiments, the PD-L1-binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 17.

In some embodiments, the CD47-binding domain can bind to a cell (e.g., cancer cell) expressing CD47 and/or block the interaction between CD47 and signal regulatory protein α (SIRPα) . In some embodiments, the CD47-binding domain is or comprises a SIRPα extracellular domain. In some embodiments, the SIRPα extracellular domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 3. In some embodiments, the SIRPα extracellular domain comprises one or more amino acid mutations at positions corresponding to H24, I31, E54, G55, H56, and/or D73 of SEQ ID NO: 3. In some embodiments, the SIRPα extracellular domain comprises one or more of the following: (a) the amino acid that corresponds to H24 of SEQ ID NO: 3 is R; (b) the amino acid that corresponds to I31 of SEQ ID NO: 3 is T; (c) the amino acid that corresponds to E54 of SEQ ID NO: 3 is A; (d) the amino acid that corresponds to G55 of SEQ ID NO: 3 is K; (e) the amino acid that corresponds to H56 of SEQ ID NO: 3 is Q; and (f) the amino acid that corresponds to D73 of SEQ ID NO: 3 is I. In some embodiments, the CD47-binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 18.

In some embodiments, the TIGIT ligand-binding domain is linked to the N-terminus of a CH2 domain in the Fc, optionally via a hinge region. In some embodiments, the PD-L1-binding domain is linked to the N-terminus of TIGIT ligand-binding domain, optionally via a first linker peptide. In some embodiments, the CD47-binding domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a second linker peptide. In some embodiments, the CD47-binding domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a first linker peptide. In some embodiments, the PD-L1-binding domain is linked to the C-terminus of the CD47-binding domain, optionally via a second linker peptide. In some embodiments, the PD-L1-binding domain is linked to the N-terminus of a CH2 domain in the Fc, optionally via a hinge region. In some embodiments, the TIGIT ligand-binding domain is linked to the N-terminus of the PD-L1 binding domain, optionally via a first linker peptide. In some embodiments, the CD47-binding domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a second linker peptide. In some embodiments, the CD47-binding domain is linked to the N-terminus of a CH2 domain in the Fc, optionally via a hinge region. In some embodiments, the PD-L1-binding domain is linked to the N-terminus of the CD47-binding domain, optionally via a first linker peptide. In some embodiments, the TIGIT ligand-binding domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a second 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 optionally with S228P mutation according to EU numbering.

In one aspect, the disclosure is related to a protein complex, comprising (a) a first polypeptide comprising from N-terminus to C-terminus: a first PD-L1-binding domain, an optional first linker peptide, a first TIGIT ligand-binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and a first CD47-binding domain; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second PD-L1-binding domain, an optional third linker peptide, a second TIGIT ligand-binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and a second CD47-binding domain. In some embodiments, the first PD-L1-binding domain and/or the second PD-L1-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 17. In some embodiments, the first TIGIT ligand-binding domain and/or the second TIGIT ligand-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 1. In some embodiments, the first CD47-binding domain and/or the second CD47-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 3 or 18. In some embodiments, the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 8. In some embodiments, the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 9. In some embodiments, the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 10. In some embodiments, the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 11. In some embodiments, the first linker peptide and/or the third linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 5. In some embodiments, the first polypeptide and/or the second polypeptide comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 12, 13, 19, or 20.

In one aspect, the disclosure is related to a protein complex, comprising (a) a first polypeptide comprising from N-terminus to C-terminus: a first TIGIT ligand-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 a first CD47-binding domain; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second TIGIT ligand-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 a second CD47-binding domain. In some embodiments, the first TIGIT-binding domain and/or the second TIGIT-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 1. In some embodiments, the first PD-L1-binding domain and/or the second PD-L1-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 17. In some embodiments, the first CD47-binding domain and/or the second CD47-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 3 or 18. In some embodiments, the first hinge region and/or the second hinge region comprise a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 9. In some embodiments, the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 10. In some embodiments, the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 11. In some embodiments, the first linker peptide and/or the third linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 5. In some embodiments, the first polypeptide and/or the second polypeptide comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 14 or 21.

In one aspect, the disclosure is related to a protein complex, comprising (a) a first polypeptide comprising from N-terminus to C-terminus: a first TIGIT ligand-binding domain, an optional first hinge region, a first Fc region, an optional first linker peptide, a first CD47-binding domain, an optional second linker peptide, and a first PD-L1-binding domain; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second TIGIT ligand-binding domain, an optional second hinge region, a second Fc region, an optional third linker peptide, a second CD47-binding domain, an optional fourth linker peptide, and a second PD-L1-binding domain. In some embodiments, the first TIGIT-binding domain and/or the second TIGIT-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 1. In some embodiments, the first CD47-binding domain and/or the second CD47-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 3 or 18. In some embodiments, the first PD-L1-binding domain and/or the second PD-L1-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 17. In some embodiments, the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 8. In some embodiments, the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 9. In some embodiments, the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 10. In some embodiments, the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 11. In some embodiments, the first linker peptide and/or the third linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 5. In some embodiments, the second linker peptide and/or the fourth linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 4. In some embodiments, the first polypeptide and/or the second polypeptide comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 15 or 22.

In one aspect, the disclosure is related to a protein complex, comprising (a) a first polypeptide comprising from N-terminus to C-terminus: a first PD-L1-binding domain, an optional first linker peptide, a first CD47-binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and a first TIGIT ligand-binding domain; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second PD-L1-binding domain, an optional third linker peptide, a second CD47-binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and a second TIGIT ligand-binding domain. In some embodiments, the first TIGIT-binding domain and/or the second TIGIT-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 1. In some embodiments, the first CD47-binding domain and/or the second CD47-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 3 or 18. In some embodiments, the first PD-L1-binding domain and/or the second PD-L1-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 17. In some embodiments, the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 8. In some embodiments, the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 9. In some embodiments, the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 10. In some embodiments, the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 11. In some embodiments, the first linker peptide and/or the third linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 5. In some embodiments, the first polypeptide and/or the second polypeptide comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 16.

In one aspect, the disclosure is related to a nucleic acid comprising a polynucleotide encoding the protein complex described herein. In some embodiments, the nucleic acid is a DNA (e.g., cDNA) or RNA (e.g., mRNA) . In one aspect, the disclosure is related to a vector comprising one or more of the nucleic acids described herein. In one aspect, the disclosure is related to a cell comprising the vector described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure is related to a cell comprising one or more of the nucleic acids described herein.

In one aspect, the disclosure is related to a method of producing a protein complex, the method comprising (a) culturing the cell described herein under conditions sufficient for the cell to produce the protein complex; and (b) collecting the protein complex produced by the cell.

In one aspect, the disclosure is related to a protein conjugate comprising the protein complex described herein, covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

In one aspect, the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex or the protein conjugate described herein, to the subject. In some embodiments, the subject has a cancer cell expressing PVR, Nectin-2, 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 carcinoma, endometrial carcinoma, stomach cancer, cholangiocarcinoma, head and neck cancer, blood cancer, or a combination thereof.

In one aspect, the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex or the protein conjugate described herein.

In one aspect, the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex or the protein conjugate described herein.

In one aspect, the disclosure is related to a pharmaceutical composition comprising the protein complex described herein and a pharmaceutically acceptable carrier.

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, forming a dimer or a multimer.

As used herein, the term “TIGIT ligand-binding domain” refers to a protein domain that can bind to a TIGIT ligand (e.g., PVR or Nectin-2) . In some embodiments, the TIGIT ligand-binding domain can be an anti-PVR or anti-Nectin-2 antibody, an antigen-binding fragment thereof (e.g., a scFv or a VHH) , or a PVR-or Nectin-2-binding protein or a portion thereof. In some embodiments, the TIGIT ligand-binding domain can have one or more self-stabilizing domains. In some embodiments, the TIGIT ligand-binding domain comprises or consists of a TIGIT extracellular domain. The TIGIT can be a wildtype TIGIT, a human TIGIT, a polypeptide derived from a wildtype TIGIT (e.g., with mutations) , or a portion thereof (e.g., the extracellular region of TIGIT, or IgV domain of TIGIT) . In some embodiments, the polypeptide derived from a wildtype TIGIT can have one or more mutations. In some embodiments, the TIGIT extracellular domain comprises or consists of substantially the entire extracellular region of TIGIT or the variant thereof. In some embodiments, the TIGIT extracellular domain comprises or consists of the IgV domain of TIGIT or the variant thereof. In some embodiments, the IgV domain has one or more mutations. In some embodiments, the TIGIT extracellular domain comprises or consists of amino acids 22-137 of human TIGIT protein (NCBI Accession No.: NP_776160.2; SEQ ID NO: 42) . In some embodiments, the TIGIT extracellular domain has one or more mutations.

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 can be an anti-CD47 antibody, an antigen-binding fragment thereof (e.g., a scFv or a VHH) , or a CD47 binding protein or a portion thereof. In some embodiments, the CD47-binding domain can have one or more self-stabilizing domains. In some embodiments, the CD47-binding domain comprises or consists of a SIRPα extracellular domain. The SIRPα can be a wildtype SIRPα, a human SIRPα, a polypeptide derived from a wildtype SIRPα (e.g., with mutations) , or a portion thereof (e.g., the extracellular region of SIRPα, or IgV domain of SIRPα) . In some embodiments, the polypeptide derived from a wildtype SIRPα can have one or more mutations. In some embodiments, the SIRPα extracellular domain comprises or consists of substantially the entire extracellular region of SIRPα or the variant thereof. In some embodiments, the SIRPα extracellular domain comprises or consists of the IgV domain of SIRPα or the variant thereof. In some embodiments, the IgV domain has one or more mutations. In some embodiments, the SIRPα extracellular domain comprises or consists of amino acids 31-148 of human SIRPα protein (NCBI Accession No.: AAH26692.1; SEQ ID NO: 41) . In some embodiments, the SIRPα extracellular domain has one or more mutations (e.g., mutations at positions corresponding to H24, I31, E54, G55, H56, and/or D73 of SEQ ID NO: 3) .

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., a scFv or a VHH) , or a PD-L1-binding protein or a portion thereof. In some embodiments, the PD-L1-binding domain can have one or more self-stabilizing domains. In some embodiments, the PD-L1-binding domain comprises or consists of a PD-1 extracellular domain. The PD-1 can be a wildtype PD-1, a human PD-1, a polypeptide derived from a wildtype PD-1 (e.g., with mutations) , or a portion thereof (e.g., the extracellular region of PD-1, or IgV domain of PD-1) . In some embodiments, the polypeptide derived from a wildtype PD-1 can have one or more mutations. In some embodiments, the PD-1 extracellular domain comprises or consists of substantially the entire extracellular region of PD-1 or the variant thereof. In some embodiments, the PD-1 extracellular domain comprises or consists of a portion of the extracellular region of PD-1 or the variant thereof. In some embodiments, the PD-1 extracellular domain comprises or consists of the IgV domain of PD-1 or the variant thereof. In some embodiments, the IgV domain has one or more mutations. In some embodiments, the PD-1 extracellular domain comprises or consists of amino acids 26-170 or 35-170 of human PD-1 protein (NP_005009.2; SEQ ID NO: 35) . In some embodiments, the PD-1 extracellular domain has one or more mutations (e.g., a mutation at a position corresponding to S39 of SEQ ID NO: 36) . In some embodiments, the PD-1 extracellular domain comprises one or more PD-L1 surface interaction sequences described herein, optionally the one or more PD-L1 surface interaction sequences are fused to the N-terminus of the PD-1 extracellular domain (e.g., any of the PD-1 extracellular domains described herein) .

As used herein, the term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine. Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues.The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation. The term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. A hematologic cancer is a cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of hematologic cancer include e.g., leukemia, lymphoma, and multiple myeloma etc.

As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

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

As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.

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

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1E show schematic structures of TgPS_v1 formats including TgPS-C1_v1, TgPS-C2_v1, TgPS-D_v1, TgPS-E_v1, and TgPS-F_v1, respectively.

FIG. 2A shows the binding ability of TgPS_v1 proteins to TIGIT ligand PVR detected by ELISA.

FIG. 2B shows the binding ability of TgPS_v1 proteins to TIGIT ligand Nectin-2 detected by ELISA.

FIG. 2C shows the binding ability of TgPS_v1 proteins to PD-L1 detected by ELISA.

FIG. 2D shows the binding ability of TgPS_v1 proteins to CD47 detected by ELISA.

FIG. 3A shows whole cell binding ability of TgPS_v1 proteins to PD-L1 tf CHO-S cells.

FIG. 3B shows whole cell binding ability of TgPS_v1 proteins to CD47 tf CHO-S cells.

FIG. 4A shows RBC-binding curves of TgPS_v1 proteins.

FIG. 4B shows platelet-binding curves of TgPS_v1 proteins.

FIG. 5A shows whole cell binding results of TgPS_v1 proteins to CellTrace-CFSE⁺ OE19 cells in a mixture of OE19 cells and PD-L1 tf OE19 cells.

FIG. 5B shows whole cell binding results of TgPS_v1 proteins to CellTrace-Violet⁺ PD-L1 tf OE19 cells in a mixture of OE19 cells and PD-L1 tf OE19 cells.

FIG. 6 shows whole cell binding results of TgPS_v1 proteins to activated T cells.

FIGS. 7A-7B show the blocking effect of TgPS_v1 proteins on the interaction between TIGIT and PVR using PVR tf CHO-S cells.

FIGS. 8A-8B show the blocking effect of TgPS_v1 proteins on the interaction between PD-1 and PD-L1 using PD-L1 tf CHO-S cells.

FIGS. 9A-9B show the blocking effect of TgPS_v1 proteins on the interaction between SIRPα and CD47 using CD47 tf CHO-S cells.

FIG. 10 is a picture showing RBC hemagglutination activity induced by TgPS_v1 proteins.

FIG. 11A shows macrophage-mediated phagocytosis on FaDu cells induced by TgPS_v1 proteins.

FIG. 11B shows macrophage-mediated phagocytosis on PD-L1 tf OE19 induced by TgPS_v1 proteins.

FIG. 11C shows macrophage-mediated phagocytosis on RBCs induced by TgPS_v1 proteins.

FIG. 12A shows TgPS_v1 proteins induced IL-2 expression in MLR assay. “TIGIT+PD-1+SIRPα” represents a combination of TIGIT/G4Fc, PD-1/G1Fc, and SIRPα/G4Fc.

FIG. 12B shows TgPS_v1 proteins induced IFN-γ expression in MLR assay. “TIGIT+PD-1+SIRPα” represents a combination of TIGIT/G4Fc, PD-1/G1Fc, and SIRPα/G4Fc.

FIG. 13 is a table showing the summary (f) of in vitro studies described in detail in FIGS. 2A-10.

FIG. 14 is a table showing the summary (II) of in vitro studies described in detail in FIGS. 11A-12B.

FIGS. 15A-15D show schematic structures of TgPS_v2 formats including TgPS-C1_v2, TgPS-C2_v2, TgPS-D_v2, and TgPS-E_v2, respectively.

FIG. 16A shows the binding ability of TgPS_v2 proteins to TIGIT ligand PVR detected by ELISA.

FIG. 16B shows the binding ability of TgPS_v2 proteins to PD-L1 detected by ELISA.

FIG. 16C shows the binding ability of TgPS_v2 proteins to CD47 detected by ELISA.

FIGS. 17A-17D show the simultaneous binding ability of TgPS_2 proteins to PVR, PD-L1, and CD47 detected by Bio-Layer Interferometry (BLI) .

FIG. 18A shows whole cell binding ability of TgPS_v2 proteins to PVR tf CHO-S cells.

FIG. 18B shows whole cell binding ability of TgPS_v2 proteins to PD-L1 tf CHO-S cells.

FIG. 18C shows whole cell binding ability of TgPS_v2 proteins to CD47 tf CHO-Scells.

FIG. 19A shows RBC-binding curves of TgPS_v2 proteins.

FIG. 19B shows platelet-binding curves of TgPS_v2 proteins.

FIGS. 20A-20B show the blocking effect of TgPS_v1 and TgPS_v2 proteins on the interaction between TIGIT and PVR using PVR tf CHO-S cells.

FIGS. 21A-21B show the blocking effect of TgPS_v1 and TgPS_v2 proteins on the interaction between PD1 and PD-L1 using PD-L1 tf CHO-S cells.

