PD-l1-binding-peptide-based multifunctional molecule

Abstract

The present disclosure relates to the field of biomedicines. Disclosed is a PD-L1-binding-peptide-based multifunctional molecule. Specifically, the present disclosure relates to a fusion protein, wherein a motif capable of binding to an additional antigen or receptor is inserted at a specific position of an anti-PD-L1 single-domain antibody.

Description

Multifunctional molecules based on PD-L1 binding polypeptides

[0001] Cross-reference to Related Applications

[0002] The present disclosure is based on and claims priority to Chinese Patent Application No. 202410652619.4, filed on May 24, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to the field of biomedicine, and discloses a multifunctional molecule based on PD-L1 binding polypeptides. Specifically, the present disclosure relates to a fusion protein, which inserts a motif that can bind to additional antigens or receptors at a specific position of an anti-PD-L1 single-domain antibody. BACKGROUND

[0004] Programmed death receptor-1 (PD-1) is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. The first members of the family, CD28 and ICOS, were discovered by their ability to enhance T cell proliferation upon addition of monoclonal antibodies. Two cell surface glycoprotein ligands for PD-1, PD-L1 and PD-L2, have been identified, and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1.

[0005] Previous results have shown that PD-L1, which is highly expressed by tumor cells, plays an important role in the immune escape of tumors by increasing the apoptosis of T cells. Researchers found that the P815 tumor cell line transfected with the PD-L1 gene can resist specific CTL lysis in vitro, and has stronger tumorigenicity and invasiveness after being inoculated into mice. These biological properties can be reversed by blocking PD-L1. Recently, immunotherapy using antibodies to block the PD1 / PD-L1 interaction has achieved amazing therapeutic effects in the clinic, showing sustained tumor inhibition and improved patient survival time. In addition, clinical results have shown that immunotherapy blocking PD-1 / PD-L1 has excellent therapeutic effects on other immune suppression-related diseases.

[0006] WO2017020801 discloses a PD-L1 blocking type heavy chain single domain antibody, WO2018133873 discloses the complex crystal structure of the single domain antibody binding to PD-L1, the results show that the CDR2 of the single domain antibody does not participate in the binding of the target, replacing it with the CDR2 sequence of other single domain antibodies, antibody heavy chains, or directly replacing it with other non-functional amino acids has little effect on the binding of the single domain antibody to PD-L1 or its blocking to PDL1 / PD1; but it does not disclose the effect on the binding characteristics of the single domain antibody after directly inserting other protein targeting molecules in CDR2.

[0007] Integrins are proteins that function as cell surface receptors, mediating cell adhesion, supporting cell movement by adhesion and traction, and bidirectional signaling between cells and their environment. Integrins are important in the regulation of various biological signaling pathways that control important cellular processes such as cell survival, migration, proliferation, differentiation, metastasis, and tumor invasion. Integrin heterodimers are formed by two different alpha and beta subunits through non-covalent bonds, all mammalian cells contain 18 different alpha subunits and 8 beta subunits, which combine in various ways to form 24 integrin protein heterodimers with unique ligand recognition and tissue-specific characteristics.

[0008] In recent years, the integrin family has been shown to be a key mediator of tissue fibrosis. Among the 24 known integrin heterodimers, 5 av integrins (avb1, avb3, avb5, avb6 and avb8) transduce mechanical and biochemical signals of fibrotic extracellular matrix into cells, activate latent TGFp, and subsequently regulate fibroblast adhesion, migration and growth. The av integrin represented by avb6 interacts with the RGD (arginine-glycine-aspartic acid) motif of the latent associated peptide (LAP) of transforming growth factor beta (TGF-β), and plays a key role in the regulation of TGF-β signaling. Imbalance of TGF-β expression and response is associated with a variety of disease processes, including fibrotic diseases and chronic inflammation. In addition, avb6 is overexpressed in many solid tumors, and several studies have noted that avb6 expression is a negative prognostic indicator for a variety of cancers, including colorectal cancer, non-small cell lung cancer, gastric cancer, and cervical cancer.

[0009] LAP is the amino-terminal domain of the TGF-β precursor peptide, which can form a latent TGF-β complex and act as a ligand for integrin avb6. When integrin avb6 binds to the latent TGF-β complex outside the cell through the RGD motif in LAP, and binds to the intracellular cytoskeleton at the same time, active TGF-β is released from the latent complex, and this activation is an important physiological role of integrin avb6 in vivo.

[0010] There is a need in the art to develop bispecific binding molecules that target both PD-L1 and integrins. SUMMARY

[0011] The present disclosure utilizes protein engineering techniques to obtain a bispecific binding molecule, such as a fusion protein, by inserting a motif that can bind an additional target at a specific region of a selected PD-L1 single domain antibody variable region, while maintaining the PD-L1 binding activity.

[0012] In a first aspect, the present disclosure relates to a fusion protein comprising an immunoglobulin single variable domain capable of binding PD-L1, the immunoglobulin single variable domain comprising a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3.

[0013] The fusion protein further comprises a polypeptide inserted in the CDR2 region or the FR3 region of the immunoglobulin single variable domain, the polypeptide comprising a motif that can bind a second antigen or receptor.

[0014] In a second aspect, the present disclosure provides a nucleic acid molecule encoding the fusion protein of the first aspect of the present disclosure.

[0015] In a third aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule of the second aspect operably linked to an expression control element.

[0016] In a fourth aspect, the present disclosure provides a recombinant cell comprising the nucleic acid molecule of the second aspect and / or the expression vector of the third aspect, and capable of expressing the fusion protein.

[0017] In a fifth aspect, the present disclosure further provides a pharmaceutical composition comprising the fusion protein of the first aspect and / or the nucleic acid molecule of the second aspect and / or the expression vector of the third aspect and / or the recombinant cell of the fourth aspect, and a pharmaceutically acceptable carrier.

[0018] In a sixth aspect, the present disclosure further provides a method of treating and / or preventing cancer, comprising administering to a subject in need thereof an effective amount of the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, and / or the pharmaceutical composition of the fifth aspect.

[0019] In a seventh aspect, the present disclosure further provides a method of treating and / or preventing a fibrotic disease or disorder, comprising administering to a subject in need thereof an effective amount of the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, and / or the pharmaceutical composition of the fifth aspect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 shows ELISA binding activity of molecules to PD-L1;

[0021] Figure 2 shows blocking activity of molecules to PD-1 and PD-L1 binding;

[0022] Figure 3 shows blocking activity of molecules to CD80 and PD-L1 binding;

[0023] Figure 4a-e shows ELISA binding activity of molecules to integrin ανβ6;

[0024] Figure 5a-c shows ELISA binding activity of molecules to integrin ανβ8;

[0025] Figure 6 shows binding activity of molecules to ανβ6 and ανβ8 overexpressing cells;

[0026] Figure 7a-c shows blocking activity of molecules to PD-1 and PD-L1 based on cells;

[0027] Figure 8 shows inhibitory activity of molecules to TGFβ;

[0028] Figure 9 shows binding activity of molecules to tumor cells Karpas299, Capan2;

[0029] Figure 10 shows binding activity of molecules to tumor cells Capan2, HCC70;

[0030] Figure 11 shows results of serum stability test of molecules. DETAILED DESCRIPTION

[0031] The present application will be readily understood by the following detailed description in conjunction with the accompanying drawings, and wherein like reference numerals designate like structural elements in the drawings and the following detailed description, in which the principles of the present application are utilized, and by its embodiments that are illustrated and described herein.

[0032] TERMS DEFINITION

[0033] Unless otherwise indicated or defined, all terms used have the ordinary meaning that would be understood by one of skill in the art, which would be in line with the present art. Reference is made, for example, to standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nded.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nded.), Gower Medical Publishing, London, New York (1989), as well as the general prior art cited herein; and furthermore, all methods, steps, techniques and procedures not specifically detailed are performed and known in the art per se, unless otherwise indicated. Reference is also made, for example, to standard handbooks, the above-mentioned general prior art, and other references cited therein.

[0034] The terms "antibody" or "immunoglobulin", which are used interchangeably unless otherwise indicated, are used herein as general terms to include both heavy chain antibodies and conventional 4-chain antibodies, whether referring to full-length antibodies, individual chains thereof, as well as all portions, domains or fragments thereof, including but not limited to antigen binding domains or fragments, such as, for example, VHH domains or VH / VL domains, respectively. Furthermore, the term "sequence" as used herein (for example, in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is to be understood generally to include both the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding said sequence, unless a more defined interpretation is required herein.

[0035] The term "domain" (of a polypeptide or protein) as used herein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of a protein, and in many cases can be added, removed or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0036] The term "immunoglobulin domain" as used herein refers to a globular region of an antibody chain (for example, of a chain of a conventional 4-chain antibody or of a chain of a heavy chain antibody), or to a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized by its maintenance of the immunoglobulin fold characteristic of antibody molecules.

[0037] The term "immunoglobulin variable domain" as used herein refers to an immunoglobulin domain essentially consisting of four "framework regions" designated as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, in the art and herein below, which are separated by three "complementarity determining regions" or "CDRs" designated as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively, in the art and herein below. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer antibody specificity for an antigen due to the possession of an antigen binding site.

[0038] The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin variable domain that is capable of specifically binding to an epitope of an antigen without pairing with another immunoglobulin variable domain. One example of an immunoglobulin single variable domain of the present disclosure is a "domain antibody", such as an immunoglobulin single variable domain VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") of Camelidae, as defined below.

[0039] A "VHH domain", also known as single-domain antibody, heavy chain single-domain antibody, VHH, VHH antibody fragment and VHH antibody, is the variable domain of an antigen-binding immunoglobulin termed "heavy chain antibody" (i.e. "antibody devoid of light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain present in a conventional 4-chain antibody (which is referred to herein as "VH domain") and from the light chain variable domain present in a conventional 4-chain antibody (which is referred to herein as "VL domain"). A VHH domain specifically binds an epitope without the need for other antigen binding domains (in contrast to a VH or VL domain in a conventional 4-chain antibody, where the epitope is recognized by the VL domain together with the VH domain). A VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. In some embodiments, a VHH domain comprises or consists of the variable region domain of the heavy chain of a heavy chain antibody.

[0040] In the context of the present disclosure, the terms "single-domain antibody", "heavy chain single-domain antibody", "VHH domain", "VHH", "VHH antibody fragment", and "VHH antibody" are used interchangeably.

[0041] A VHH of the present application can also be comprised in a larger polypeptide / protein. Examples of polypeptides / proteins comprising a VHH of the present application include, but are not limited to, fusion proteins.

[0042] The amino acid residues applied for VHH domains of Camelidae can be numbered according to the general numbering scheme for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), e.g. as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999).

[0043] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an antigenic epitope. The CDRs of a heavy chain and light chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (https: / / www.imgt.org / ), and North CDR definitions based on affinity propagation clustering with a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

[0044] In the present disclosure, the Kabat assignment system is used to determine the CDR regions, unless otherwise specified:

[0045] Unless otherwise indicated, in the disclosure, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the manners described above.

[0046] The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibody.

[0047] The term "epitope" or "antigenic epitope" generally refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also known in the art as an "antigenic determinant". Epitopes or antigenic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, an epitope often comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction exist across protein amino acid residues that are separated from one another.

[0048] The term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigen-binding molecule or antigen-binding protein can bind. Specificity of an antigen-binding protein can be determined based on its affinity and / or avidity. Affinity, as represented by the dissociation equilibrium constant (KD) of an antigen for an antigen-binding protein, is a measure of the strength of binding between an epitope and an antigen-binding site on the antigen-binding protein: the smaller the KD value, the stronger the binding between the epitope and the antigen-binding protein (or, affinity can also be expressed as the association constant (KA), which is 1 / KD). As will be appreciated by those skilled in the art, affinity can be determined in known ways, depending on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen-binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and the relevant antigen. Avidity is related to both the affinity between the epitope and the antigen-binding site on its antigen-binding protein, and the number of relevant binding sites present on the antigen-binding protein. Binding affinity can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or bio-layer interferometry assay or MSD assay or surface plasmon resonance (SPR) method.

[0049] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, i.e., CH2, CH3, and optionally CH4 domains. For example, in native antibodies, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of a heavy chain derived from an IgG, IgA, and IgD class antibody; or the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of a heavy chain derived from an IgM and IgE class antibody. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering scheme (also referred to as EU index) as set forth in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The term "Fc region" herein does not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, and the heavy chain constant region CH1 and light chain constant region CL, but can in some cases include the hinge region N-terminal to the heavy chain constant region. In some embodiments, the Fc region of the present disclosure is from IgGl, IgG2, IgG3, or IgG4. In some embodiments, the Fc region of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 15 or 16, or an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence.