FIGS. 22A-22B show the blocking effect of TgPS_v1 and TgPS_v2 proteins on the interaction between SIRPα and CD47 using CD47 tf CHO-S cells.

FIG. 23 shows the blocking effect of TgPS_v2 proteins on the interaction between PD-1 and PD-L1 using NFAT-Luc signaling blocking assays.

FIG. 24A shows macrophage-mediated phagocytosis on FaDu cells induced by TgPS_v1 and TgPS_v2 proteins.

FIG. 24B shows macrophage-mediated phagocytosis on PD-L1 tf OE19 induced by TgPS_v1 and TgPS_v2 proteins.

FIG. 25A shows macrophage-mediated phagocytosis on RBCs induced by TgPS_v1 and TgPS_v2 proteins.

FIG. 25B shows macrophage-mediated phagocytosis on platelets induced by TgPS_v1 and TgPS_v2 proteins.

FIG. 26A shows TgPS_v2 proteins induced IL-2 expression in MLR assay. “3 combo (G1Fc) ” represents a combination of TIGIT/G1Fc, PD-1-mt13/G1Fc, and SIRPα-mt15/G1Fc. “3 combo (G4Fc) ” represents a combination of TIGIT/G4Fc, PD-1-mt13/G4Fc, and SIRPα-mt15/G4Fc.

FIG. 26B shows TgPS_v2 proteins induced IFN-γ expression in MLR assay. “3 combo (G1Fc) ” represents a combination of TIGIT/G1Fc, PD-1-mt13/G1Fc, and SIRPα-mt15/G1Fc. “3 combo (G4Fc) ” represents a combination of TIGIT/G4Fc, PD-1-mt13/G4Fc, and SIRPα-mt15/G4Fc.

FIG. 27 is a table showing the summary (f) of in vitro studies described in detail in FIGS. 16A-23.

FIG. 28 is a table showing the summary (II) of in vitro studies described in detail in FIGS. 24A-26B.

FIG. 29 lists the amino acid sequences of proteins used in this disclosure.

DETAILED DESCRIPTION

Immunotherapy has emerged as a promising approach in the treatment of various cancers in human populations by means of enhancing the recognition and elimination of abnormal cells, including cancer cells. While immunotherapy has shown impressive clinical successes, a significant proportion of patients either do not respond or develop resistance over time. The limitations and challenges, that need to be addressed, include loss of antigen expression, impaired T cell function, activation of alternative immune checkpoints, and tumor immune evasion. Co-inhibitory molecules expressed on immune cells (e.g., T cells) play a crucial role in regulating immune responses and maintaining immune homeostasis. Targeting these co-inhibitory molecules can be a promising strategy for enhancing T cell function and unleashing robust anti-tumor immune responses.

Signal regulatory protein α (SIRPα) is a regulatory membrane glycoprotein from SIRP family. It 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 broadly expressed transmembrane protein CD47. This interaction negatively regulates the effector function of innate immune cells such as host cell phagocytosis. SIRPα diffuses laterally on the macrophage membrane and accumulates at a phagocytic synapse to bind CD47, thereby inhibiting the cytoskeleton-intensive process of phagocytosis by the macrophage. CD47 provides a “do not eat” signal by binding to the N-terminus of signal regulatory protein alpha (SIRPα) . CD47 has been found to be overexpressed in many different tumor cells. Targeting CD47 and/or SIRPα can be useful for cancer immunotherapy. However, given that CD47 is also expressed on red blood cells (RBCs) and platelets, inhibiting the CD47/SIRPα interaction may cause the phagocytosis of RBCs and platelets.

Programmed Cell Death Ligand 1 (PD-L1) is a trans-membrane protein that is considered to be a co-inhibitory factor in the immune response. It can combine with Programmed Cell Death Protein 1 (PD-1) to reduce the proliferation of PD-1 positive cells, inhibit their cytokine secretion and induce apoptosis. PD-L1 also plays an important role in various malignancies where it can attenuate the host immune response to tumor cells. Thus, PD-1/PD-L1 axis is responsible for cancer immune escape and makes a huge effect on cancer therapy.

T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) has emerged as an attractive immuno-oncology target. In native cells, TIGIT expression levels are generally low. However, upon activation, both T cells and nature killer (NK) cells have been observed to up-regulate TIGIT expression. Typically, TIGIT interacts with four ligands, CD155 (also known as PVR) , and CD112 (also known as Nectin-2 or PVRL2) , CD113 (also known as Nectin-3, PVRL3) and CD114 (Nectin-4) , those are expressed on antigen-presenting cells and tumor cells. TIGIT functions as an immune checkpoint receptor that suppresses the activities of effector T cells and NK cells while promoting regulatory T cells (Treg) functions. For example, TIGIT can inhibit CD8⁺ T cell or NK cell-mediated killing of tumor cells. TIGIT expression has been observed in various tumor-infiltrating immune cells, and its upregulation is associated with immune evasion and tumor progression. As a result, targeting TIGIT through blockade therapy has emerged as a promising approach to enhance anti-tumor immune responses and overcome immunosuppression.

The present disclosure provides protein complexes binding to CD47, PD-L1, and a TIGIT ligand (e.g., PVR or Nectin-2) . These protein complexes can target the CD47/SIRPα pathway, the PD-1/PD-L1 pathway, and the TIGIT/PVR pathway simultaneously. The results indicate that the protein complexes can effectively bind to CD47-expressing cancer cells and block the interaction between endogenous SIRPα and CD47, thereby inducing innate immune response (e.g., phagocytosis of cancer cells by macrophages) . On the other hand, the protein complexes showed minimal binding to red blood cells or platelets, thereby inhibiting the clearance of host cells as observed by the anti-CD47 antibody Magrolimab. In addition, the results indicate that the protein complexes can selectively bind to PD-L1-expressing cancer cells, block the interaction between endogenous PD-1 and PD-L1, and induce phagocytic activities against PD-L1⁺ tumor cells. Further, by binding to the TIGIT ligand (e.g., PVR) , the protein complexes can also compete with endogenous TIGIT, exhibiting a similar TIGIT-PVR blocking activity as observed by the anti-TIGIT antibody Tiragolumab. As a result, the protein complexes can bind to activated T cells, and synergistically induce higher cytokine (e.g., IL-2 and IFN-γ) expression than a combination of individual functional domains.

Therefore, the protein complexes described herein can be used for cancer treatment with enhanced tumor immunogenicity and antigen presentation through increased phagocytosis by macrophages (e.g., by inactivation of CD47-mediated inhibition of phagocytosis) ; and enhanced T cell activation through inhibition of PD-1/PD-L1 as well as TIGIT/PVR signaling pathways.

TIGIT extracellular domain

TIGIT (T-cell immunoglobulin and ITIM domain) is a type I transmembrane protein that is expressed on various immune cells, including T cells, regulatory T cells (Tregs) , natural killer (NK) cells, and subsets of dendritic cells. It is also found in certain non-immune tissues. TIGIT belongs to the immunoglobulin superfamily and consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The extracellular region is responsible for ligand binding and is composed of an immunoglobulin variable (IgV) domain, followed by an Ig constant (IgC) domain. The IgV domain of TIGIT is responsible for binding to its ligands, primarily CD155 (PVR) and CD112 (Nectin-2) . The cytoplasmic region of TIGIT contains an immunoreceptor tyrosine-based inhibition motif (ITIM) . Upon TIGIT activation, the ITIM motif recruits phosphatases, leading to the inhibition of downstream signaling pathways and dampening of immune responses. TIGIT also interacts with CD226 (DNAM-1) , another protein of the immunoglobulin superfamily, which serves as a co-stimulatory receptor. The binding of TIGIT to CD226 can inhibit CD226-mediated activation signals, further modulating immune cell functions. TIGIT expression has been observed in various tumor-infiltrating immune cells, and its upregulation is associated with immune evasion and tumor progression. As a result, targeting TIGIT through blockade therapy has emerged as a promising approach to enhance anti-tumor immune responses and overcome immunosuppression.

A detailed description of TIGIT and its function can be found, e.g., in Manieri, N.A., et al. "TIGIT: a key inhibitor of the cancer immunity cycle. " Trends in Immunology 38.1 (2017) : 20-28; and H., et al. "TIGIT as an emerging immune checkpoint. " Clinical & Experimental Immunology 200.2 (2020) : 108-119; each of which is incorporated herein by reference in its entirety.

According to UniProt identifier Q495A1, the extracellular region of human TIGIT corresponds to amino acids 22-141 of SEQ ID NO: 42, the transmembrane region of human TIGIT corresponds to amino acids 142-162 of SEQ ID NO: 42, and the cytoplasmic region of human TIGIT corresponds to amino acids 163-244 of SEQ ID NO: 42. The TIGIT extracellular region also has an IgV domain, which corresponds to amino acids 22-124 of the human TIGIT protein (NP_776160.2; SEQ ID NO: 42) . The signal peptide corresponds to amino acids 1-21 of SEQ ID NO: 42.

In some embodiments, the protein complex described herein comprises one or more TIGIT ligand-binding domains. In some embodiments, the TIGIT ligand-binding domain is or comprises a TIGIT extracellular domain. As used herein, the “TIGIT extracellular domain” refers to the entire or a portion of the extracellular region of TIGIT or the variant thereof, wherein the portion of the extracellular region can bind to TIGIT ligands. The TIGIT extracellular domain can have one or more protein domains that can fold independently and form self-stabilizing structures. In some embodiments, the TIGIT extracellular domain comprises or consists of an IgV domain.

In some embodiments, the amino acid sequences of the TIGIT extracellular domain described herein are provided. In some embodiments, the TIGIT extracellular domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to amino acids 22-137 of human TIGIT protein (NCBI Accession No.: NP_776160.2; SEQ ID NO: 42) . In some embodiments, the TIGIT extracellular domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1. In some embodiments, the TIGIT ligand-binding domain or TIGIT extracellular domain described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 1, and also includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid mutations.

In some embodiments, the TIGIT ligand-binding domain or TIGIT extracellular domain described herein includes the IgV domain of human TIGIT protein (wildtype or mutated) . In some embodiments, the TIGIT ligand-binding domain or TIGIT extracellular domain described herein includes the IgV domain of mouse TIGIT protein (wildtype or mutated) .

PD-1 extracellular domain

PD-1 (Programmed cell death protein 1) and its ligand PD-L1 (Programmed death-ligand 1) are key players in immune regulation and have garnered significant attention in the field of immunology and cancer immunotherapy. PD-1 is a cell surface receptor primarily expressed on activated T cells, B cells, natural killer (NK) cells, and myeloid cells. PD-L1, on the other hand, is a ligand expressed on various immune cells, as well as non-immune cells, such as tumor cells. PD-1 and PD-L1 interactions play a critical role in maintaining immune homeostasis and preventing excessive immune activation. Under normal conditions, the binding of PD-1 to PD-L1 provides a negative regulatory signal that attenuates immune responses and helps prevent autoimmunity and tissue damage. Therefore, the PD-1/PD-L1 pathway is essential for suppressing anti-tumor immune responses and contributing to the induction and maintenance of immune tolerance within the tumor microenvironment.

However, tumor cells and certain immune cells can exploit the PD-1/PD-L1 pathway to evade immune surveillance and promote immune tolerance. In the tumor microenvironment, cancer cells often upregulate PD-L1 expression, enabling them to bind to PD-1 on immune cells. This engagement effectively inhibits anti-tumor immune responses and promotes immune escape. The discovery of the PD-1/PD-L1 pathway′srole in immune evasion led to the development of PD-1/PD-L1 blockade therapies, also known as immune checkpoint inhibitors. These therapies aim to block the interaction between PD-1 and PD-L1, thereby unleashing the immune system′sability to recognize and eliminate tumor cells. PD-1/PD-L1 blockade has shown remarkable clinical success, leading to durable responses and improved outcomes in various types of cancer. By releasing the "brakes" on the immune system, these therapies revitalize anti-tumor immune responses, enhance T cell activation and infiltration into tumors, and promote tumor cell killing.

PD-1 (Programmed cell death protein 1) , also referred to as CD279, is a 55-kDa transmembrane protein containing 288 amino acids with an extracellular N-terminal domain (IgV-Like) , a membrane-permeating domain and a cytoplasmic tail located at the N and C ends, respectively, with two tyrosine bases. PD-1 is an inhibitor of both adaptive and innate immune responses, and is expressed on activated T, natural killer (NK) and B lymphocytes, macrophages, dendritic cells (DCs) and monocytes.

PD-L1 (Programmed death-ligand 1) , also referred to as CD279 and B7-H1, belongs to the B7 series and is a 33-kDa type 1 transmembrane glycoprotein that contains 290 amino acids with IgV and IgC domains in its extracellular region. PD-L1 is commonly expressed on macrophages, certain activated T cells, and B cells, dendritic cells (DCs) , and some epithelial cells, especially in the presence of inflammatory conditions. Furthermore, tumor cells express PD-L1 as an “adaptive immune mechanism” to evade anti-tumor responses. PD-L1 is often associated with an immune environment characterized by the presence of CD8 T cells, production of Th1 cytokines, chemokines, interferons, and specific gene expression patterns. For example, lymphocyte derived IFN-γ has been shown to induce PD-L1 upregulation and promotes progression of ovarian cancer. Inhibition of IFN-γ receptor 1 can decrease PD-L1 expression in mouse models of acute myeloid leukemia through the MEK/extracellular signal-regulated kinase (ERK) and MYD88/TRAF6 pathways. Moreover, IFN-γ induces the expression of protein kinase D isoform 2 (PKD2) , and inhibiting PKD2 activity suppresses PD-L1 expression, thereby enhancing the development of a potent anti-tumor immune response. NK cells secrete IFN-γ via the Janus kinase (JAK) 1, JAK2 and signal transducer and activator of transcription (STAT) 1 pathways, which subsequently upregulates the expression of PD-L1 on the surface of tumor cells. Studies on melanoma cells have also revealed that T cells secrete IFN-γ via the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 pathways. IFN-γ secreted by T cells and NK cells have been found to lead to the induction of PD-L1 expression on the surface of target cells, including tumor cells.

PD-L1 acts as a pro-tumorigenic factor in cancer cells by engaging with its receptors and initiating proliferative and survival signaling pathways. This finding further supports the implication of PD-L1 in subsequent tumor progression. In addition, PD-L1 has been shown to exert non-immune proliferative effects on diverse tumor cells. For example, PD-L1 has been observed to induce epithelial-to-mesenchymal transition (EMT) and promote stem cell-like features in renal cancer cells. This indicates that the intrinsic pathway of PD-L1 contributes to kidney cancer progression.

The use of PD-1 blockade to enhance anti-tumor immunity originated from observations in chronic infection models, where preventing PD-1 interactions reversed T-cell exhaustion. Similarly, blockade of PD-1 prevents T-cell PD-1/tumor cell PD-L1 or T-cell PD-1/tumor cell PD-L2 interaction, leading to restoration of T-cell mediated anti-tumor immunity.

A detailed review of PD-L1 and its functions can be found in Han, Y., et al. "PD-1/PD-L1 pathway: current research in cancer. " American Journal of Cancer Research 10.3 (2020) : 727; and Liu, J., et al. "PD-1/PD-L1 checkpoint inhibitors in tumor immunotherapy. " Frontiers in Pharmacology 12 (2021) ; each of which is incorporated by reference in its entirety.

According to UniProt identifier Q15116, the extracellular region of human PD-1 corresponds to amino acids 24-170 of SEQ ID NO: 35, the transmembrane region of human PD-1 corresponds to amino acids 171-191 of SEQ ID NO: 35, and the cytoplasmic region of human PD-1 corresponds to amino acids 192-288 of SEQ ID NO: 35. The PD-1 extracellular region also has an IgV domain, which corresponds to amino acids 35-145 of the human PD-1 protein (NP_005009.2; SEQ ID NO: 35) . The signal peptide corresponds to amino acids 1-23 of SEQ ID NO: 35.

In some embodiments, the protein complex described herein comprises one or more PD-L1-binding domains. In some embodiments, the PD-L1-binding domain comprises or consists of a PD-1 extracellular domain. As used herein, the “PD-1 extracellular domain” refers to the entire or a portion of the extracellular region of PD-1 or the variant thereof, wherein the portion of the extracellular region can bind to PD-L1. The PD-1 extracellular domain can have one or more protein domains that can fold independently and form self-stabilizing structures. In some embodiments, the PD-1 extracellular domain comprises or consists of an IgV domain.

In some embodiments, the amino acid sequences of the PD-1 extracellular domain described herein are provided. In some embodiments, the PD-1 extracellular domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to amino acids 26-170 or 35-170 of human PD-1 protein (NP_005009.2; SEQ ID NO: 35) . In some embodiments, the PD-L1-binding domain or PD-1 extracellular domain described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2 or 36. In some embodiments, the PD-L1-binding domain or PD-1 extracellular domain described herein includes one or more one or more mutations (e.g., a mutation at a position corresponding to S39 of SEQ ID NO: 36) . In some embodiments, the PD-L1-binding domain or PD-1 extracellular domain described herein includes a histidine (H) at a position that corresponds to S39 of SEQ ID NO: 36.

In some embodiments, the PD-L1-binding domain or PD-1 extracellular domain described herein includes a PD-L1 surface interaction sequence (e.g., any one of the PD-L1 surface interaction sequences described herein) . In some embodiments, the PD-L1 surface interaction sequence is fused at the N-terminus of the PD-L1-binding domain or PD-1 extracellular domain described herein (e.g., any one of SEQ ID NOs: 37, 38, 39, and 40) . In some embodiments, the PD-L1-binding domain or PD-1 extracellular domain described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 17.

In some embodiments, the PD-L1-binding domain or PD-1 extracellular domain includes a PD-L1 surface interaction sequence comprising about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 amino acids, wherein the PD-L1 surface interaction sequence includes two or more histidine residues. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 50 amino acids. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 50 amino acids. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 5-15, 5-20, 5-30, 5-40, 10-15, 10-20, 10-30, 10-40, 15-20, 15-30, or 15-40 amino acids. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 5-15 amino acids.

In some embodiments, the PD-L1 surface interaction sequence comprises or consists of about or at least 2, 3, 4, 5, or 6 histidine residues. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of at most 2, 3, 4, 5, or 6 histidine residues. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 2-3, 2-4, 2-5 or 2-6 histidine residues. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 2-4 histidine residues.

In some embodiments, the PD-L1 surface interaction sequence comprises or consists of about or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 positively charged amino acid residues (e.g., histidine, lysine or arginine) . In some embodiments, the PD-L1 surface interaction sequence comprises or consists of at most 2, 3, 4, 5, 6, 7, 8, 9, or 10 positively charged amino acid residues (e.g., histidine, lysine or arginine) . In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 2-3, 2-4, 2-5, 2-6, 2-10, 3-10, or 5-10 positively charged amino acid residues (e.g., histidine, lysine or arginine) . In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 2-4 positive amino acid residues (e.g., histidine, lysine or arginine) .

In some embodiments, the PD-L1 surface interaction sequence is selected from the group consisting of: SHGHGGG (SEQ ID NO: 37) , SHHGHGHGGGG (SEQ ID NO: 38) , SHGHHGHGGGG (SEQ ID NO: 39) , and SHGHGHHGGGG (SEQ ID NO: 40) .

In some embodiments, the PD-L1-binding domain or PD-1 extracellular domain described herein includes the IgV domain of human PD-1 protein (wildtype or mutated) . In some embodiments, the PD-L1-binding domain or PD-1 extracellular domain described herein includes the IgV domain of mouse PD-1 protein (wildtype or mutated) .

SIRPα extracellular domains

Signal regulatory protein α (SIRPα, SIRPa, Sirpa, or CD172A) is a transmembrane protein. It has an extracellular region comprising three Ig-like domains and a cytoplasmic region containing immunoreceptor tyrosine-based inhibition motifs that mediate binding of the protein tyrosine phosphatases SHP1 and SHP2. Tyrosine phosphorylation of SIRPα is regulated by various growth factors and cytokines as well as by integrin-mediated cell adhesion to extracellular matrix proteins. SIRPα is predominantly found in myeloid cells, including macrophages and dendritic cells, where it is expressed at high levels. By contrast, its expression in T, B, NK, and NKT cells is relatively low.

The extracellular region of SIRPα can interact with its ligand CD47. The interaction of SIRPα on macrophages with CD47 on red blood cells prevents phagocytosis of Ig-opsonized red blood cells by macrophages in vitro and in vivo. When CD47, CD47 expressed on a neighboring cell, binds to SIRPα on phagocytes, it triggers the phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) comprised in SIRPα. This phosphorylation event facilitates the recruitment of SHP-1 and SHP-2 phosphatases to the SIRPα cytoplasmic domain. Consequently, one downstream effect of SIRPα expression in myeloid cells is preventing myosin-IIA accumulation at the phagocytic synapse, leading to the inhibition of phagocytosis. Thus, CD47-SIRPα interaction functions as a negative immune checkpoint to send a “don’t eat me” signal to ensure that healthy autologous cells are not inappropriately phagocytosed.

However, CD47 overexpression has been observed in a wide range of tumor types, including but not limited to acute myeloid leukemia, non-Hodgkin′slymphoma, bladder cancer, and breast cancer. The negative regulation of macrophages can be attenuated by inhibiting the binding of CD47 to SIRPα. As a result, agents that hinder the interaction between CD47 and SIRPα can enhance both antibody-dependent cellular phagocytosis (ADCP) and, in certain instances, trigger antibody-dependent cellular cytotoxicity (ADCC) , thereby leading to recognition and elimination of cancer cells. The mechanism of blocking the engagement between CD47 and SIRPα can be implicated in treating various types of tumors and cancers, e.g., solid tumors, hematologic malignancies (e.g., relapsed, or refractory hematologic malignancies) , acute myeloid leukemia, non-Hodgkin’s lymphoma, breast cancer, bladder cancer, ovarian cancer, and small cell lung cancer tumors.

In addition, SIRPα functions by inhibiting the clearance of CD47-expressing host cells, such as red blood cells and platelets, by macrophages in vivo. Moreover, CD47-SIRPα interactions also play an essential role in the successful engraftment of hematopoietic stem cells. Therefore, inhibiting the interaction between CD47 and SIRPα can inadvertently lead to the destruction of healthy red blood cells, which may result in anemia and provoke inflammation. Therefore, it is crucial to carefully modulate the interaction of a SIRPα-targeting agent with CD47, aiming for limited or controlled effects on red blood cells.

A detailed description of SIRPα and its function can be found, e.g., 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, which are incorporated by reference herein in the entirety.

Human SIRPα is a member of 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.

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. Their corresponding regions of specific amino acid ranges (NP_542970.1) include amino acids 32-137, amino acids 148-247, and amino acids 254-348. The region of amino acids 1-30 is for signal peptides. Human SIRPα also has a long intracellular domain that comprises two putative immunoreceptor tyrosine-based inhibition motifs (ITIM) . Activation of SIRPα ITIMs delivers inhibitory signals that negatively regulate cell responses.

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

In some embodiments, the SIRPα extracellular domain described herein includes 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 No.: AAH26692.1; SEQ ID NO: 41) . In some embodiments, the CD47-binding domain or SIRPα extracellular domain described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 3. In some embodiments, the CD47-binding domain or SIRPα extracellular domain described herein includes 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 D73 of SEQ ID NO: 3. In some embodiments, the CD47-binding domain or SIRPα extracellular domain described herein includes one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following: (a) the amino acid that corresponds to H24 of SEQ ID NO: 3 is R; (b) the amino acid that corresponds to I31 of SEQ ID NO: 3 is T; (c) the amino acid that corresponds to E54 of SEQ ID NO: 3 is A; (d) the amino acid that corresponds to G55 of SEQ ID NO: 3 is K; (e) the amino acid that corresponds to H56 of SEQ ID NO: 3 is Q; and (f) the amino acid that corresponds to D73 of SEQ ID NO: 3 is I. In some embodiments, the CD47-binding domain or SIRPα extracellular domain described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 18.

In some embodiments, the CD47-binding domain or SIRPα extracellular domain described herein includes the IgV domain of human SIRPα protein (wildtype or mutated) . In some embodiments, the CD47-binding domain or SIRPα extracellular domain described herein includes the IgV domain of mouse SIRPα protein (wildtype or mutated) .

Protein complexes targeting TIGIT ligands, PD-L1, and CD47

The disclosure provides protein complexes that can specifically bind to a TIGIT ligand (e.g., PVR or Nectin-2) . In some embodiments, these protein complexes can block TIGIT/PVR and/or TIGIT/Nectin-2 signaling pathways. In some embodiments, these protein complexes can block the immunosuppressive signaling of TIGIT on NK, T, and Treg cells. The disclosure also provides protein complexes that can specifically bind to PD-L1. In some embodiments, these protein complexes can block PD-1/PD-L1 signaling pathway thus increase immune response. In some embodiments, these protein complexes can induce T cell activation, proliferation, and/or cytokine release. The disclosure also provides protein complexes that can specifically bind to CD47. In some embodiments, these protein complexes can block SIRPα/CD47 signaling pathway thus increase immune response. In some embodiments, these protein complexes can initiate phagocytosis.

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

In some embodiments, the protein complex or the protein construct comprises or consists of an Fc, a first domain that specifically binds to a TIGIT ligand (e.g., PVR or Nectin-2) , a second domain that specifically binds to PD-L1, and a third domain that specifically binds to CD47.

In some embodiments, the first domain can bind to a cell (e.g., cancer cell) expressing one or more TIGIT ligands (e.g., PVR and/or Nectin-2) and/or block the interaction between TIGIT and the one or more TIGIT ligands. In some embodiments, the first domain comprises all or a portion of the extracellular region of TIGIT. In some embodiments, the TIGIT is human TIGIT extracellular domain, optionally with one or more mutations. In some embodiments, the first domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 1.

In some embodiments, the second domain can bind to a cell (e.g., cancer cell) expressing PD-L1 and/or stimulate T cell activation and proliferation. In some embodiments, the second domain comprises all or a portion of the extracellular region of PD-1. In some embodiments, the PD-1 is human PD-1 extracellular domain with one or more mutations (e.g., any of the PD-1 mutations described herein) . 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: 2, 17, or 36.

In some embodiments, the third domain can bind to a cell (e.g., cancer cell) expressing CD47 and/or block the interaction between CD47 and signal regulatory protein α (SIRPα) . In some embodiments, the third domain comprises all or a portion of the extracellular region of SIRPα. In some embodiments, the SIRPα is human SIRPα extracellular domain with one or more mutations (e.g., any of the SIRPα mutations described herein) . In some embodiments, the first domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 3 or 18.

In some embodiments, the Fc is human IgG1Fc. In some embodiments, the Fc is human IgG4Fc. In some embodiments, the first domain is linked to the N-terminus of a CH2 domain in the Fc, optionally via a hinge region (e.g., any of the hinge regions described herein) . In some embodiments, the second domain is linked to the N-terminus of a CH2 domain in the Fc, optionally via a hinge region (e.g., any of the hinge regions described herein) . In some embodiments, the third domain is linked to the N-terminus of a CH2 domain in the Fc, optionally via a hinge region (e.g., any of the hinge regions described herein) . In some embodiments, the hinge region is a human IgG4 hinge region optionally with S228P mutation according to EU numbering. In some embodiments, the first domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a linker peptide (e.g., any of the linker peptides described herein) . In some embodiments, the second domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a linker peptide (e.g., any of the linker peptides described herein) . In some embodiments, the third domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a linker peptide (e.g., any of the linker peptides described herein) .

In some embodiments, the order of the first domain, the second domain, the third domain, and the Fc described herein, from N-terminus to C-terminus, can be any of the following: first domain-second domain-Fc-third domain; first domain-third domain-Fc-second domain; second domain-first domain-Fc-third domain; second domain-third domain-Fc-first domain; third domain-first domain-Fc-second domain; third domain-second domain-Fc-first domain; first domain-Fc-second domain-third domain; first domain-Fc-third domain-second domain; second domain-Fc-first domain-third domain; second domain-Fc-third domain-first domain; third domain-Fc-first domain-second domain; and third domain-Fc-second domain-first domain.

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.

In some embodiments, the one or more TIGIT ligand-binding domains, the one or more CD47-binding domains, and the one or more PD-L1-binding domains are linked to the Fc region through any of the linker peptides or the hinge region sequences as described herein.

Some embodiments of the protein complexes are shown in FIGS. 1A-1E and FIGS. 15A-15D. They are described in detail below.

TgPS-C1_v1 and TgPS-C1_v2

In one aspect, the disclosure is related to a protein complex including a first polypeptide and a second polypeptide. The first polypeptide includes, preferably from N-terminus to C-terminus: a first PD-L1-binding domain, an optional first linker peptide, a first TIGIT ligand-binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and a first CD47-binding domain. The second polypeptide includes, preferably from N-terminus to C-terminus: a second PD-L1-binding domain, an optional third linker peptide, a second TIGIT ligand-binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and a second CD47-binding domain. A schematic structure of an exemplary protein complex having a TgPS-C1_v1 format and a TgPS-C1_v2 format are shown in FIG. 1A and FIG. 15A, respectively.

In any of the protein complexes described herein, the first and/or the second TIGIT ligand-binding domains can include a TIGIT extracellular domain (e.g., any of the TIGIT extracellular domains described herein) . In some embodiments, the TIGIT extracellular domain includes amino acids 22-137 of human TIGIT protein (SEQ ID NO: 42) . In some embodiments, the first and the second TIGIT ligand-binding domains are identical. In some embodiments, the first and the second TIGIT ligand-binding domains are different. In some embodiments, the first and/or the second TIGIT ligand-binding domains include 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. In some embodiments, the TIGIT extracellular domain includes of the IgV domain of TIGIT (e.g., human TIGIT) .

In any of the protein complexes described herein, the first and/or the second PD-L1-binding domains include a PD-1 extracellular domain (e.g., any of the PD-1 extracellular domains described herein) . In some embodiments, the PD-1 extracellular domain includes amino acids 26-170 or 35-170 of human PD-1 protein (SEQ ID NO: 35) . In some embodiments, the PD-1 extracellular domain comprises one or more PD-L1 surface interaction sequences described herein (e.g., any of SEQ ID NOs: 37-40) that are fused to the N-terminus of the PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain includes one or more mutations (e.g., a mutation at a position corresponding to S39 of SEQ ID NO: 36) . In some embodiments, the first and/or the second PD-L1-binding domains are identical. In some embodiments, the first and/or the second PD-L1-binding domains are different. In some embodiments, the first and/or the second PD-L1-binding domains include 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, 17, or 36.

In any of the protein complexes described herein, the first and/or the second CD47-binding domains can include a SIRPα extracellular domain (e.g., any of the SIRPα extracellular domains described herein) . In some embodiments, the SIRPα extracellular domain includes amino acids 31-148 of human SIRPα protein (SEQ ID NO: 41) . In some embodiments, the SIRPα extracellular domain includes one or more (e.g., 1, 2, 3, 4, 5, or 6) mutations (e.g., mutations at positions corresponding to H24, I31, E54, G55, H56, and/or D73 of SEQ ID NO: 3) . In some embodiments, the first and the second CD47-binding domains are identical. In some embodiments, the first and the second CD47-binding domains are different. In some embodiments, the first and/or the second CD47-binding domain include 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 or 18. In some embodiments, the SIRPα extracellular domain includes 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 D73 of SEQ ID NO: 3) .

In some embodiments, the first and/or the second hinge region can include all or a portion of the hinge region of an immunoglobulin, e.g., human IgG1 hinge region (SEQ ID NO: 8) . In some embodiments, the first and/or the second hinge region include 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. In some embodiments, the first and the second hinge regions are identical. In some embodiments, the first and the second hinge regions are different.

In some embodiments, the first and/or the second Fc region can be identical and can form a Fc homodimer. In some embodiments, the first and/or the second Fc region include all or a portion of the Fc region of an immunoglobulin, e.g., human IgG1 Fc region (SEQ ID NO: 9) . In some embodiments, the first and/or the second Fc region include 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.

In some embodiments, the first and/or the third linker peptide described herein include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4. In some embodiments, the second and/or fourth linker peptide described herein include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 5. In some embodiments, the first, the second, the third, and/or the fourth linker peptides described herein include 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, or 6) repeats of GSG (SEQ ID NO: 31) or GGGGS (SEQ ID NO: 33) .