[0050] As used herein, the term "and / or" means any one of the items, or any two or more of the items.

[0051] As used herein, the term "comprises" or "comprising" means including, but not limited to, the recited items, integers or steps, unless otherwise specified herein. As used herein, the term "comprises" or "comprising" also encompasses the case where the recited items, integers or steps are combined with other items, integers or steps, unless otherwise specified herein.

[0052] The term "administering" generally refers to the method of giving a dose of a compound or pharmaceutical composition to a subject (e.g., a patient). Administration can be by any suitable means, including parenterally, intrapulmonary, and intranasally, as well as intralesionally, if local treatment is desired. Parenteral infusions include, e.g., intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In the present disclosure, the term "about" generally refers to a variance within a range of 0.5-10% above or below the indicated numerical value, e.g., within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the indicated numerical value, unless otherwise specified. As used in the present disclosure, numerical values mentioned are to be considered as modified by "about," unless otherwise indicated. If in doubt, or for error ranges not recognized by the general understanding in the art for a particular value or parameter, "about" means ±5% of the value or parameter.

[0053] The term "effective amount" refers to that amount or dose of an antibody or fragment or composition or combination of the present disclosure, which when administered to a patient, either as a single dose or as multiple doses, produces the intended effect in the patient in need of treatment or prevention. Depending on the intended effect, this can include both "therapeutically effective amounts" and "prophylactically effective amounts."

[0054] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Generally, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0055] An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0056] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The cancer can be in an early, intermediate, or advanced stage or be a metastatic cancer.

[0057] The term "fibrotic disease or disorder" is a pathological state in which there is a prevalent increase in fibrous connective tissue within an organ or tissue, with a decrease in parenchymal cells, and a progressive course leading to destruction of organ architecture and loss of function, culminating in failure.

[0058] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active compound is administered.

[0059] The term "pharmaceutical composition" refers to a composition that is in a form that is effective for the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that are unacceptable for the subject to which the composition is administered.

[0060] As used herein, "treatment" refers to slowing, interrupting, arresting, reversing, stopping, reducing, or otherwise impeding the progression or severity of an existing symptom, disorder, condition, or disease.

[0061] The term "therapeutic agent" as described herein encompasses any active substance or active agent that is effective in the prevention or treatment of the disease to be treated.

[0062] The term "pharmaceutical combination" or "combination product" refers to either a non- fixed combination or a fixed combination, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the fusion protein of the present application, and (ii) the other therapeutic agent) are administered to a patient as separate entities, either simultaneously, without specific time limitations, or sequentially with no specific time limitations, wherein such administration provides therapeutically effective levels of the two or more active agents in the body of the patient. The term "fixed combination" means that the two or more active agents are administered to a patient as a single entity. Preferably, the dosages and / or time intervals of the two or more active agents are selected so that the use of the combination results in a therapeutic effect that is greater than the effect induced by administration of any of the agents alone. The individual components can each be present in the form of separate formulations that can be administered simultaneously or sequentially without specific time limitations.

[0063] DETAILED DESCRIPTION

[0064] Fusion protein

[0065] In one aspect, the present disclosure relates to a fusion protein comprising an immunoglobulin single variable domain;

[0066] The fusion protein further comprises a polypeptide that specifically binds a second antigen or receptor inserted in the immunoglobulin single variable domain, e.g., inserted in the CDR2 region or FR3 region, the polypeptide comprising a motif that can bind the second antigen or receptor.

[0067] In some embodiments, the immunoglobulin single variable domain is an immunoglobulin single variable domain capable of binding to Programmed Cell Death Ligand 1 (PD-L1). In some embodiments, the immunoglobulin single variable domain capable of binding to Programmed Cell Death Ligand 1 (PD-L1) comprises a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3.

[0068] In some embodiments, the immunoglobulin single variable domain is a heavy chain single domain antibody VHH. In some embodiments, the heavy chain single domain antibody of the present disclosure is a VHH antibody comprising or consisting of a heavy chain variable region of a heavy chain antibody, which heavy chain variable region of the heavy chain single domain antibody typically has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementarity determining regions 1-3.

[0069] In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity thereto. For example, the immunoglobulin single variable domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0070] In some embodiments, the immunoglobulin single variable domain is the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity thereto. For example, the immunoglobulin single variable domain is an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0071] In some embodiments, the immunoglobulin single variable domain is a humanized VHH.

[0072] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5-10, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 5-10. For example, the immunoglobulin single variable domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 5-10.

[0073] In some embodiments, the immunoglobulin single variable domain is an amino acid sequence as set forth in any one of SEQ ID NOs: 5-10, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity thereto. For example, the immunoglobulin single variable domain is an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 5-10.

[0074] In some embodiments, the immunoglobulin single variable domain comprises a variable region structure of Envafolimab (also known as KN035). In some embodiments, the immunoglobulin single variable domain is a variable region structure of Envafolimab.

[0075] In some embodiments, the fusion protein comprises 1 or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the aforementioned immunoglobulin single variable domains; the amino acid sequences of the plurality of immunoglobulin single variable domains can be identical, can each be different, or can be partially identical. In some embodiments, the immunoglobulin single variable domains are connected in series. In some embodiments, the immunoglobulin single variable domains are connected to each other via a linker.

[0076] Unless otherwise specified, the position of the variable region in the immunoglobulin single variable domain (single-domain antibody, VHH) described in the present disclosure is described using the Kabat numbering system. For illustration only and not by way of limitation, taking the VHH as set forth in SEQ ID NO: 4 as an example, the result of numbering according to the Kabat numbering system is as follows (the CDR regions are defined according to the Kabat assignment system, which have been highlighted):

[0077] Thus, in some embodiments, the immunoglobulin single variable domain comprises the three complementarity determining regions (CDRs) contained in the heavy chain variable region of a heavy chain antibody as set forth in any one of SEQ ID NOs: 4-10, which CDR1, CDR2, CDR3 can be defined according to any CDR assignment system, for example, which CDR1, CDR2, and CDR3 can be defined according to the Kabat, Chothia, AbM, or IMGT assignment system, or a combination thereof, for example, which CDR1, CDR2, and CDR3 are defined according to the Kabat assignment system.