In some embodiments, the first and/or the second polypeptide include 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: 12 or 19.

TgPS-C2_v1 and TgPS-C2_v2

In one aspect, the disclosure is related to a protein complex including a first polypeptide and a second polypeptide. The first polypeptide includes, preferably from N-terminus to C-terminus: a first PD-L1-binding domain, an optional first linker peptide, a first TIGIT ligand-binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and a first CD47-binding domain. The second polypeptide includes, preferably from N-terminus to C-terminus: a second PD-L1-binding domain, an optional third linker peptide, a second TIGIT ligand-binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and a second CD47-binding domain. A schematic structure of an exemplary protein complex having a TgPS-C2_v1 format and a TgPS-C2_v2 format are shown in FIG. 1B and FIG. 15B, respectively.

In some embodiments, the first and/or the second hinge region can include all or a portion of the hinge region of an immunoglobulin, e.g., human IgG4 hinge region (SEQ ID NO: 10) . In some embodiments, the first and/or the second hinge region include 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: 10. In some embodiments, the first and the second hinge regions are identical. In some embodiments, the first and the second hinge regions are different.

In some embodiments, the first and/or the second Fc region can be identical and can form a Fc homodimer. In some embodiments, the first and/or the second Fc region include all or a portion of the Fc region of an immunoglobulin, e.g., human IgG4 Fc region (SEQ ID NO: 11) . In some embodiments, the first and/or the second Fc region include 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: 11. In some embodiments, the first and/or the second hinge region include a proline at position 228 according to EU numbering.

In some embodiments, the first and/or the third linker peptide described herein include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4. In some embodiments, the second and/or fourth linker peptide described herein include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 5. In some embodiments, the first, the second, the third, and/or the fourth linker peptides described herein include 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, or 6) repeats of GSG (SEQ ID NO: 31) or GGGGS (SEQ ID NO: 33) .

In some embodiments, the first and/or the second polypeptide include 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: 13 or 20.

TgPS-D_v1 and TgPS-D_v2

In one aspect, the disclosure is related to a protein complex including a first polypeptide and a second polypeptide. The first polypeptide includes, preferably from N-terminus to C-terminus: a first TIGIT ligand-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 a first CD47-binding domain. The second polypeptide includes, preferably from N-terminus to C-terminus: a second TIGIT ligand-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 a second CD47-binding domain. A schematic structure of an exemplary protein complex having a TgPS-D_v1 format and a TgPS-D_v2 format are shown in FIG. 1C and FIG. 15C, respectively.

In some embodiments, the first and/or the second polypeptide include 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 or 21.

TgPS-E_v1 and TgPS-E_v2

In one aspect, the disclosure is related to a protein complex including a first polypeptide and a second polypeptide. The first polypeptide includes, preferably from N-terminus to C-terminus: a first TIGIT ligand-binding domain, an optional first hinge region, a first Fc region, an optional first linker peptide, a first CD47-binding domain, an optional second linker peptide, and a first PD-L1-binding domain. The second polypeptide includes, preferably from N-terminus to C-terminus: a second TIGIT ligand-binding domain, an optional second hinge region, a second Fc region, an optional third linker peptide, a second CD47-binding domain, an optional fourth linker peptide, and a second PD-L1-binding domain. A schematic structure of an exemplary protein complex having a TgPS-E_v1 format and a TgPS-E_v2 format are shown in FIG. 1D and FIG. 15D, respectively.

In some embodiments, the first and/or the third linker peptide described herein include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 5. In some embodiments, the second and/or fourth linker peptide described herein include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4. In some embodiments, the first, the second, the third, and/or the fourth linker peptides described herein include 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, or 6) repeats of GSG (SEQ ID NO: 31) or GGGGS (SEQ ID NO: 33) .

In some embodiments, the first and/or the second polypeptide include 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 or 22.

TgPS-F_v1

In one aspect, the disclosure is related to a protein complex including a first polypeptide and a second polypeptide. The first polypeptide includes, preferably from N-terminus to C-terminus: a first PD-L1-binding domain, an optional first linker peptide, a first CD47-binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and a first TIGIT ligand-binding domain. The second polypeptide includes, preferably from N-terminus to C-terminus: a second PD-L1-binding domain, an optional third linker peptide, a second CD47-binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and a second TIGIT ligand-binding domain. A schematic structure of an exemplary protein complex having a TgPS-F_v1 format is shown in FIG. 1E.

In any of the protein complexes described herein, the first and/or the second PD-L1-binding domains include a PD-1 extracellular domain (e.g., any of the PD-1 extracellular domains described herein) . In some embodiments, the PD-1 extracellular domain includes amino acids 26-170 of human PD-1 protein (SEQ ID NO: 35) . In some embodiments, the first and/or the second PD-L1-binding domains are identical. In some embodiments, the first and/or the second PD-L1-binding domains are different. In some embodiments, the first and/or the second PD-L1-binding domains include 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.

In any of the protein complexes described herein, the first and/or the second CD47-binding domains can include a SIRPα extracellular domain (e.g., any of the SIRPα extracellular domains described herein) . In some embodiments, the SIRPα extracellular domain includes amino acids 31-148 of human SIRPα protein (SEQ ID NO: 41) . In some embodiments, the first and the second CD47-binding domains are identical. In some embodiments, the first and the second CD47-binding domains are different. In some embodiments, the first and/or the second CD47-binding domain include 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. In some embodiments, the SIRPα extracellular domain includes the IgV domain of SIRPα (e.g., human SIRPα) .

In some embodiments, the first and/or the second polypeptide include 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.

Characteristics of protein complexes

In some embodiments, the protein complex can comprise any TIGIT ligand-binding domains, PD-L1-binding domains, CD47-binding domains as described herein. The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The length of a reference sequence aligned for comparison purposes is at least 80%of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between 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, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The protein complex described herein can include an Fc of an antibody. These antibodies can 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., human IgG4 Fc region) .

In some embodiments, the protein complex described herein is linked to the Fc region through 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.

The protein complex described herein can block the binding of endogenous TIGIT derived from immune cells to TIGIT ligands (e.g., PVR and Nectin-2) . In some embodiments, by binding to TIGIT ligands, the protein complex described herein can inhibit the interaction between one or more TIGIT ligands (e.g., those expressed on tumor cells) to endogenous TIGIT that is expressed on immune cells (e.g., myeloid cells, macrophages, and/or dendritic cells) . This consequently result in the interruption of TIGIT/TIGIT ligand signaling pathways (e.g., TIGIT/PVR and TIGIT/Nectin-2 signaling pathways) , thereby enhancing T cell activation and upregulating immune response.

The protein complex described herein can block the engagement between PD-L1 and endogenous PD-1 that are expressed on immune cells. In some embodiments, by binding to PD-L1, the protein complex described herein can inhibit the binding of PD-L1 (e.g., expressed on tumor cells) to endogenous PD-1 derived from immune cells (e.g., T cells) , thereby blocking PD-1/PD-L1 pathway, upregulating immune response, promoting T cell proliferation and cytokine production.

The protein complex described herein can block the engagement between CD47 and endogenous SIRPα that are expressed on immune cells. In some embodiments, by binding to CD47, the protein complex described herein can inhibit the binding of CD47 (e.g., that is expressed on tumor cells) to endogenous SIRPα that is expressed on immune cells (e.g., myeloid cells, macrophages, and dendritic cells) , thereby blocking CD47/SIRPα pathway and further enhancing immune responses and phagocytosis.

In some embodiments, the protein complex described herein can increase 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 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

In some implementations, the protein complex 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 a TIGIT ligand (e.g., a human TIGIT ligand, a monkey TIGIT ligand, or a mouse TIGIT ligand) with a dissociation rate (koff) of less than 0.1 s^-1, less than 0.01 s^-1, less than 0.001 s^-1, less than 0.0001 s^-1, or less than 0.00001 s^-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s^-1, greater than 0.001 s^-1, greater than 0.0001 s^-1, greater than 0.00001 s^-1, or greater than 0.000001 s^-1. In some embodiments, kinetic association rates (kon) is greater than 1 × 10²/Ms, greater than 1 × 10³/Ms, greater than 1 × 10⁴/Ms, greater than 1 × 10⁵/Ms, or greater than 1 × 10⁶/Ms. In some embodiments, kinetic association rates (kon) is less than 1 × 10⁵/Ms, less than 1 × 10⁶/Ms, or less than 1 × 10⁷/Ms. Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some embodiments, KD is less than 1 × 10^-6M, less than 1 × 10^-7M, less than 1 × 10^-8 M, less than 1 × 10^-9 M, or less than 1 × 10^-10 M. In some embodiments, the KD is less than 300 nM, 200 nM, 100 nM, 50nM, 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, KD is greater than 1 × 10^-7M, greater than 1 × 10^-8M, 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.

General techniques for measuring the affinity include, e.g., ELISA, RIA, and surface plasmon resonance (SPR) . In some embodiments, the protein complex described herein can bind to monkey CD47, and/or mouse CD47. In some embodiments, the protein complex described herein cannot bind to monkey CD47, and/or mouse CD47. In some embodiments, the protein complex described herein can bind to monkey PD-L1, and/or mouse PD-L1. In some embodiments, the protein complex described herein cannot bind to monkey PD-L1, and/or mouse PD-L1. In some embodiments, the protein complex described herein can bind to a monkey TIGIT ligand, and/or a mouse TIGIT ligand. In some embodiments, the protein complex described herein cannot bind to a monkey TIGIT ligand, and/or a mouse TIGIT ligand.

In some embodiments, thermal stabilities are determined. The protein complex described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃. In some embodiments, Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃.

In some embodiments, the protein complex described herein has a tumor growth inhibition percentage (TGI%) that is 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 complex described herein has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI%can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:
TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100

Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

In some embodiments, the tumor inhibitory effects of the protein complex described herein are comparable to an anti-CD47 reference antibody, e.g., Magrolimab (Hu5F9-G4) , and/or an anti-TIGIT reference antibody, e.g., Tiragolumab. Magrolimab and Tiragolumab are described e.g., in Sikic et al. "First-in-human, first-in-class phase I trial of the anti-CD47 antibody Hu5F9-G4 in patients with advanced cancers. " Journal of Clinical Oncology 37.12 (2019) : 946; and Rousseau, A., et al. "Anti-TIGIT therapies for solid tumors: a systematic review. " ESMO open 8.2 (2023) : 101184; each of which is incorporated herein by reference in its entirety. In some embodiments, the tumor inhibitory effects of the protein complex described herein are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, or 5 folds more than any of the anti-CD47 reference antibodies or anti-TIGIT reference antibodies described herein.

In some embodiments, the tumor inhibitory effects of the protein complex described herein are comparable to an anti-PD-L1 reference antibody, e.g., Atezolizumab (MPDL3280A) , or an anti-PD-1 antibody, e.g., Pembrolizumab. MPDL3280A is described e.g., in Powles, T. et al. "MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. " Nature 515.7528 (2014) : 558-562, which is incorporated herein by reference in its entirety.

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

Some other modifications to the Fc region can be made. For example, a cysteine residue (s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric fusion protein thus generated may have any increased half-life in vitro and/or in vivo.

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

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

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

In some embodiments, the protein complex described herein can bind to a TIGIT ligand (e.g., PVR-ECD/Fc, PVR-ECD/His, or Nectin-2-ECD/Fc) with an affinity that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%as compared to that of a reference protein (e.g., TIGIT/G4Fc or TIGIT/G1Fc) . In some embodiments, the protein complex described herein can bind to PD-L1 (e.g., PD-L1-ECD/Fc or PD-L1-ECD/His) with an affinity that is at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500%as compared to that of a reference protein (e.g., PD-1/G4Fc or PD-1-mt13/G4Fc) . In some embodiments, the protein complex described herein can bind to CD47 (e.g., CD47-ECD/His) with an affinity that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 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%, or at least 100%as compared to that of a reference protein (e.g., SIRPα/G4Fc, SIRPα-mt15/G4Fc, or SIRPα-mt15/G1Fc) .

In some embodiments, the protein complex described herein can bind to human PD-L1-expressing tumor cells (e.g., human PD-L1 tf CHO-S cells) with an affinity that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%as compared to that of a reference protein (e.g., PD-1/G4Fc, PD-1-mt13/G4Fc, or PD-1-mt13/G1Fc) . In some embodiments, the protein complex described herein can bind to human CD47-expressing tumor cells (e.g., human CD47 tf CHO-S cells) with an affinity that is at least 5%, 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%, at least 120%, at least 130%, at least 140%, or at least 150%as compared to that of a reference protein (e.g., SIRPα/G4Fc, SIRPα-mt15/G4Fc, or SIRPα-mt15/G1Fc) or a reference anti-CD47 antibody (e.g., Magrolimab analog) . In some embodiments, the protein complex described herein can bind to tumor cells expressing a TIGIT ligand (e.g., human PVR tf CHO-S cells) with an affinity that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, or at least 120%, at least 130%, at least 140%, or at least 150%as compared to that of a reference protein (e.g., TIGIT/G4Fc or TIGIT/G1Fc) .

In some embodiments, the protein complex described herein can bind to human PD-L1-expressing tumor cells (e.g., PD-L1 tf OE19) with an affinity that is at least 10%, at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, or at least 300%as compared to that of a reference protein (e.g., PD-1/G4Fc) . In some embodiments, the protein complex described herein can selectively bind to cells expressing PD-L1 (e.g., PD-L1 tf OE19) , e.g., in a mixture of cells of cells expressing PD-L1 and untransfected cells. In some embodiments, the selectivity of the protein complex is comparable or stronger than a reference protein (e.g., PD-1/G4Fc) .

In some embodiments, the protein complex described herein can bind to RBC cells or platelets (e.g., from human donors) with an affinity that is 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 that of an anti-CD47 reference antibody (e.g., Magrolimab analog) or a reference protein (e.g., SIRPα/G4Fc, SIRPα/G1Fc, SIRPα-mt15/G4Fc or SIRPα-mt15/G1Fc) .

In some embodiments, the protein complex described herein can bind to activated T cells with an affinity that is at least 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%, at least 130%, at least 140%, at least 150%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, or at least 300%as compared to that of a reference protein (e.g., TIGIT/G4Fc, TIGIT/G1Fc, PD-1/G4Fc, PD-1/G1Fc, SIRPα/G4Fc, or SIRPα/G1Fc) .

In some embodiments, the protein complex described herein can block the interaction between a TIGIT ligand (e.g., human PVR or fragments thereof) and TIGIT (e.g., human TIGIT or fragments thereof) . In some embodiments, the protein complex described herein can block the interaction between human TIGIT ligand-expressing cells (e.g., PVR tf CHO-Scells) and human TIGIT. 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%as compared to that of a reference anti-TIGIT antibody (e.g., Tiragolumab analog) or a reference protein (e.g., TIGIT/G4Fc or TIGIT/G1Fc) .

In some embodiments, the protein complex described herein can block the interaction between PD-L1 (e.g., human PD-L1 or fragments thereof) and PD-1 (e.g., human PD-1 or fragments thereof) . In some embodiments, the protein complex described herein can block the interaction between human PD-L1-expressing cells (e.g., PD-L1 tf CHO-S cells) and human PD-1. In some embodiments, the blocking ability of 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%as compared to that of a reference protein (e.g., PD-1/G1Fc, PD-1-mt13/G4Fc, or PD-1-mt13/G1Fc) .

In some embodiments, the protein complex described herein can block the interaction between PD-1 and PD-L1, thereby increasing the signal of an NFAT-Luc signaling blocking assay 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%, or at least 100%as compared to that of a reference protein (e.g., PD-1-mt13/G4Fc or PD-1-mt13/G1Fc) .

In some embodiments, the protein complex described herein can block the interaction between CD47 (e.g., human CD47 or fragments thereof) and SIRPα (e.g., human SIRPα or fragments thereof) . In some embodiments, the protein complex described herein can block the interaction between human CD47-expressing cells (e.g., CD47 tf CHO-S cells) and human SIRPα. In some embodiments, the blocking ability of the protein complex described herein is at least 20%, 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%as compared to that of an anti-CD47 reference antibody (e.g., Magrolimab) or a reference protein (e.g., SIRPα/G1Fc, SIRPα-mt15/G4Fc, or SIRPα-mt15/G1Fc) .

In some embodiments, the protein complex described herein does not induce hemagglutination. In some embodiments, the protein complex described herein can induce hemagglutination at a minimal concentration that is greater than 500-fold, 2000-fold, 5000-fold, 20000-fold, or 50000-fold as compared to that of an anti-CD47 reference antibody (e.g., Magrolimab analog) .

In some embodiments, the protein complex described herein can induce phagocytosis of tumor cells by macrophages (e.g., mouse RAW264.7 cells) . In some embodiments, the ability of the protein complex described herein to induce phagocytosis of tumor cells, e.g., FaDu cells or PD-L1-expressing tumor cells (e.g., PD-L1 tf OE19) , by macrophages is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%as compared to that of a reference anti-CD47 antibody (e.g., Magrolimab analog) or a reference protein (e.g., SIRPα/G1Fc, SIRPα-mt15/G4Fc, or SIRPα-mt15/G1Fc) . In some embodiments, the protein complex described herein has a weaker ability (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, 20%or 10%) to induce phagocytosis of RBC cells or platelets by macrophages (e.g., mouse RAW264.7 cells) than an anti-CD47 reference antibody (e.g., Magrolimab analog) or a reference protein (e.g., SIRPα/G1Fc, SIRPα-mt15/G4Fc, or SIRPα-mt15/G1Fc) .

In some embodiments, the protein complex described herein can induce phagocytosis of PD-L1-expressing tumor cells by mouse macrophages (e.g., RAW264.7 cells) . In some embodiments, the protein complex described herein can induce phagocytosis of PD-L1-expressing tumor cells by human macrophages (e.g., MDM cells) . In some embodiments, the protein complex described herein can induce phagocytosis of CD47-expressing tumor cells by mouse macrophages (e.g., RAW264.7 cells) . In some embodiments, the protein complex described herein can induce phagocytosis of CD47-expressing tumor cells by human macrophages (e.g., MDM cells) .

Endogenous expression of CD47 on a variety of cell types, including red blood cells, creates a formidable “antigen sink” that may limit the efficacy of CD47-targeting therapies. Thus, the weaker ability of the protein complex described herein to induce phagocytosis of RBC cells and/or platelets may increase the in vivo efficacy of the protein complex. In addition, the protein complex may be administered with a lower dose level and/or less frequent dosage schedule with similar efficacy than an anti-CD47 reference antibody (e.g., Magrolimab analog) .

In some embodiments, the protein complex described herein can enhance T cell response (e.g., in an MLR assay) . The principle of a mixed lymphocyte reaction (MLR) is that T cells from one donor will proliferate in the presence of APCs from a different donor. This is caused by the recognition of an HLA mismatch between two unrelated donors, which provokes an immune response from the T cells. MLR is often used as a means of inducing generalized stimulation/activation of T cells in culture. In some embodiments, the protein complex can increase the T cell proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%than control molecules used herein or combinations thereof. In some embodiments, the protein complex described herein can increase cytokine (e.g., IFN-γ and/or IL-2) production by at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, or 10000-fold than control molecules used herein or combinations thereof. In some embodiments, the protein complex described herein can increase cytokine (e.g., IFN-γ and/or IL-2) production by at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold than a combination of individual functional domains (e.g., TIGIT/Fc+PD-1/Fc+SIRPα/Fc) .

In some embodiments, the protein complex described herein does not induce cytokine storm in human. In some embodiments, the protein complex described herein is not a superagonist. Details of cytokine storm and superagonist can be found, e.g., in Shimabukuro-Vornhagen, A. et al. "Cytokine release syndrome. " Journal for ImmunoTherapy of Cancer 6.1 (2018) : 1-14, which is incorporated herein by reference in its entirety.

In some embodiments, the protein complex described herein can inhibit tumor growth. In some embodiments, the protein complex (e.g., any of the protein complexes described herein) can significantly inhibit tumor growth as compared the vehicle control. In some embodiments, the protein complex described herein can inhibit tumor growth with a TGI value that 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-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, as compared to that of a reference antibody or a reference protein in a mouse xenograft model. In some embodiments, the tumor cells are subcutaneously inoculated in immunocompromised or immunodeficient mice to generate the xenograft model. For example, the immunodeficient mice can be NPG^TM mice (NOD. Cg-PrkdcscidIl2rgtm1Vst/Vst mice) which are the NOD/SCID mice with the knock-out interleukin-2 gamma chain receptor. In some embodiments, the tumor volume of the mice can be analyzed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days post inoculation. In some embodiments, the TGI value of mice treated with the protein complex described herein can be 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%.

Methods of making protein complexes

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

Screening can be performed to increase binding affinity of the CD47-binding domains, PD-L1-binding domains, and/or TIGIT ligand-binding domains. Any combination of deletions, insertions, and/or combinations can be made to arrive at a variant that has increased binding affinity for the target. The amino acid changes introduced into the variant can also alter 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 such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.

The CD47-binding domains, PD-L1-binding domains, and/or TIGIT ligand-binding domains can be derived from any species of animal, 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) , chicken, goats, and rodents (e.g., rats, mice, hamsters, and rabbits) .

The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant polypeptides or fragments thereof by recombinant techniques.

As used herein, a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable to deliver and express one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-Atail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

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

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

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

As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HEK293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

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

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

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

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

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

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

The polypeptides can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

Methods of Treatment

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

In one aspect, the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.

In one aspect, the disclosure features methods that include administering a therapeutically effective amount of protein constructs or polypeptides disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer) , e.g., breast cancer (e.g., triple-negative breast cancer) , carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN) , renal cell carcinoma (RCC) , triple-negative breast cancer (TNBC) , or colorectal carcinoma. In some embodiments, the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, especially Non-Hodgkin′slymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the protein constructs or the polypeptides, vector comprising the polynucleotide encoding the protein constructs or the polypeptides, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

An effective amount can be administered in one or more administrations. By way of example, an effective amount of the protein constructs or the polypeptides is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro. As is understood in the art, an effective amount may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of the protein constructs or the polypeptides used.

Effective amounts and schedules for administering the protein constructs or the polypeptides, the polynucleotides encoding the protein constructs or the polypeptides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the protein constructs or the polypeptides, the polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of polynucleotides, and/or compositions disclosed herein used and other drugs being administered to the mammal.

A typical daily dosage of an effective amount of the protein constructs and/or the polypeptides is 0.1 mg/kg to 100 mg/kg (mg per kg of patient weight) . In some embodiments, the dosage can 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 can 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 to 10 mg/kg, about 1 to 5 mg/kg, or about 2 to 5 mg/kg.

In any of the methods described herein, the protein constructs or the polypeptides 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) .

In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the protein constructs or the polypeptides. In some embodiments, the one or more additional therapeutic agents are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the protein constructs or the polypeptides in the subject.

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

In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

In some embodiments, the additional therapeutic agent can comprise 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, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

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

In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

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) .

Pharmaceutical Compositions and Routes of Administration

Also provided herein are pharmaceutical compositions that contain the protein constructs, or the polypeptides described herein. The pharmaceutical compositions can be formulated in any manner known in the art.

Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . The compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the agents can be prolonged 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, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) .

Compositions containing the protein constructs, or the 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 a predetermined quantity of active compound for ease of administration and uniformity of dosage) .

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration) . Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, the agents can be formulated in aqueous solutions, preferably in physiologically compatible buffers to reduce discomfort at the site of injection. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the protein constructs or the polypeptides can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) . One can, for example, determine the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) : the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) . Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

Exemplary doses include milligram or microgram amounts of any of the protein constructs or the polypeptides described herein per kilogram of the subject’s weight (e.g., about 1 μg/kg to about 500 mg/kg; about 100 μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10 μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; about 1 μg/kg to about 50 μg/kg; about 1 mg/kg to about 10 mg/kg; or about 1 mg/kg to about 5 mg/kg) . While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents can vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life in vivo.

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

EXAMPLES

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

Example 1. Design of Fc-Based Designer Biologics (FBDB^TM) with TIGIT × PD-1 × SIRPα domains

The following examples demonstrate the application and efficacy of Fc-Based Designer Biologics (FBDB^TM) with TIGIT × PD-1 × SIRPα domains in enhancing the immune response. These triple-targeting formats of FBDB^TM were developed and optimized to enhance the synergistic power between innate immunity (e.g., targeting the SIRPα/CD47 and TIGIT/PVR pathways) and adaptive immunity (e.g., targeting the PD-1/PD-L1 and TIGIT/PVR pathways) .

Each format contains at least three distinct types of immune modules that are directly or indirectly connected to the Fc region of an IgG (e.g., human IgG4 or human IgG1) . The three immune modules are: (1) one or more PD-1 extracellular domains (ECDs) as PD-1 decoys that can block the PD-1/PD-L1 pathway to enhance the T cell function and guide the TIGIT × PD-1 × SIRPα molecules to the PD-L1-expressing tumors; (2) one or more SIRPα extracellular domains as SIRPα decoys that can stimulate the antigen-presentation by inducing phagocytosis; and (3) one or more TIGIT extracellular domains as TIGIT decoys that can interact with all the native TIGIT ligands and block the immunosuppressive signaling of TIGIT on NK, T, and Treg cells. For example, the one or more PD-1 extracellular domains can disrupt 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 functions; the one or more SIRPα extracellular domains can disrupt the interaction between CD47 expressed on tumor cells and SIRPα present on macrophages, thereby enabling macrophages to overcome the “don′t eat me” inhibitory brake; and the TIGIT extracellular domains can block the inhibitory signal on effector T cell and NK cells while inhibiting the activation signal on regulatory T cells by blocking the interaction between TIGIT and PVR.

TgPS is a triple-targeting FBDB^TM fusion protein platform designed with wildtype or variant domains of TIGIT × PD-1 × SIRPα that can be arranged in diverse alignments, resulting in the generation of a wide range of fusion proteins. For example, a group of fusion proteins named “TgPS_v1” was developed that includes five formats, i.e., TgPS-C1_v1, TgPS-C2_v1, TgPS-D_v1, TgPS-E_v1, and TgPS-F_v1, as shown in FIGS. 1A-1E, respectively. Each format includes a wildtype TIGIT extracellular domain (TIGIT-ECD-WT; SEQ ID NO: 1) , a wildtype PD-1 extracellular domain (PD-1-ECD-WT; SEQ ID NO: 2) , and a SIRPα extracellular domain (SIRPα-ECD-WT; SEQ ID NO: 3) . Mutations can also be introduced to the wildtype PD-1 extracellular domain and the wildtype SIRPα extracellular domain, to generate PD-1-ECD-mt13 (SEQ ID NO: 17) and SIRPα-ECD-mt15 (SEQ ID NO: 18) , respectively. As a result, another group of fusion proteins named “TgPS_v2” was developed that includes four formats, i.e., TgPS-C1_v2, TgPS-C2_v2, TgPS-D_v2, and TgPS-E_v2, as shown in FIGS. 15A-15D, respectively. Each format includes TIGIT-ECD-WT, PD-1-ECD-mt13, and SIRPα-ECD-mt15.

TgPS-C1_v1 (schematic structure shown in FIG. 1A) contains two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 12. Specifically, each polypeptide chain includes, from N-terminus to C-terminus, a PD-1-ECD-WT (SEQ ID NO: 2) , a TIGIT-ECD-WT (SEQ ID NO: 1) , a human IgG1 Fc region containing the hinge region (SEQ ID NO: 6) , and a SIRPα-ECD-WT (SEQ ID NO: 3) . The PD-1-ECD-WT is connected to the N-terminus of the TIGIT-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4) . The SIRPα-ECD-WT is connected to the C-terminus of the human IgG1 Fc via a A3S2 (G4S) ₂ linker peptide (SEQ ID NO: 5) .

TgPS-C2_v1 (schematic structure shown in FIG. 1B) contains two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 13. Specifically, each polypeptide chain includes, from N-terminus to C-terminus, a PD-1-ECD-WT (SEQ ID NO: 2) , a TIGIT-ECD-WT (SEQ ID NO: 1) , a human IgG4 (S228P) Fc region containing the hinge region (SEQ ID NO: 7) , and a SIRPα-ECD-WT (SEQ ID NO: 3) . The PD-1-ECD-WT is connected to the N-terminus of the TIGIT-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4) . The SIRPα-ECD-WT is connected to the C-terminus of the human IgG4 (S228P) Fc via a A3S2 (G4S) ₂ linker peptide (SEQ ID NO: 5) .

TgPS-D_v1 (schematic structure shown in FIG. 1C) contains two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 14. Specifically, each polypeptide chain includes, from N-terminus to C-terminus, a TIGIT-ECD-WT (SEQ ID NO: 1) , a PD-1-ECD-WT (SEQ ID NO: 2) , a human IgG4 (S228P) Fc region containing the hinge region (SEQ ID NO: 7) , and a SIRPα-ECD-WT (SEQ ID NO: 3) . The TIGIT-ECD-WT is connected to the N-terminus of the PD-1-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4) . The SIRPα-ECD-WT is connected to the C-terminus of the human IgG4 (S228P) Fc via a A3S2 (G4S) ₂ linker peptide (SEQ ID NO: 5) .

TgPS-E_v1 (schematic structure shown in FIG. 1D) contains two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 15. Specifically, each polypeptide chain includes, from N-terminus to C-terminus, a TIGIT-ECD-WT (SEQ ID NO: 1) , a human IgG4 (S228P) Fc region containing the hinge region (SEQ ID NO: 7) , a SIRPα-ECD-WT (SEQ ID NO: 3) , and a PD-1-ECD-WT (SEQ ID NO: 2) . The SIRPα-ECD-WT is connected to the C-terminus of the human IgG4 (S228P) Fc via a A3S2 (G4S) ₂ linker peptide (SEQ ID NO: 5) . The PD-1-ECD-WT is connected to the C-terminus of the SIRPα-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4) .

TgPS-F_v1 (schematic structure shown in FIG. 1E) contains two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 16. Specifically, each polypeptide chain includes, from N-terminus to C-terminus, a PD-1-ECD-WT (SEQ ID NO: 2) , a SIRPα-ECD-WT (SEQ ID NO: 3) , a human IgG4 (S228P) Fc region containing the hinge region (SEQ ID NO: 7) , and a TIGIT-ECD-WT (SEQ ID NO: 1) . The PD-1-ECD-WT is connected to the N-terminus of the SIRPα-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4) . The TIGIT-ECD-WT is connected to the C-terminus of the human IgG4 (S228P) Fc via a A3S2 (G4S) ₂ linker peptide (SEQ ID NO: 5) .

TgPS-C1_v2 (schematic structure shown in FIG. 15A) contains two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 19. Specifically, each polypeptide chain includes, from N-terminus to C-terminus, a PD-1-ECD-mt13 (SEQ ID NO: 17) , a TIGIT-ECD-WT (SEQ ID NO: 1) , a human IgG1 Fc region containing the hinge region (SEQ ID NO: 6) , and a SIRPα-ECD-mt15 (SEQ ID NO: 18) . The PD-1-ECD-mt13 is connected to the N-terminus of the TIGIT-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4) . The SIRPα-ECD-mt15 is connected to the C-terminus of the human IgG1 Fc via a A3S2 (G4S) ₂ linker peptide (SEQ ID NO: 5) .

TgPS-C2_v2 (schematic structure shown in FIG. 15B) contains two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 20. Specifically, each polypeptide chain includes, from N-terminus to C-terminus, a PD-1-ECD-mt13 (SEQ ID NO: 17) , a TIGIT-ECD-WT (SEQ ID NO: 1) , a human IgG4 (S228P) Fc region containing the hinge region (SEQ ID NO: 7) , and a SIRPα- ECD-mt15 (SEQ ID NO: 18) . The PD-1-ECD-mt13 is connected to the N-terminus of the TIGIT-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4) . The SIRPα-ECD-mt15 is connected to the C-terminus of the human IgG4 (S228P) Fc via a A3S2 (G4S) ₂ linker peptide (SEQ ID NO: 5) .

TgPS-D_v2 (schematic structure shown in FIG. 15C) contains two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 21. Specifically, each polypeptide chain includes, from N-terminus to C-terminus, a TIGIT-ECD-WT (SEQ ID NO: 1) , a PD-1-ECD-mt13 (SEQ ID NO: 17) , a human IgG4 (S228P) Fc region containing the hinge region (SEQ ID NO: 7) , and a SIRPα-ECD-mt15 (SEQ ID NO: 18) . The TIGIT-ECD-WT is connected to the N-terminus of the PD-1-ECD-mt13 via a (GSG) ₅ linker peptide (SEQ ID NO: 4) . The SIRPα-ECD-mt15 is connected to the C-terminus of the human IgG4 (S228P) Fc via a A3S2 (G4S) ₂ linker peptide (SEQ ID NO: 5) .