[0078] In some embodiments, the immunoglobulin single variable domain comprises or consists of a heavy chain variable region of a heavy chain antibody comprising the three complementarity determining regions (CDRs) contained in the amino acid sequence as set forth in any one of SEQ ID NOs: 4-10, which CDR1, CDR2, CDR3 can be defined according to any CDR assignment system, for example, which CDR1, CDR2, and CDR3 can be defined according to the Kabat, Chothia, AbM, or IMGT assignment system, or a combination thereof, preferably, which CDR1, CDR2, and CDR3 are defined according to the Kabat.

[0079] In some embodiments, the immunoglobulin single variable domain comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the immunoglobulin single variable domain of the present disclosure, e.g., a VHH, comprises or consists of a heavy chain variable region of a heavy chain antibody comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, the VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and / or the VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0080] In some embodiments, the polypeptide that specifically binds to a second antigen or receptor is inserted after amino acid position 50, 51, 52, 52A, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 in the CDR2 region of the immunoglobulin single variable domain; preferably, the polypeptide is inserted after amino acid position 50, 51, 52, 52A, 53, 54, or 55 in the CDR2 region; more preferably, the polypeptide is inserted after amino acid position 53 or 54 in the CDR2 region, for example, Thr at position 53 or Ser at position 54.

[0081] In some embodiments, the polypeptide that specifically binds to a second antigen or receptor is inserted after amino acid position 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 82A, 82B, 82C, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 in the FR3 region of the immunoglobulin single variable domain; preferably, the polypeptide is inserted after amino acid position 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 in the FR3 region; more preferably, the polypeptide is inserted after amino acid position 70, 71, 72, 73, or 74 in the FR3 region; even more preferably, the polypeptide is inserted after amino acid position 71 or 73 in the FR3 region, e.g., Gln at position 71 or Asn at position 73.

[0082] In the present disclosure, when referring to a position in the CDR2 or FR3 region, the position is determined according to the Kabat numbering scheme, unless specified otherwise; for example, reference can be made to the positions in the VHH shown in SEQ ID NO: 4 numbered according to the Kabat numbering system.

[0083] In some embodiments, the N-terminus and / or C-terminus of the polypeptide that specifically binds to a second antigen or receptor each independently further comprises a spacer peptide consisting of 1-4 amino acids. For example, the N-terminus and C-terminus of the polypeptide each independently comprises a spacer peptide consisting of 1-4 amino acids.

[0084] In some embodiments, the spacer peptide is selected from one or more of A, G, S, P, AA, AS, GG, GS, SS, PP, ASGS (SEQ ID NO: 62), GSAS (SEQ ID NO: 63), and GGGS (SEQ ID NO: 64).

[0085] In some embodiments, the spacer peptide at the N-terminus of the polypeptide that specifically binds to a second antigen or receptor is selected from one or more of A, P, and PP; in some embodiments, the spacer peptide at the N-terminus of the polypeptide that specifically binds to a second antigen or receptor is A, P, or PP.

[0086] In some embodiments, the spacer peptide at the C-terminus of the polypeptide that specifically binds to a second antigen or receptor is selected from one or more of A, AA, AS, and ASGS; in some embodiments, the spacer peptide at the C-terminus of the polypeptide that specifically binds to a second antigen or receptor is A, AA, AS, or ASGS.

[0087] In some embodiments, the spacer peptide at the N-terminus of the polypeptide that specifically binds to the second antigen or receptor is A, and the spacer peptide at the C-terminus is selected from one or more of G, S, P, AA, AS, GG, GS, SS, PP, ASGS, GSAS, and GGGS.

[0088] In some embodiments, the spacer peptide at the C-terminus of the polypeptide that specifically binds to the second antigen or receptor is PP, and the spacer peptide at the N-terminus is selected from one or more of A, G, S, P, AA, AS, GG, GS, SS, ASGS, GSAS, and GGGS.

[0089] In other embodiments, the spacer peptides at the N-terminus and C-terminus of the polypeptide are selected from the following combinations:

[0090] (a) N-terminus: A, C-terminus: AA;

[0091] (b) N-terminus: A, C-terminus: AS;

[0092] (c) N-terminus: A, C-terminus: A;

[0093] (d) N-terminus: PP, C-terminus: AS;

[0094] (e) N-terminus: PP, C-terminus: ASGS;

[0095] (f) N-terminus: PP, C-terminus: A; and

[0096] (g) N-terminus: PP, C-terminus: AA.

[0097] In the present disclosure, the polypeptide that specifically binds to the second antigen or receptor inserted in the immunoglobulin single variable domain comprises a motif that can bind to the second antigen or receptor. "Motif" refers to a polypeptide sequence that has biological significance and / or exerts biological effects or participates in some biological processes.

[0098] In some embodiments, the second antigen or receptor is an integrin, preferably αvβ6 and / or αvβ8.

[0099] In some embodiments, the inserted polypeptide comprises an Arg-Gly-Asp (RGD) motif; the RGD motif can specifically bind to αvβ6 and / or αvβ8.

[0100] In some embodiments, the polypeptide comprising an RGD motif is derived from Pro-TGF-β, preferably the polypeptide is derived from Pro-TGF-β1 (e.g., from the protein with Uniprot accession number P01137 or a natural variant thereof), Pro-TGF-β2 (e.g., from the protein with Uniprot accession number P61812 or a natural variant thereof), or Pro-TGF-β3 (e.g., from the protein with Uniprot accession number P10600 or a natural variant thereof). In some embodiments, the RGD motif is a fragment of at least 5, 6, 7, 8, 9, 10, or 11 contiguous amino acids in Pro-TGF-β1, Pro-TGF-β2, or Pro-TGF-β3 that comprises RGD. In some embodiments, the RGD motif is a fragment of 9, 10, or 11 contiguous amino acids in Pro-TGF-β1, Pro-TGF-β2, or Pro-TGF-β3 that comprises RGD. In some embodiments, the RGD is located at positions 3-5 of the contiguous amino acid fragment in Pro-TGF-β1, Pro-TGF-β2, or Pro-TGF-β3 that comprises RGD. In some embodiments, the RGD motif is a fragment of 11 contiguous amino acids in Pro-TGF-β1, Pro-TGF-β2, or Pro-TGF-β3 that comprises RGD, and the RGD is located at positions 3-5 of the contiguous amino acid fragment.