TgPS-E_v2 (schematic structure shown in FIG. 15D) contains two identical polypeptide chains, and each polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 22. Specifically, each polypeptide chain includes, from N-terminus to C-terminus, a TIGIT-ECD-WT (SEQ ID NO: 1) , a human IgG4 (S228P) Fc region containing the hinge region (SEQ ID NO: 7) , a SIRPα-ECD-mt15 (SEQ ID NO: 18) , and a PD-1-ECD-mt13 (SEQ ID NO: 17) . The SIRPα-ECD-mt15 is connected to the C-terminus of the human IgG4 (S228P) Fc via a A3S2 (G4S) ₂ linker peptide (SEQ ID NO: 5) . The PD-1-ECD-mt13 is connected to the C-terminus of the SIRPα-ECD-mt15 via a (GSG) ₅ linker peptide (SEQ ID NO: 4) .

The expressed proteins were purified by a protein A column, followed by HPLC-SEC (high-performance liquid chromatography coupled with size-exclusion chromatography; Agilent) . Specifically, the TgPS proteins were expressed in CHO-S cells. The culture supernatant was collected and subject to protein A purification. Next, the protein A column was equilibrated with 10× column volume of an equilibration buffer (25 mM Tris, 150 mM NaCl, pH 8.0) , and the culture supernatant was then loaded to the equilibrated protein A column. The column was then washed with 6× column volume of an equilibration buffer (25 mM Tris, 150 mM NaCl, pH 8.0) . The protein sample was eluted by 6× column volume of an elution buffer (100 mM acetate, 20 mM NaCl, pH 3.0) , and pH was adjusted to 7.0-7.2 by a buffer containing 1 M HEPES, pH 8.0. The results indicate that all TgPS proteins can be expressed and harvested with a high purity.

In addition, the amino acid sequences of SEQ ID NOs: 12-16, and 19-22 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.

Example 2. The binding ability of TgPS proteins to TIGIT ligand (s) , PD-L1, and CD47 detected by ELISA

ELISA assays were performed to test the binding ability of TgPS_v1 proteins to a TIGIT ligand, e.g., a fusion protein including a recombinant human PVR extracellular domain (SEQ ID NO: 47) fused with Fc (recombinant PVR-ECD/Fc fusion protein; SEQ ID NO: 48) , as shown in FIG. 2A. Specifically, 1 μg/mL of TgPS_v1 proteins and TIGIT/G4Fc were used to coat wells of 96-well EIA microplates overnight at 4℃. TIGIT/G4Fc was used as a positive control, which includes a TIGIT extracellular domain that is fused to an IgG4 Fc, with amino acid sequence set forth in SEQ ID NO: 23. After blocking with 5%skim milk diluted in phosphate-buffered saline (PBS) , serially diluted Biotin-PVR-ECD/Fc in PBS was added and incubated at 25℃ for 1 hour. The unbound Biotin-PVR-ECD/Fc was removed by washing 3 times with PBST (1×PBS supplemented with 0.05%20) . The HRP-conjugated Avidin secondary antibody (BioLegend, Cat. No.: 405103) was added to each well and incubated at 25℃ for 1 hour. Following 3 times of washing with PBST (containing 0.1%20) to remove excess secondary antibodies, TMB substrate was subsequently added to each well and incubated. Then, the reaction was stopped, and HRP activity shown as optical density (OD) was measured at 450 nm using a spectrophotometer (Varioskan LUK^TM, Thermo Scientific, Type 3020) .

Similar experiments were performed to test the binding ability of TgPS_v1 proteins to another TIGIT ligand, e.g., a fusion protein including a recombinant human Nectin-2 extracellular domain fused with Fc (recombinant human Nectin-2-ECD/Fc fusion protein) , as shown in FIG. 2B. Specifically, 1 μg/mL of human Nectin-2-ECD/Fc (Sino Biological, Cat. No.: 10005-H02H) was used to coat wells of 96-well EIA microplates. Biotin-TgPS_v1 proteins or Biotin-TIGIT/G4Fc in PBS were added to each microplate well for ligand binding. Other materials used and the general ELISA steps conducted were identical to those described above.

As shown in FIGS. 2A-2B, TgPS-C2/D/E/F_v1 proteins can bind to TIGIT ligands, both PVR and Nectin-2, whereas TgPS-C1_v1 can only bind to PVR. TgPS-D_v1 and TgPS-E_v1 exhibited stronger TIGIT ligand-binding activity compared to TgPS-C1_v1, TgPS-C2_v1, TgPS-F_v1, and the positive control protein TIGIT/G4Fc.

ELISA assays were also performed to test the binding ability of TgPS_v1 proteins to a PD-1 ligand, PD-L1 (FIG. 2C) and a SIRPα ligand, CD47 (FIG. 2D) . Similar experiments were performed as described above. In FIG. 2C, PD-1/G4Fc was used as a positive control, which includes a PD-1 extracellular domain that is fused to an IgG4 Fc, with amino acid sequence set forth in SEQ ID NO: 24. In FIG. 2D, SIRPα/G4Fc was used as a positive control, which includes a SIRPα extracellular domain that is fused to an IgG4 Fc, with amino acid sequence set forth in SEQ ID NO: 25.

As shown in FIGS. 2C-2D, all five TgPS_v1 proteins exhibited a decent and comparable binding ability to both PD-L1 and CD47, respectively, as detected by ELISA. Specifically, all TgPS_v1 proteins bound to PD-L1 more effectively than the positive control, PD-1/G4Fc. In addition, all TgPS_v1 proteins exhibited evident binding ability to CD47 compared to the negative control, although not reaching the level of efficacy demonstrated by the positive control, SIRPα/G4Fc.

ELISA assays were also performed to test the binding ability of TgPS_v2 proteins to recombinant human PVR-ECD/His (ACROBiosystems; Cat. No.: CD5-H5223) , PD-L1-ECD/His (Sino Biological; Cat. No.: 10084-H08H) and CD47-ECD/His (Sino Biological; Cat. No.: 12283-H08H) proteins, which include a His-tag at the C-terminus of the PVR extracellular domain, the PD-L1 extracellular domain, and the CD47 extracellular domain, respectively. Specifically, 1 μg/mL PVR-ECD/His (FIG. 16A) , PD-L1-ECD/His (FIG. 16B) , or CD47-ECD/His (FIG. 16C) was separately used to coat wells of 96-well EIA microplates. TgPS_v2 proteins and corresponding control proteins in PBS were added to each well for ligand binding. The HRP-conjugated anti-human IgG, Fcγ fragment specific secondary antibody (Jackson ImmunoResearch, Cat. No.: 109-035-008) and TMB substrate were used for signal measurement. Other materials used and the general ELISA steps conducted were identical to those described above. In FIG. 16A, both TIGIT/G1Fc and TIGIT/G4Fc were used as positive controls. TIGIT/G1Fc includes a TIGIT extracellular domain that is fused to an IgG1 Fc, with amino acid sequence set forth in SEQ ID NO: 26. In FIG. 16B, PD-1- mt13/G4Fc was used as a positive control, which includes a PD-1-ECD-mt13 that is fused to an IgG4 Fc, with amino acid sequence set forth in SEQ ID NO: 27. In FIG. 16C, both SIRPα-mt15/G4Fc and SIRPα-mt15/G1Fc were used as positive controls. SIRPα-mt15/G4Fc includes a SIRPα-ECD-mt15 that is fused to an IgG4 Fc, with amino acid sequence set forth in SEQ ID NO: 28. SIRPα-mt15/G1Fc includes a SIRPα-ECD-mt15 that is fused to an IgG1 Fc, with amino acid sequence set forth in SEQ ID NO: 29.

As shown in FIG. 16A, all four TgPS_v2 proteins displayed substantial binding ability to PVR, with TgPS-D_v2 exhibiting the strongest binding among them. The PVR-binding activity of all four TgPS_v2 proteins and the positive control proteins can be ranked as follows: TgPS-D_v2 ＞ TgPS-C2_v2, TgPS-C1_v2 ＞ TgPS-E_v2, TIGIT/G1Fc ＞ TIGIT/G4Fc. Besides TgPS-E_v2, other TgPS_v2 proteins showed stronger PVR-binding activity than the positive controls (TIGIT/G4Fc and TIGIT/G1Fc) .

As shown in FIG. 16B, all four TgPS_v2 proteins displayed substantial binding ability to PD-L1, with TgPS-C2_v2 exhibiting the strongest binding ability among them. The PD-L1-binding activity of all four TgPS_v2 proteins and the positive control protein can be ranked as follows: TgPS-C2_v2 ＞ TgPS-C1_v2, TgPS-D_v2, PD-1-mt13/G4Fc ＞ TgPS-E_v2. In particular, TgPS-C1_v2, TgPS-C2_v2, and TgPS-D_v2 proteins showed stronger PD-L1-binding activity than PD-1-mt13/G4Fc.

As shown in FIG. 16C, all four TgPS_v2 proteins displayed substantial binding ability to CD47, although not reaching the level of efficacy demonstrated by the positive control proteins, SIRPα-mt15/G4Fc and SIRPα-mt15/G1Fc.

These results together suggest that the variation in TgPS fusion protein formats is likely impact their ligand specific binding ability detected by ELISA.

Example 3. Simultaneous binding of TgPS_2 proteins to PVR, PD-L1, and CD47 detected by Bio-Layer Interferometry (BLI)

A three-way binding assay was performed using RED96 instrument with anti-human IgG Fc (AHC) biosensor tips. experiments were run with the 96-well black sample plates (200 μL/well) at 30℃ and 1000 RPM. TgPS_v2 proteins, recombinant human PVR, PD-L1, and CD47 proteins were prepared in an assay buffer (PBS, pH 7.4, 0.05%20) . The three-way binding assay was performed as follows: (1) setting a baseline by running with the assay buffer for 60 seconds; (2) loading 10 μg/mL each of the TgPS_v2 proteins to AHC biosensor tips for 600 seconds; (3) setting a baseline by running with the assay buffer for an additional 60 seconds; (4) letting association of serially diluted human CD47 protein (100 nM) for 180 seconds; (5) letting dissociation in the assay buffer for 5 seconds; (6) repeating steps (1) - (5) , whereas the associated protein was replaced by human PD-L1 (1000 nM) ; and (7) repeating steps (1) - (5) , whereas the associated protein was replaced by human PVR (1500 nM) . The biosensor tips were regenerated with 10 mM glycine pH 1.5 to repeat the measurements. Details of the three-way binding assays are summarized in the table below.

Table 1. Three-way binding of TgPS_v2 proteins

Three ways binding of TgPS molecules

As shown in FIGS. 17A-17D, the three-way binding data showed that TgPS_v2 proteins are competent to interact with PVR, PD-L1 and CD47 simultaneously. The three-arm format of TgPS_v2 proteins did not affect the ligand-binding activity of each arm. The results indicate that TgPS_v2 proteins may possess the potential to target PVR⁺PD-L1⁺CD47⁺ tumor cells.

Example 4. Whole cell binding ability of TgPS proteins to PVR tf CHO-S cells, PD-L1 tf CHO-S cells, and CD47 tf CHO-S cells

Whole cell binding ability of TgPS_v1 and TgPS_v2 proteins was tested by separately incubating 3 × 10⁴ cells from each type, namely cells transfected to express PVR (PVR tf cells) , cells transfected to express PD-L1 (PD-L1 tf cells) , or cells transfected to express CD47 (CD47 tf cells) , with serially diluted TgPS proteins or corresponding control proteins in FACS buffer (PBS supplemented with 4%FBS) at 4℃ for 30 minutes. After washing the cells with FACS buffer, the whole cell binding was detected by incubating the cells with R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ fragment specific antibody (Jackson ImmunoResearch, Cat. No: 109-115-098) at 4℃ for an additional 30 minutes. Then, the cells were subject to flow cytometry analysis using a CytoFLEX flow cytometer (Beckman Coulter Inc. ) .

As shown in FIGS. 3A-3B, all five TgPS_v1 proteins were able to bind to PD-L1 tf CHO-S cells and CD47 tf CHO-S cells, respectively. Specifically, in FIG. 3A, TgPS-C2_v1 showed the strongest PD-L1 binding activity among the TgPS_v1 proteins and PD-1/G4Fc. The binding activity to PD-L1 tf CHO-S cells can be ranked as follows: TgPS-C2_v1 ＞ TgPS-D_v1 ＞ TgPS-E_v1, TgPS-C1_v1, PD-1/G4Fc ＞ TgPS-F_v1. In FIG. 3B, all TgPS_v1 proteins exhibited substantial CD47-binding activity, although not reaching the level demonstrated by SIRPα/G4Fc.

As shown in FIGS. 18A-18C, all TgPS_v2 proteins were able to bind to PVR tf cells, PD-L1 tf cells, and CD47 tf cells, respectively. Specifically, in FIG. 18A, TgPS-D_v2 showed the strongest PVR-binding activity. The PVR-binding activity among all four TgPS_v2 proteins and the positive control proteins can be ranked as follows: TgPS-D_v2 ＞ TgPS-E_v2 ＞ TgPS-C2_v2 ＞ TgPS-C1_v2, TIGIT/G1Fc, TIGIT/G4Fc. In FIG. 18B, TgPS-C2_v2 exhibited the strongest PD-L1-binding ability. The PD-L1-binding activity among all four TgPS_v2 proteins and the positive control proteins can be ranked as follows: TgPS-C2_v2 ＞ TgPS-C1_v2, TgPS-D_v2, PD-1-mt13/G1Fc ＞ PD-1-mt13/G4Fc ＞ TgPS-E_v2. PD-1-mt13/G1Fc was used as a positive control, which includes a PD-1-ECD-mt13 that is fused to an IgG4 Fc, with amino acid sequence set forth in SEQ ID NO: 30. In FIG. 18C, all four TgPS_v2 proteins exhibited a comparable binding ability to CD47 tf cells, although not reaching the level of efficacy demonstrated by the positive control proteins including the Magrolimab analog (an anti-CD47 antibody analog; heavy chain sequence: SEQ ID NO: 43 and light chain sequence: SEQ ID NO: 44) , SIRPα-mt15/G1Fc, and SIRPα-mt15/G4Fc.

Overall, these results suggest that the variation in TgPS fusion protein formats can impact their ligand specific binding ability detected by whole cell binding assays.

Example 5. The binding ability of TgPS proteins to RBCs and platelets

The binding ability of TgPS_v1 and TgPS_v2 proteins to red blood cells (RBCs) and platelets was tested by incubating 3 × 10⁵ RBCs (FIG. 4A) or 5 × 10⁵ platelets (FIG. 4B) freshly obtained from healthy human donors with serially diluted TgPS proteins or corresponding control proteins in modified FACS buffer (1 × PBS supplemented with 4%FBS) at 4℃ for 30 minutes. After washing the cells with FACS buffer, the binding with RBCs or platelets was detected by incubating the RBCs or platelets with R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ fragment specific antibody (Jackson ImmunoResearch, Cat. No: 109-115-098) at 4℃ for an additional 30 minutes. After the incubation, the RBCs or platelets were subject to flow cytometry analysis using a CytoFLEX flow cytometer (Beckman Coulter Inc. ) .

As shown in FIG. 4A, none of the TgPS_v1 proteins bound to RBCs at any of the concentrations tested, whereas the positive control, Magrolimab analog, demonstrated evident RBC binding. FIG. 4B showed that no platelet binding was detected for TgPS-C1/C2/D/E_v1 proteins at any of the concentrations tested. TgPS-F_v1 showed a detectable level of binding to platelets at its highest concentration tested (1000 nM) . These results suggest that TgPS_v1 proteins are less likely to induce binding to RBCs or platelet compared to the positive control, Magrolimab analog. SIRPα/G1Fc was used as a positive control, which includes a SIRPα extracellular domain that is fused to an IgG1 Fc, with amino acid sequence set forth in SEQ ID NO: 32.