[0101] In other embodiments, the polypeptide comprising an RGD motif is derived from foot-and-mouth disease virus (FMDV), e.g., from the protein with Uniprot accession number P03305 or a natural variant thereof. In some embodiments, the RGD motif is a fragment of at least 5, 6, 7, 8, 9, 10, or 11 contiguous amino acids in FMDV that comprises RGD. In some embodiments, the RGD motif is a fragment of 9, 10, or 11 contiguous amino acids in FMDV that comprises RGD. In some embodiments, the RGD is located at positions 3-5 of the contiguous amino acid fragment in FMDV that comprises RGD. In some embodiments, the RGD motif is a fragment of 11 contiguous amino acids in FMDV that comprises RGD, and the RGD is located at positions 3-5 of the contiguous amino acid fragment.

[0102] In some embodiments, the polypeptide comprising an RGD sequence comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 11-14. In other embodiments, the polypeptide comprising an RGD sequence has 1, 2, or 3 substitutions, deletions, or additions of amino acids compared to the amino acid sequence of any one of SEQ ID NOs: 11-14; provided that the polypeptide is capable of specifically binding αvβ6 and / or αvβ8.

[0103] In some embodiments, the inserted polypeptide and the spacer peptide can be selected from the following combinations:

[0104] In some embodiments, the inserted polypeptide and the spacer peptide combination comprises a polypeptide selected from the group consisting of any one of the amino acid sequences set forth in SEQ ID NOs: 17-22 or the inserted polypeptide has 1, 2, or 3 amino acid substitutions, deletions, or additions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 17-22; provided that the polypeptide is capable of specifically binding to ανβ6 and / or ανβ8.

[0105] In some embodiments, the fusion protein comprises or is an amino acid sequence set forth in any one of SEQ ID NOs: 23-31, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity thereto. For example, the immunoglobulin single variable domain comprises or is an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 23-31.

[0106] In some embodiments, the fusion protein comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 23-31.

[0107] In some embodiments, the fusion protein further comprises an immunoglobulin Fc region.

[0108] In some embodiments, the immunoglobulin Fc region is or is from a human immunoglobulin Fc region, preferably a Fc region of human IgGl, human IgG2, human IgG3, or human IgG4. In some embodiments, the immunoglobulin Fc region comprises or is an amino acid sequence set forth in SEQ ID NO: 15 or 16, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity thereto. For example, the immunoglobulin Fc region comprises or is an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15 or 16. In some specific embodiments, the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO: 15 or 16. In some specific embodiments, the immunoglobulin Fc region can or can not comprise EPKSS.

[0109] In some embodiments, the immunoglobulin Fc region is a variant of a human immunoglobulin Fc region, e.g., a variant of a human IgGl Fc region. In some embodiments, the Fc region comprising an intact hinge region comprises a C220S mutation in the hinge region.

[0110] In some embodiments, the immunoglobulin Fc region variant can further comprise a mutation that reduces or abrogates effector function. The effector function can be antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), etc. Specifically, the mutation that reduces or abrogates effector function attenuates or abrogates the binding between the immunoglobulin and FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, FcyRIIIb, and / or Clq.

[0111] The mutation that attenuates or abrogates effector function can be any known mutation or combination of mutations in the art, e.g., described in Esohe E.I., J Immunol 2000; 164: 4178-4184; Hutchins, J.T., Proc. Natl. Acad. Sci. USA 1995, 92, 11980-11984; Xu D., Cell Immunol. 2000, 200, 16-26; Hezareh, M., J. Virol. 2001, 75, 12161-12168; Schlothauer, T., Protein Eng. Des. Sel. 2016, 29, 457-466; Chu, S.Y., Mol. Immunol. 2008, 45, 3926-3933; Sazinsky, S.L., Proc. Natl. Acad. Sci. USA 2008, 105, 20167-20172; Oganesyan, V., Acta Crystallogr. Sect. D Biol. Crystallogr. 2008, 64 Pt 6, 700-704; An, Z., MAbs 2009, 1, 572-579; Moore, G.L., Methods 2019, 154, 38-50; Schlothauer, T., Protein Eng. Des. Sel. 2016, 29, 457-466; Strohl, W., US20150337053; Engelberts, P.J., EBioMedicine 2020, 52, 102625, etc.

[0112] In some embodiments, the immunoglobulin Fc region variant can further comprise a mutation that reduces or abrogates effector function selected from the group consisting of D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R, S298G / T299A, L234F / L235E / P331S, L234F / L235E / D265A, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236del / S267K, L234A / L235A / P329G, L234F / L235E / D265A, or V234A / G237A / P238S / H268A / V309L / A330S / P331S. Preferably, the immunoglobulin Fc variant can further comprise a mutation that reduces or abrogates effector function selected from the group consisting of D265A, P331G, D265A / P331G (hereinafter abbreviated as AG), L234F / L235E / P331S (hereinafter abbreviated as FES), L234F / L235E / D265A (hereinafter abbreviated as FEA), or L234A / L235A (hereinafter abbreviated as AA).

[0113] In other embodiments, the immunoglobulin Fc region variant can further comprise a mutation that modulates its ability to bind to FcRn. For example, the Fc region variant improves the binding to FcRn at acidic pH conditions without affecting the binding to FcRn at neutral pH conditions, thereby obtaining a longer half-life compared to the parent.

[0114] The mutation that modulates the ability to bind to FcRn and / or prolongs the half-life can be any known mutation or combination of mutations in the art, for example as described in Dall'Acqua W, J. Immunol. 2002; 169: 5171-5180; Dall'Acqua W, J. Biol. Chem. 2006b; 281 :23514-23524; Hinton PR, J. Immunol. 2006; 176:346-356; Petkova, J. Exp. Med. 2006; 203:275-280; Yeung, J. Immunol. 2009; 182:7663-7671; Zalevsky, Nat. Biotechnol. 2010; 28: 157-159; Monnet, MAbs. 2014; 6:422-436, among others.

[0115] In some embodiments, the immunoglobulin Fc region variant can comprise a mutation selected from the group of mutations that modulate the ability to bind to FcRn and / or prolong half-life: M252Y / S254T / T256E, T250Q / M428L, N434A, M428L / N434S, N315D / A330V / N361D / A378V / N434Y, E294D / T307P / N434Y, V259I / N315D / N434Y, T307A / N315D / A330V / E382V / N389T / N434Y, or L234F / L235E / D265A. Preferably, the immunoglobulin Fc region variant can comprise a mutation selected from the group of mutations that modulate the ability to bind to FcRn and / or prolong half-life: M252Y / S254T / T256E (hereinafter abbreviated as YTE) or M428L / N434S (hereinafter abbreviated as LS).