As shown in FIGS. 19A-19B, all TgPS_v2 proteins showed a detectable RBC-and platelet-binding activity, but it was notably weaker as compared to the positive control proteins, including Magrolimab analog, SIRPα-mt15/G1Fc, and SIRPα-mt15/G4Fc. These results suggest that the possible risk of TgPS_v2 proteins to induce RBC or platelet binding is low.

Example 6. Selective binding ability of TgPS_v1 proteins to PD-L1-overexpressing tumor cells

The selective binding ability of TgPS_v1 proteins to PD-L1-overexpressing tumor cells was tested by incubating 2 × 10⁴ CellTrace^TM-CFSE (Thermo Fisher Scientific, Cat. No: C34554) -labeled OE19 (PVR⁺CD47⁺PD-L1^low) cells and 2 × 10⁴ of CellTrace^TM-Violet (Thermo Fisher Scientific, Cat. No: C34557) -labeled PD-L1 tf OE19 cells (PVR⁺CD47⁺PD-L1⁺) with serially diluted TgPS proteins or corresponding control proteins in FACS buffer (PBS supplemented with 4%FBS) at 4℃ for 30 minutes. After washing the cells with FACS buffer, the binding was detected by incubating with R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ fragment specific antibody (Jackson ImmunoResearch, Cat. No: 109-115-098) at 4℃ for an additional 30 minutes. After the incubation, the cells were subject to flow cytometry analysis using a CytoFLEX flow cytometer (Beckman Coulter Inc. ) . The cell populations were gated based on PE signals, and the percentages of different cell types were calculated using the Kaluza analysis software (Beckman Coulter Inc. ) .

As shown in FIG. 5A, little or no binding signal was detected between any of the tested proteins and OE19 cells. By contrast, in FIG. 5B, all TgPS_v1 proteins preferentially bound to PD-L1 tf OE19 cells in the mixture of OE19 cells and PD-L1 tf OE19 cells (at a ratio of 1: 1) . This selective binding to PD-L1 tf OE19 cells observed for all TgPS_v1 proteins was even stronger than that of the positive control, PD-1/G4Fc.

Example 7. The binding ability of TgPS_v1 proteins to activated T cell

The binding ability of TgPS_v1 proteins to activated T cells were tested by incubating 3 × 10⁴ Dynabeads^TM Human T-Activator CD3/CD28 (Thermo Fisher Scientific, Cat. No: 11131D) -activated T cells (bead-to-cell ratio = 1: 1) with the serially diluted TgPS_v1 proteins and corresponding control proteins in FACS buffer (PBS supplemented with 4%FBS) at 4℃ for 30 minutes. The cells were washed with FACS buffer, and the binding was detected by incubating the cells with R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ fragment specific antibody (Jackson ImmunoResearch, Cat. No: 109-115-098) at 4℃ for an additional 30 minutes. Then, the cells were subject to flow cytometry analysis using a CytoFLEX flow cytometer (Beckman Coulter Inc. ) .

As shown in FIG. 6, all TgPS_v1 proteins were able to bind to activated T cells that express PVR (low) , PD-L1 (low) and CD47 (high) . In particular, TgPS-C1_v1 exhibited the strongest binding efficacy among the tested TgPS_v1 proteins and control proteins.

Example 8. Blocking effect of TgPS proteins on the interaction between TIGIT and PVR

The blocking effect of TgPS_v1 and TgPS_v2 proteins on the interaction between TIGIT and PVR was determined as follows. 3 × 10⁴ PVR tf CHO-S cells were co-incubated with serially diluted TgPS proteins or corresponding control proteins, along with 250 nM of Biotin-TIGIT/G4Fc proteins in FACS buffer (PBS supplemented with 4%FBS) , at 4℃ for 30 minutes. After washing, 0.3 μg streptavidin-PE (or “SA-PE” ; eBioscience, Cat. No: 12- 4317-87) was added to each well, and PE signals from the cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc. ) .

The blocking activity in FIG. 7B was calculated as follows:

[1- (Mean-PE value of each tested protein at 1000 nM) / (the subtract difference of Mean-PE value between SA-PE only group and Biotin-TIGIT/G4Fc group) ] ×100%.

The blocking activity in FIG. 20B was calculated as follows:

[1- (Mean-PE value of each tested protein at 125 nM) / (the subtract difference of Mean-PE value between SA-PE only group and Biotin-TIGIT/G4Fc group) ] ×100%.

The calculated value of blocking activity increases (FIG. 7B and FIG. 20B) as the TIGIT/PVR binding declines (FIG. 7A and FIG. 20A) . The SA-PE only group was used as the staining background control.

As shown in FIGS. 7A-7B, all TgPS_v1 proteins were able to block the interaction between TIGIT and PVR, with TgPS-D_v1 and TgPS-E_v1 showing a similar blocking efficacy to Tiragolumab analog (an anti-TIGIT antibody analog; heavy chain sequence: SEQ ID NO: 45 and light chain sequence: SEQ ID NO: 46) and TIGIT/G4Fc. The blocking activity can be ranked as follows: TgPS-E_v1, TIGIT/G4Fc ＞ Tiragolumab analog (anti-TIGIT) , TgPS-D_v1 ＞ TgPS-C2_v1, TgPS-C1_v1 ＞ TgPS-F_v1. Notably, the blocking activity of TgPS_v1 proteins against TIGIT/PVR binding aligns with their PVR-binding ability.

As shown in FIGS. 20A-20B, all TgPS_v2 proteins were able to block the interaction between TIGIT and PVR. Taking TgPS-C1/C2/D/E_v1 together with all four TgPS_v2 proteins in this analysis, the blocking activity can be ranked as follows: TgPS-E_v1, TgPS-E_v2, Tiragolumab analog (anti-TIGIT) , TgPS-D_v2 ＞ TgPS-D_v1 ＞ TgPS-C2_v1, TgPS-C2_v2, TIGIT/G1Fc, TIGIT/G4Fc ＞ TgPS-C1_v1, TgPS-C1_v2. The similar trend and ranking observed among different formats of TgPS_v1 proteins and TgPS_v2 proteins are likely attributed to the presence of the same TIGIT arm in all TgPS fusion proteins.

Example 9. Blocking effect of TgPS proteins on the interaction between PD-1 and PD-L1

The blocking effect of TgPS_v1 and TgPS_v2 proteins on the interaction between PD-1 and PD-L1 was determined as follows. 3 × 10⁴ cells PD-L1 tf CHO-S cells were co-incubated with serially diluted TgPS proteins or corresponding control proteins, along with 2 μg/mL Biotin-PD-1/G4Fc in FACS buffer (PBS supplemented with 4%FBS) , at 4℃ for 30 minutes. After washing, 0.3 μg streptavidin-PE (or “SA-PE” ; eBioscience, Cat. No: 12-4317-87) was added to each well, and PE signals from the cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc. ) . The blocking activity in FIG. 8B and FIG. 21B was calculated as follows:

[1- (Mean-PE value of each tested molecule at 125 nM) / (the subtract difference of Mean-PE value between SA-PE only group and Biotin-PD-1/G4Fc group) ] ×100%.

The calculated value of blocking activity increases (FIG. 8B and FIG. 21B) as the PD-1/PD-L1 binding declines (FIG. 8A and FIG. 21A) . PD-1/G1Fc was used as a positive control, which includes a PD-1 extracellular domain that is fused to an IgG1 Fc, with amino acid sequence set forth in SEQ ID NO: 34. The SA-PE only group was used as the staining background control.

The signaling blocking effect of TgPS_v2 proteins on the interaction between PD-1 and PD-L1 was also testified by NFAT-Luc signaling blocking assays (FIG. 23) . Briefly, 4 × 10⁴ PD-L1 tf aAPC/CHO-K1 cells and 5 × 10⁴ PD-1 tf NFAT-Luc Jurkat cells were co-incubated with the serially diluted TgPS_v2 proteins or corresponding control proteins at 37℃ for 4 hours. Then, steadylite^TM plus reporter gene assay system reagent (PerkinElmer, Cat. No: 6066759) was added to each reaction. After a 15-minute incubation, the reaction mixture was transferred into 96-well regular white plate (Greiner Bio-One, Cat. No: 655098) and the luciferase activity was measured by a spectrophotometer (Varioskan LUK^TM, Thermo Scientific, Type 3020) .

As shown in FIGS. 8A-8B, all TgPS_v1 proteins were able to block the interaction between PD-1 and PD-L1, with TgPS-C1_v1 and TgPS-C2_v1 showing similar blocking efficacy to the positive control PD-1/G1Fc. The blocking activity can be ranked as follows: TgPS-C2_v1, TgPS-C1_v1, PD-1/G1Fc ＞ TgPS-F_v1, TgPS-D_v1, TgPS-E_v1.

As shown in FIGS. 21A-21B, all TgPS_v2 proteins were able to block the interaction between PD-1 and PD-L1. Taking TgPS-C1/C2/D/E_v1 together with all four TgPS_v2 proteins in this analysis, the blocking activity can be ranked as follows: TgPS-C2_v1, TgPS-C2_v2, PD-1-mt13/G4Fc, PD-1-mt13/G1Fc ＞ TgPS-C1_v2 ＞ TgPS-D_v2 ＞ TgPS-E_v2 ＞ TgPS-C1_v1 ＞ TgPS-D_v1 ＞ TgPS-E_v1. Specifically, among all the tested proteins, TgPS-C2_v1 and TgPS-C2_v2 showed a stronger blocking activity against PD-1/PD-L1 binding than other TgPS_v1 and TgPS_v2 proteins, and a similar blocking activity as compared to PD-1-mt13/G1Fc and PD-1-mt13/G4Fc. Overall, the blocking effect of TgPS_v2 proteins was stronger than that of TgPS_v1 proteins, and this result is likely attributed to the presence of PD-1-ECD-mt13 in TgPS_v2 proteins.

The results shown in FIG. 23 reinforced the findings, at least partly demonstrated in FIGS. 21A-21B, that all TgPS_v2 proteins were able to block the interaction between PD-1 and PD-L1, and TgPS-C2_v2 showed stronger blocking activity against PD-1/PD-L1 binding than other TgPS_v1 and TgPS_v2 proteins, as well as PD-1-mt13/G1Fc and PD-1-mt13/G4Fc. The blocking activity can be ranked as follows: TgPS-C2_v2 ＞ TgPS-C1_v2 ＞ TgPS-D_v2 ＞ PD-1-mt13/G1Fc ＞ PD-1-mt13/G4Fc, TgPS-E_v2. The results also indicate that TgPS-E_v2 did not block the negative signaling of PD-1-PD-L1 interaction.

Example 10. Blocking effect of TgPS proteins on the interaction between SIRPα and CD47

The blocking effect of TgPS_v1 and TgPS_v2 proteins against the interaction between SIRPα and CD47 was determined as follows. 3 × 10⁴ cells of CD47 tf CHO-S cells were co-incubated with the serially diluted TgPS molecules or corresponding control proteins, along with 1 μg/mL Biotin-SIRPα/Fc in FACS buffer (PBS supplemented with 4%FBS) , at 4℃ for 30 minutes. After washing, 0.3 μg streptavidin-PE (or “SA-PE” ; eBioscience, Cat. No: 12-4317-87) was added to each well, and PE signals from the cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc. ) .

The blocking activity in FIG. 9B was calculated as follows:

[1- (Mean-PE value of each tested molecule at 125 nM) / (the subtract difference of Mean-PE value between SA-PE only group and Biotin-SIRPα/G4Fc group) ] ×100%.

The blocking activity in FIG. 22B was calculated as follows:

[1- (Mean-PE value of each tested molecule at 15.6 nM) / (the subtract difference of Mean-PE value between SA-PE only group and Biotin-SIRPα/G4Fc group) ] ×100%.

The calculated value of blocking activity increases (FIG. 9B and FIG. 22B) as the SIRPα/CD47 binding declines (FIG. 9A and FIG. 22A) . The SA-PE only group was used as the staining background control.

As shown in FIGS. 9A-9B, all five TgPS_v1 proteins were able to block the interaction between SIRPα and CD47, although not reaching the level of blocking efficacy demonstrated by the positive control proteins, including Magrolimab analog and SIRPα/G1Fc. Specifically, the blocking activity can be ranked as follows: Magrolimab analog, SIRPα/G1Fc ＞ TgPS-C1_v1, TgPS-C2_v1, TgPS-D_v1, TgPS-F_v1 ＞ TgPS-E_v1.

As shown in FIGS. 22A-22B, all TgPS_v2 molecules were able to block the interaction between SIRPα and CD47. Taking TgPS-C1/C2/D/E_v1 together with all four TgPS_v2 proteins in this analysis, the blocking activity can be ranked as follows: SIRPα-mt15/G4Fc, SIRPα-mt15/G1Fc, Magrolimab analog (anti-CD47) ＞ TgPS-C1_v2 ＞ TgPS-C2_v2, TgPS-D_v2, TgPS-E_v2 ＞ TgPS-C1_v1, TgPS-C2_v1, TgPS-D_v1 ＞ TgPS-E_v1. Although not reaching the level of blocking efficacy demonstrated by the positive control proteins, TgPS_v2 proteins overall showed a stronger blocking activity than TgPS_v1 proteins. This result is likely attributed to the presence of SIRPα-ECD-mt15 in TgPS_v2 proteins.

Example 11. Hemagglutination (HA) activity induced by TgPS_v1 proteins

To determine the HA activity induced by TgPS_v1 proteins, a 10%RBC solution was prepared from whole blood of a healthy donor. The RBCs were washed twice with 0.9%NaCl buffer, and then diluted to 10%by volume in 0.9%NaCl buffer. The 10%RBC solution were then incubated with serially diluted TgPS_v1 proteins and corresponding control proteins in a round-bottom 96-well plate at room temperature overnight. An image of the plate was captured on the next day. As a result, agglutinated RBCs coated the wells evenly, whereas non-agglutinated cells formed a distinct red dot at the bottom of each well.

FIG. 10 is a picture of the 96-well plate captured on the next day after overnight incubation. The image showed that only Magrolimab analog induced evident HA activity, whereas none of the five TgPS_v1 proteins induced HA activity within the indicated concentration range.

Example 12. TgPS proteins induced macrophage-mediated phagocytosis on tumor cells (FaDu) and PD-L1 tf tumor cells (PD-L1 tf OE19)

To determine macrophage-mediated phagocytosis on tumor cells and RBCs induced by TgPS_v1 and TgPS_v2 proteins, phagocytosis assays were performed as follows. FaDu cells (FIG. 11A) and PD-L1-overexpressing tumor cells (PD-L1 tf OE19) (FIG. 11B) were labeled with 5 μM or 10 μM CellTrace^TM-CFSE (Thermo Fisher Scientific, Cat. No: C34554) at room temperature for 10 minutes, followed by washing with complete culture medium. Then, 1 × 10⁵ -5 × 10⁵ cells/well of CFSE-labeled FaDu cells or CFSE-labeled PD-L1 tf OE19 (target cells) were added to each well and incubated with serially diluted TgPS proteins and corresponding control proteins at 37℃ for 30 minutes. Afterwards, 5 × 10⁴ mouse macrophage RAW264.7 cells were added to each well and incubated at 37℃ for 2 hours. The RAW264.7 cells were stained with PE-Cyanine7 conjugated F4/80 antibody (Invitrogen, Cat. No: 25-4801-82) . The phagocytic activity of the tested proteins was evaluated by calculating the percentage of CFSE⁺ F4/80⁺ macrophages (indicating macrophages phagocytosed CFSE-labeled target cells) from the total F4/80 signals (indicating total macrophages) by a CytoFLEX flow cytometer (Beckman Coulter Inc. ) .

An shown in FIG. 11A, TgPS-C1_v1 was able to induce substantial phagocytic activity against FaDu cells (PVR^medPD-L1^medCD47^med) . TgPS-C2_v1 and TgPS-F_v1 induced slight phagocytic activity at higher concentrations. In FIG. 11B, TgPS-C1_v1. TgPS-C2_v1, and TgPS-D_v1 showed substantial phagocytic activity against PD-L1 tf OE19 cells (PVR^medPD-L1^highCD47^low) at a similar level of efficacy to the positive control protein SIRPα/G1Fc. These results suggested that some of the TgPS_v1 proteins can promote macrophages-mediated phagocytosis on PD-L1-overexpressing tumor cells.

An shown in FIG. 24A, among all the tested TgPS_v1 and TgPS_v2 proteins, TgPS-C1_v2 and TgPS-C2_v2 induced a stronger phagocytic activity against FaDu cells than other TgPS_v1 and TgPS_v2 proteins. Overall, TgPS_v2 proteins were able to promote macrophage-mediated phagocytosis more effectively than TgPS_v1 proteins. This result is likely attributed to the presence of SIRPα-ECD-mt15 in TgPS_v2 proteins. In FIG. 24B, TgPS-C1/C2/D/E_v1 and all four TgPS_v2 proteins exhibited a substantial efficacy on promoting phagocytosis on PD-L1 tf OE19 cells, with TgPS-C1_v2 and TgPS-C1_v1 showing the strongest ability. The phagocytosis-inducing activity can be ranked as follows: SIRPα-mt15/G1Fc, Magrolimab analog (anti-CD47) ＞ TgPS-C1_v2, TgPS-C1_v1 ＞ TgPS-C2_v2, TgPS-E_v2, SIRPα-mt15/G4Fc ＞ TgPS-C2_v1, TgPS-D_v1, TgPS-E_v1 ＞ TgPS-D_v2.

Example 13. TgPS proteins induced macrophage-mediated phagocytosis on RBCs and/or platelets

The phagocytosis assay with RBCs and platelets were conducted following the steps and materials that are described in Example 12. Specifically, For TgPS_v1 proteins, the phagocytosis assay was conducted only with RBCs, and the results are shown in FIG. 11C. For TgPS_v2 proteins, the phagocytosis on both RBCs and platelets were tested.

As shown in FIG. 11C, the positive control proteins, Magrolimab analog (anti-CD47) and SIRPα/G1Fc, induced evident phagocytic activities against RBCs. By contrast, TgPS_v1 proteins induced minor or no phagocytic activities against RBCs. Specifically, only TgPS-C1_v1 showed minimal phagocytosis-promoting effect at higher concentrations. The data indicated that TgPS_v1 proteins are less likely to induce adverse events of phagocytosis on RBCs.

As shown in FIGS. 25A-25B, the positive control proteins Magrolimab analog and SIRPα-mt15/G1Fc induced evident phagocytic activities against RBCs and platelets. All TgPS_v1 and TgPS_v2 proteins tested only induced minimal or no phagocytic activities against RBCs and platelets at higher concentrations. The data, reinforcing and supplementing the findings described in FIG. 11C, suggested that TgPS_v1 and TgPS_v2 proteins are less likely to induce adverse events of phagocytosis on RBCs and platelets.

Example 14. Evaluation of TgPS protein-induced T cell responses by MLR assays

Mixed lymphocyte reaction (MLR) assays were performed to determine the enhancement of T cell responses by TgPS_v1 and TgPS_v2 proteins. Specifically, 1 × 10⁵ total T cells and 1 × 10⁴ dendritic cells (DCs) were co-incubated with 100 nM, 10 nM, or 1 nM of the TgPS proteins, or 100 nM of corresponding control proteins. After an incubation period of five days, culture supernatant was collected to measure IL-2 and IFN-γ expressions using Human IL-2 ELISA MAX^TM Deluxe Set (BioLegend, Cat. No: 431805) and Human IFN-γ ELISA MAX^TM Deluxe Set (BioLegend, Cat. No: 430105) , respectively.

As shown in FIG. 12A, TgPS-C1_v1, TgPS-C2_v1, TgPS-D_v1 induced IL-2 expression and TgPS-C1_v1 induced the highest IL-2 expression level. In particular, cells treated with TIGIT/Fc+PD-1/Fc+SIRPα/Fc showed a lower IL-2 expression level as compared to that of TgPS-C1_v1. As shown in FIG. 12B, TgPS-C1_v1 induced more IFN-γ expression compared to other TgPS_v1 proteins and TIGIT/Fc+PD-1/Fc+SIRPα/Fc combination.

As shown in FIG. 26A, TgPS-C1_v2, TgPS-C2_v2, and TgPS-D_v2 induced IL-2 expression. In particular, TgPS-C1_v2 and TgPS-C2_v2 induced the highest IL-2 expression level. In addition, cells treated with TIGIT/Fc+PD-1-mt13/Fc+SIRPα-mt15/Fc combination did not induce IL-2 expression. As shown in FIG. 26B, TgPS-C2_v2 induced more IFN-γ expression compared to TgPS-C1_v2, TgPS-D_v2 and TIGIT/Fc+PD-1-mt13/Fc+SIRPα-mt15/Fc combination.

These results suggested that some formats among TgPS_v1 and TgPS_v2 proteins were able to promote T cell activation and response.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not 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

A protein complex, comprising:

(a) an Fc;

(b) a TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains) ligand-binding domain;

(c) a PD-L1 (programmed death-ligand 1) -binding domain; and

(d) a CD47-binding domain.
The protein complex of claim 1, wherein the TIGIT ligand-binding domain can bind to a cell (e.g., cancer cell) expressing a TIGIT ligand (e.g., PVR or Nectin-2) and/or block the interaction between TIGIT and the TIGIT ligand.
The protein complex of claims 1-2, wherein the TIGIT ligand-binding domain is or comprises a TIGIT extracellular domain.
The protein complex of claim 3, wherein the TIGIT extracellular 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: 1.
The protein complex of any one of claims 1-4, wherein the PD-L1-binding domain can bind to a cell (e.g., cancer cell) expressing PD-L1 and/or block the interaction between PD-1 (programmed cell death protein 1) and PD-L1.
The protein complex of any one of claims 1-5, wherein the PD-L1-binding domain is or comprises a PD-1 extracellular domain.
The protein complex of claim 6, wherein the PD-1 extracellular 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: 2.
The protein complex of claim 6, wherein the PD-1 extracellular domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 36.
The protein complex of claim 8, wherein the amino acid that corresponds to S39 of SEQ ID NO: 36 is H.
The protein complex of claim 8 or 9, wherein the PD-1 extracellular domain further comprises a PD-L1 surface interaction sequence, wherein the PD-L1 surface interaction sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 37, 38, 39, or 40.
The protein complex of any one of claims 1-6 and 8-10, wherein the PD-L1-binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 17.
The protein complex of claim 1-11, wherein the CD47-binding domain can bind to a cell (e.g., cancer cell) expressing CD47 and/or block the interaction between CD47 and signal regulatory protein α (SIRPα) .
The protein complex of any one of claims 1-12, wherein the CD47-binding domain is or comprises a SIRPα extracellular domain.
The protein complex of claim 13, wherein the SIRPα extracellular domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 3.
The protein complex of claim 14, wherein the SIRPα extracellular domain comprises one or more amino acid mutations at positions corresponding to H24, I31, E54, G55, H56, and/or D73 of SEQ ID NO: 3.
The protein complex of claim 14 or 15, wherein the SIRPα extracellular domain comprises one or more of the following:

(a) the amino acid that corresponds to H24 of SEQ ID NO: 3 is R;

(b) the amino acid that corresponds to I31 of SEQ ID NO: 3 is T;

(c) the amino acid that corresponds to E54 of SEQ ID NO: 3 is A;

(d) the amino acid that corresponds to G55 of SEQ ID NO: 3 is K;

(e) the amino acid that corresponds to H56 of SEQ ID NO: 3 is Q; and

(f) the amino acid that corresponds to D73 of SEQ ID NO: 3 is I.
The protein complex of any one of claims 1-16, 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: 18.
The protein complex of any one of claims 1-17, wherein the TIGIT ligand-binding domain is linked to the N-terminus of a CH2 domain in the Fc, optionally via a hinge region.
The protein complex of claim 18, wherein the PD-L1-binding domain is linked to the N-terminus of TIGIT ligand-binding domain, optionally via a first linker peptide.
The protein complex of claim 19, wherein the CD47-binding domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a second linker peptide.
The protein complex of claim 18, wherein the CD47-binding domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a first linker peptide.
The protein complex of claim 21, wherein the PD-L1-binding domain is linked to the C-terminus of the CD47-binding domain, optionally via a second linker peptide.
The protein complex of any one of claims 1-17, wherein the PD-L1-binding domain is linked to the N-terminus of a CH2 domain in the Fc, optionally via a hinge region.
The protein complex of claim 23, wherein the TIGIT ligand-binding domain is linked to the N-terminus of the PD-L1 binding domain, optionally via a first linker peptide.
The protein complex of claim 24, wherein the CD47-binding domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a second linker peptide.
The protein complex of any one of claims 1-17, wherein the CD47-binding domain is linked to the N-terminus of a CH2 domain in the Fc, optionally via a hinge region.
The protein complex of claim 26, wherein the PD-L1-binding domain is linked to the N-terminus of the CD47-binding domain, optionally via a first linker peptide.
The protein complex of claim 27, wherein the TIGIT ligand-binding domain is linked to the C-terminus of a CH3 domain in the Fc, optionally via a second linker peptide.
The protein complex of any one of claims 1-28, wherein the Fc is human IgG1 Fc.
The protein complex of any one of claims 1-28, wherein the Fc is human IgG4 Fc.
The protein complex of any one of claims 18-28 and 30, wherein the hinge region is a human IgG4 hinge region optionally with S228P mutation according to EU numbering.
A protein complex, comprising

(a) a first polypeptide comprising from N-terminus to C-terminus: a first PD-L1-binding domain, an optional first linker peptide, a first TIGIT ligand-binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and a first CD47-binding domain; and

(b) a second polypeptide comprising from N-terminus to C-terminus: a second PD-L1-binding domain, an optional third linker peptide, a second TIGIT ligand-binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and a second CD47-binding domain.
The protein complex of claim 32, wherein the first PD-L1-binding domain and/or the second PD-L1-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 17.
The protein complex of claim 32 or 33, wherein the first TIGIT ligand-binding domain and/or the second TIGIT ligand-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 1.
The protein complex of claim 32-34, wherein the first CD47-binding domain and/or the second CD47-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 3 or 18.
The protein complex of any one of claims 32-35, wherein the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 8.
The protein complex of any one of claims 32-36, wherein the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 9.
The protein complex of any one of claims 32-35, wherein the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 10.
The protein complex of any one of claims 32-35 and 38, wherein the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 11.
The protein complex of any one of claims 32-39, wherein the first linker peptide and/or the third linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 4.
The protein complex of any one of claims 32-40, wherein the second linker peptide and/or the fourth linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 5.
The protein complex of any one of claims 32-41, wherein the first polypeptide and/or the second polypeptide comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 12, 13, 19, or 20.
A protein complex, comprising

(a) a first polypeptide comprising from N-terminus to C-terminus: a first TIGIT ligand-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 a first CD47-binding domain; and

(b) a second polypeptide comprising from N-terminus to C-terminus: a second TIGIT ligand-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 a second CD47-binding domain.
The protein complex of claim 43, wherein the first TIGIT-binding domain and/or the second TIGIT-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 1.
The protein complex of claim 43 or 44, wherein the first PD-L1-binding domain and/or the second PD-L1-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 17.
The protein complex of any one of claims 43-45, wherein the first CD47-binding domain and/or the second CD47-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 3 or 18.
The protein complex of any one of claims 43-46, wherein the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 8.
The protein complex of any one of claims 43-47, wherein the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 9.
The protein complex of any one of claims 43-46, wherein the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 10.
The protein complex of any one of claims 43-46 and 49, wherein the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 11.
The protein complex of any one of claims 43-50, wherein the first linker peptide and/or the third linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 4.
The protein complex of any one of claims 43-51, wherein the second linker peptide and/or the fourth linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 5.
The protein complex of any one of claims 43-52, wherein the first polypeptide and/or the second polypeptide comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 14 or 21.
A protein complex, comprising

(a) a first polypeptide comprising from N-terminus to C-terminus: a first TIGIT ligand-binding domain, an optional first hinge region, a first Fc region, an optional first linker peptide, a first CD47-binding domain, an optional second linker peptide, and a first PD-L1-binding domain; and

(b) a second polypeptide comprising from N-terminus to C-terminus: a second TIGIT ligand-binding domain, an optional second hinge region, a second Fc region, an optional third linker peptide, a second CD47-binding domain, an optional fourth linker peptide, and a second PD-L1-binding domain.
The protein complex of claim 54, wherein the first TIGIT-binding domain and/or the second TIGIT-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 1.
The protein complex of claim 54 or 55, wherein the first CD47-binding domain and/or the second CD47-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 3 or 18.
The protein complex of any one of claims 54-56, wherein the first PD-L1-binding domain and/or the second PD-L1-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 17.
The protein complex of any one of claims 54-57, wherein the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 8.
The protein complex of any one of claims 54-58, wherein the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 9.
The protein complex of any one of claims 54-57, wherein the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 10.
The protein complex of any one of claims 54-57 and 60, wherein the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 11.
The protein complex of any one of claims 54-61, wherein the first linker peptide and/or the third linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 5.
The protein complex of any one of claims 54-62, wherein the second linker peptide and/or the fourth linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 4.
The protein complex of any one of claims 54-63, wherein the first polypeptide and/or the second polypeptide comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 15 or 22.
A protein complex, comprising

(a) a first polypeptide comprising from N-terminus to C-terminus: a first PD-L1-binding domain, an optional first linker peptide, a first CD47-binding domain, an optional first hinge region, a first Fc region, an optional second linker peptide, and a first TIGIT ligand-binding domain; and

(b) a second polypeptide comprising from N-terminus to C-terminus: a second PD-L1-binding domain, an optional third linker peptide, a second CD47-binding domain, an optional second hinge region, a second Fc region, an optional fourth linker peptide, and a second TIGIT ligand-binding domain.
The protein complex of claim 65, wherein the first TIGIT-binding domain and/or the second TIGIT-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 1.
The protein complex of claim 65 or 66, wherein the first CD47-binding domain and/or the second CD47-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 3 or 18.
The protein complex of any one of claims 65-67, wherein the first PD-L1-binding domain and/or the second PD-L1-binding domain comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 17.
The protein complex of any one of claims 65-68, wherein the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 8.
The protein complex of any one of claims 65-69, wherein the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 9.
The protein complex of any one of claims 65-68, wherein the first hinge region and/or the second hinge region comprise a sequence that is at least 80%identical to SEQ ID NO: 10.
The protein complex of any one of claims 65-68 and 71, wherein the first Fc region and/or the second Fc region comprise a sequence that is at least 80%identical to SEQ ID NO: 11.
The protein complex of any one of claims 65-72, wherein the first linker peptide and/or the third linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 4.
The protein complex of any one of claims 65-73, wherein the second linker peptide and/or the fourth linker peptide comprise a sequence that is at least 80%identical to SEQ ID NO: 5.
The protein complex of any one of claims 65-74, wherein the first polypeptide and/or the second polypeptide comprise a sequence that is at least 80%, 90%, 95%, or 100%identical to SEQ ID NO: 16.
A nucleic acid comprising a polynucleotide encoding the protein complex of any one of claims 1-75.
The nucleic acid of claim 76, wherein the nucleic acid is a DNA (e.g., cDNA) or RNA (e.g., mRNA) .
A vector comprising one or more of the nucleic acids of claim 76 or 77.
A cell comprising the vector of claim 78.
The cell of claim 79, wherein the cell is a CHO cell.
A cell comprising one or more of the nucleic acids of claim 76 or 77.
A method of producing a protein complex, the method comprising

(a) culturing the cell of any one of claims 79-81 under conditions sufficient for the cell to produce the protein complex; and

(b) collecting the protein complex produced by the cell.
A protein conjugate comprising the protein complex of any one of claims 1-75, covalently bound to a therapeutic agent.
The protein conjugate of claim 83, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of claims 1-75, or the protein conjugate of claims 83 or 84, to the subject.
The method of claim 85, wherein the subject has a cancer cell expressing PVR, Nectin-2, CD47 and/or PD-L1.
The method of claim 85 or 86, wherein the cancer is breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, diffuse large B-cell lymphoma, mesothelioma, lung cancer, ovarian cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, cholangiocarcinoma, head and neck cancer, blood cancer, or a combination thereof.
A method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex of any one of claims 1-75, or the protein conjugate of claim 83 or 84.
A method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex of any one of claims 1-75, or the protein conjugate of claim 83 or 84.
A pharmaceutical composition comprising the protein complex of any one of claims 1-75, and a pharmaceutically acceptable carrier.