[0116] In some embodiments, the immunoglobulin Fc region variant can comprise a combination of mutations selected from the group consisting of:

[0117] 1) D265A / P331G;

[0118] 2) D265A / P331G, M252Y / S254T / T256E;

[0119] 3) L234F / L235E / P331S, M252Y / S254T / T256E;

[0120] 4) L234F / L235E / D265A, M252Y / S254T / T256E;

[0121] 5) L234F / L235E / D265A, M428L / N434S;

[0122] 6) D265A / P331G, M428L / N434S;

[0123] 7) L234F / L235E / P331S, M428L / N434S;

[0124] 8) L234A / L235A, M252Y / S254T / T256E; and

[0125] 9) L234A / L235A, M428L / N434S.

[0126] In some embodiments, the Fc region comprises or consists of the amino acid sequence:

[0127] (i) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 53 and comprises D265A / P331G;

[0128] (ii) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 54 and comprises D265A / P331G and M252Y / S254T / T256E;

[0129] (iii) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 55 and comprises L234F / L235E / P331S and M252Y / S254T / T256E;

[0130] (iv) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 56 and comprises L234F / L235E / P331S and M428L / N434S;

[0131] (v) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 57 and comprises L234F / L235E / D265A and M428L / N434S;

[0132] (vi) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 58 and comprises D265A / P331G and M428L / N434S;

[0133] (vii) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 59 and comprises L234F / L235E / P331S and M252Y / S254T / T256E;

[0134] (viii) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 60 and comprises L234A / L235A and M252Y / S254T / T256E; or

[0135] (ix) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 61 and comprises L234A / L235A and M428L / N434S.

[0136] In some embodiments, the Fc region comprises or consists of the amino acid set forth in any one of SEQ ID NOs: 53-61.

[0137] In some embodiments, the fusion protein of the present disclosure comprising an Fc region comprises or is the amino acid sequence set forth in any one of SEQ ID NOs: 32-51, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity thereto. For example, the fusion protein comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 32-51.

[0138] In some embodiments, the fusion protein of the present disclosure comprises the amino acid sequence set forth in any one of SEQ ID NOs: 32-51.

[0139] In some embodiments, the fusion protein of the present disclosure forms a dimer, e.g., a homo-dimer, via the Fc region. Thus, in some embodiments, the fusion protein of the present disclosure is a homo-dimer.

[0140] In other embodiments, the fusion protein of the present disclosure has at least one of the following characteristics:

[0141] (1) a KD value of less than 1 ╳ 10 -7 M, preferably less than 1 ╳ 10 -8 M, more preferably less than 1 ╳ 10 -9 M, even more preferably less than 1 ╳ 10 -10 M;

[0142] (2) blocks the interaction of PD-L1 and PD-1;

[0143] (3) blocks the interaction of PD-L1 and CD80;

[0144] (4) is capable of binding to tumor cells overexpressing PD-L1.

[0145] In some embodiments, the fusion proteins of the present disclosure have at least one of the following characteristics:

[0146] (a) a KD value of less than 1 ╳ 10 -6 M, preferably less than 1 ╳ 10 -7 M, more preferably less than 1 ╳ 10 - 8 M, even more preferably less than 1 ╳ 10 -9 M;

[0147] (b) a KD value of less than 1 ╳ 10 -6 M, preferably less than 1 ╳ 10 -7 M, more preferably less than 1 ╳ 10 - 8 M, even more preferably less than 1 ╳ 10 -9 M;

[0148] (c) is capable of binding to cells overexpressing ανβ6 and / or ανβ8;

[0149] (d) is capable of inhibiting the activation of TGF-β, preferably TGF-β1 and / or TGF-β3.

[0150] Nucleic acids, vectors and host cells comprising the same

[0151] In one aspect, the present disclosure provides nucleic acids encoding any of the above fusion proteins. The present disclosure also encompasses nucleic acids that hybridize to the above-mentioned nucleic acids under stringent conditions, or nucleic acids that have one or more substitutions (e.g., conservative substitutions), deletions or insertions compared to the above-mentioned nucleic acids, or nucleic acid sequences that have at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity compared to the above-mentioned nucleic acids.

[0152] For example, the nucleic acid of the present disclosure comprises a nucleic acid encoding an amino acid sequence selected from the amino acid sequence set forth in any one of SEQ ID NOs: 1-51, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence selected from the amino acid sequence set forth in any one of SEQ ID NOs: 1-51.

[0153] As will be apparent to those skilled in the art, the amino acid sequence of each fusion protein can be encoded by a variety of nucleic acid sequences due to codon degeneracy. Nucleic acid sequences encoding molecules of the present disclosure can be produced using methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification.

[0154] In one embodiment, nucleic acids encoding the fusion proteins of the present disclosure can be in the same vector or in different vectors. In yet another embodiment, nucleic acids encoding each chain of the fusion proteins of the present disclosure can be introduced into the same or different host cells for expression. Thus, in some embodiments, the method of producing a fusion protein of the present disclosure comprises the step of culturing host cells comprising nucleic acids encoding each chain of the molecule under conditions suitable for expression of each chain of the molecule to produce a fusion protein of the present disclosure.

[0155] In another aspect, the present disclosure provides a vector comprising the above-mentioned nucleic acid. The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host and which transfers an inserted nucleic acid molecule to and / or between host cells. The vector can include a vector primarily for insertion of DNA or RNA into a cell, a vector primarily for replication of DNA or RNA, and a vector primarily for expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell comprising the vector.

[0156] In a preferred embodiment, the vector is an expression vector. The expression vector generally comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). Selection of the elements and their sequences for expression in a particular host is a matter of choice within the skill in the art. Specific examples of control elements and other elements useful or necessary for expression of the fusion proteins of the present disclosure include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes. It will be well within the skill of the artisan to appreciate that vectors commonly employed in the art to which the present disclosure pertains can be applied to the present disclosure.

[0157] In one embodiment, the present disclosure provides a host cell comprising the nucleic acid or the vector.

[0158] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of the cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cells and progeny of the original transformant that have a non-identical genotype. Progeny can not be identical to the parent cell as a result of, e.g., mutation, however, and thus can potentially not be identical to the original transformant in nucleic acid content. Mutant progeny that have the same function or biological activity as the originally transformed cell are included herein. Host cells are any type of cell system that can be used to produce the antibody molecules of the present disclosure, including eukaryotic cells, e.g., mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cells in culture as well as cells within a transgenic animal, transgenic plant, or cultured plant tissue or animal tissue.

[0159] Compositions

[0160] In another aspect, the present disclosure provides a composition, e.g., preferably, a pharmaceutical composition, containing one or a combination of the fusion proteins of the present disclosure formulated together with a pharmaceutically acceptable carrier.

[0161] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers and isotonic and absorption delaying agents, etc. which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical (e.g., by injection or infusion) administration. Depending on the route of administration, the active compound, i.e., the antibody molecule, can be coated in a material to protect the compound from the action of acids and other natural conditions which can inactivate the compound.

[0162] The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material for a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent (100%), this amount will range from about 0.01% to about 99% of active ingredient, such as from about 0.1% to about 70%, or about 1% to about 30% of active ingredient, in combination with a pharmaceutically acceptable carrier.

[0163] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions of the present disclosure applied or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound being applied, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0164] The compositions of the present disclosure can be administered using one or more methods known in the art by one or more routes of administration. The skilled artisan will appreciate that the route and / or mode of administration will vary depending upon the desired results. Preferred routes of administration of the fusion proteins of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes, such as by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0165] Pharmaceutical combinations and kits

[0166] The present disclosure also provides a pharmaceutical combination or pharmaceutical combination product comprising a molecule of the present disclosure. Optionally, the pharmaceutical combination or pharmaceutical combination product further comprises one or more other therapeutic agents.

[0167] The present disclosure also provides a kit-of-parts comprising said pharmaceutical combination, e.g. said kit-of-parts comprises in the same package:

[0168] - a first container containing a pharmaceutical composition comprising a molecule of the present disclosure;

[0169] - optionally, a second container containing a pharmaceutical composition comprising one or more other therapeutic agents (in some embodiments, two or more other therapeutic agents are in the same container, or in separate containers, respectively).

[0170] In some embodiments, the other therapeutic agents encompass any therapeutic agent useful in the treatment of a disease described in the present disclosure. For example, if the molecule of the present disclosure is used in the treatment of a tumor, the therapeutic agent is, e.g., any of the various therapeutic agents used in the treatment of tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g. immune checkpoint inhibitors or agonists).

[0171] Methods of treatment and uses

[0172] In another aspect, the present disclosure also provides a method of treating and / or preventing a PD-L1 -related disease or disorder, comprising administering to a patient in need thereof one or more of the fusion protein, nucleic acid, vector, pharmaceutical composition or drug combination of the present disclosure.

[0173] In some embodiments, the PD-L1 -related disease or disorder refers to an indication associated with abnormal expression or activity of PD-L1, or a disease or disorder associated with abnormal activation of PD-1 / PD-L1. In some embodiments, the PD-L1 -related disease or disorder is a tumor, e.g., a cancer.

[0174] Accordingly, the present disclosure also provides a method of treating and / or preventing a cancer, e.g., a solid tumor or a non-solid tumor, comprising administering to a patient in need thereof one or more of the fusion protein, nucleic acid and vector of the present disclosure.

[0175] The present disclosure also provides a method of treating and / or preventing a cancer, e.g., a solid tumor or a non-solid tumor, comprising administering to a patient in need thereof a pharmaceutical composition of the present disclosure.

[0176] In some embodiments, the tumor is a solid tumor or a hematological tumor and a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in early, intermediate or advanced stage or metastatic cancer. In some embodiments, the tumor is a tumor immune escape.

[0177] In another aspect, the present disclosure also relates to the use of the fusion protein, nucleic acid, vector, pharmaceutical composition and / or drug combination in the manufacture of a medicament for treating and / or preventing a PD-L1 -related disease or disorder, e.g., a tumor or a cancer.

[0178] In some embodiments, the tumor or cancer refers to a PD-L1 -positive tumor or cancer. In some embodiments, the tumor or cancer refers to abnormal expression or activity of PD-L1 in a subject having the tumor or cancer. In some embodiments, the subject (particularly an adult subject) has PD-L1 expression (e.g., low, intermediate or high expression). In some embodiments, the subject has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity) of PD-L1 (e.g., compared to a healthy subject). In some embodiments, the subject has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity) of PD-L1 in a biological sample (e.g., tumor cell or tumor tissue) of the subject (e.g., compared to a biological sample of a healthy subject (e.g., a corresponding tissue or cell in a healthy subject), or compared to PD-L1 in a proximal healthy tissue or cell of the subject).

[0179] In some embodiments, a PD-L1 positive tumor refers to a tumor cell that aberrantly expresses PD-L1. In some embodiments, the aberrant expression of PD-L1 refers to the expression of PD-L1 on the cell membrane of the tumor cell. In some embodiments, the aberrant expression of PD-L1 refers to the expression of PD-L1 on the tumor cell is higher than the expression of PD-L1 in a control cell (e.g., a healthy cell of the corresponding tissue of a healthy individual, or a healthy cell adjacent to the tumor cell). In some embodiments, the aberrant activity of PD-L1 refers to the aberrant activation of the PD-L1 / PD-1 signaling pathway.

[0180] In some embodiments, the tumor or cancer refers to a PD-L1 and / or integrin positive tumor or cancer. In some embodiments, the tumor or cancer refers to an aberrant expression or activity of PD-L1 and / or integrin in a subject having the tumor or cancer. In some embodiments, the subject (particularly an adult subject) has PD-L1 and / or integrin expression (e.g., low, intermediate, or high expression). In some embodiments, the subject has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity) of PD-L1 and / or integrin (e.g., compared to a healthy subject).

[0181] In some embodiments, the subject has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity) of PD-L1 and / or integrin in a biological sample (e.g., tumor cell or tumor tissue) of the subject (e.g., compared to a biological sample of a healthy subject (e.g., a corresponding tissue or cell in a healthy subject), or compared to PD-L1 and / or integrin in an adjacent healthy tissue or cell of the subject).

[0182] In some embodiments, the PD-L1 and / or integrin positive tumor refers to the abnormal expression of PD-L1 and / or integrin in tumor cells. In some embodiments, the abnormal expression of PD-L1 and / or integrin refers to the expression of PD-L1 and / or integrin on the cell membrane of the tumor cells. In some embodiments, the abnormal expression of PD-L1 and / or integrin refers to the expression of PD-L1 and / or integrin on the tumor cells is higher than the expression of PD-L1 and / or integrin in control cells (e.g. healthy cells of the corresponding tissue of a healthy individual, or compared to healthy cells adjacent to the tumor cells). In some embodiments, the abnormal activity of PD-L1 refers to the abnormal activation of the PD-L1 / PD-1 signaling pathway. In some embodiments, the abnormal activity of integrin refers to the abnormal activation of the TGF-β signaling pathway. In some embodiments, the abnormal activity of PD-L1 and integrin refers to the abnormal activation of the PD-L1 / PD-1 signaling pathway and the abnormal activation of the TGF-β signaling pathway. In some embodiments, integrin encompasses ανβ6 and / or ανβ8.

[0183] In some embodiments, the cancer is selected from anaplastic large cell lymphoma, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer, and osteosarcoma.

[0184] In another aspect, the present disclosure also provides a method of treating and / or preventing an integrin-related disease or disorder, comprising administering to a patient in need thereof one or more of the fusion protein, nucleic acid, vector, pharmaceutical composition or drug combination of the present disclosure.

[0185] In another aspect, the present disclosure also provides a method of treating and / or preventing a fibrotic disease or disorder, comprising administering to a patient in need thereof one or more of the fusion protein, nucleic acid and vector of the present disclosure. The present disclosure also provides a method of treating and / or preventing a fibrotic disease or disorder, comprising administering to a patient in need thereof the pharmaceutical composition of the present disclosure.

[0186] In another aspect, the present disclosure also relates to the use of the fusion protein, nucleic acid, vector, pharmaceutical composition and / or drug combination for the manufacture of a medicament for the treatment and / or prevention of the relevant disease or disorder mentioned herein, such as a fibrotic disease or disorder.

[0187] In some embodiments, the fibrotic disease or disorder is selected from lung fibrosis, liver fibrosis, fibrosis of the heart or vasculature, kidney fibrosis, skin fibrosis, gastrointestinal tract fibrosis, bone marrow or hematopoietic tissue fibrosis, nervous system fibrosis, joint fibrosis, or a combination thereof.

[0188] Depending on its therapeutic use, the fusion protein, nucleic acid, vector or pharmaceutical composition of the present disclosure can also be administered in combination or association with one or more other therapies, e.g., therapeutic modalities and / or other therapeutic agents, for use in the methods or uses described herein, e.g., for the prevention and / or treatment of the relevant disease or disorder mentioned herein.

[0189] In some embodiments, when the molecules of the present disclosure, e.g., the fusion proteins of the present disclosure, nucleic acids, vectors or pharmaceutical compositions are used to treat a tumor, the therapeutic agents in combination or association with the fusion proteins, nucleic acids, vectors or pharmaceutical compositions of the present disclosure, e.g., various therapeutic agents for treating a tumor, e.g., chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulatory agents (e.g., immune checkpoint inhibitors or agonists). In some embodiments, when the molecules of the present disclosure, e.g., the fusion proteins of the present disclosure, nucleic acids, vectors or pharmaceutical compositions are used to treat a tumor, the therapeutic modalities include surgery; radiotherapy, local or focused irradiation, etc.

[0190] Reference to combination or association in the present disclosure means administration of two or more therapeutic agents or therapeutic modalities to treat a disease described herein. Such administration includes coadministration of these therapeutic agents in a substantially simultaneous manner, e.g., in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes separate administration of the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders and liquids).

[0191] In other aspects, the present disclosure also provides the molecules of the present disclosure, e.g., fusion proteins, etc., or the fusion proteins, nucleic acids, vectors, pharmaceutical compositions and / or drug combinations for use in therapy, e.g., for treating the relevant disease or disorder mentioned herein.

[0192] In other aspects, the present disclosure also provides the molecules of the present disclosure, e.g., fusion proteins, etc., or the fusion proteins, nucleic acids, vectors, pharmaceutical compositions and / or drug combinations for use in therapy, e.g., for treating the relevant disease or disorder mentioned herein.

[0193] Examples

[0194] Example 1, Preparation of fusion proteins

[0195] The VHH sequence fused with the RGD peptide segment was fused with human IgG1 Fc or its variant, respectively, and was subjected to gene synthesis and cloned into the eukaryotic expression vector pCDNA3.4 to obtain a eukaryotic expression vector of single-domain antibody-Fc fragment fusion. The positive control antibody H2A2-IgG1 is an antibody that only binds αVβ6, and the sequence is from the h2A2 HCLG sequence in the patent WO2021113697. After gene synthesis, the sequence was cloned into the eukaryotic vector pCDNA3.4 to obtain the h2A2-hIgG1 eukaryotic expression vector.

[0196] HEK293 cells were used for transient expression. First, the cell density was adjusted to 4x10 6 cells / mL with expression medium OPM-CD05 for standby, then PEI and each of the above obtained plasmids (200 μg) were mixed according to the mass ratio of 6:1, and the complex was formed after standing at room temperature for 5 min, then the complex was added to the prepared cell suspension, and placed in a 36.5±0.5℃, 7%±3% CO2, 100±10rpm condition for 4h, then the speed was adjusted to 130±10rpm for continuous culture for 7 days, and the inhibitor and feed were added according to the actual situation during the culture.

[0197] After the culture of the transient cells, centrifugation was performed at 4℃, 4000rpm for 10 min, and the cell supernatant was collected. The 0.22 μm filter membrane was used to remove the particles in the supernatant. The treated supernatant was loaded onto the Protein A affinity column Mabselect SuRe TM , which was equilibrated with the equilibration buffer (10 mM PB, pH 6.0). The same buffer was used to rinse and remove the unabsorbed impurities, and the high-salt buffer (25 mM PB, 500 mM NaCl, pH 7.0) was used to wash the weakly absorbed impurities on the column. The elution buffer (20 mM citric acid buffer, pH 3.6) was used to elute the target protein. The eluted protein was adjusted to pH 7.0 using the neutralization buffer (2 M Tris-HCl, pH 9.5), and then electrophoresis analysis was performed to analyze the purified components.

[0198] KN035 (also referred to as hu56-Fcag in the present disclosure) is a marketed PD-L1 single-domain antibody-Fc fusion protein (SEQ ID NO: 52), and the generic name is envafolimab. Antibody strain 156 is KN035 before humanization, and both are produced and prepared by Kangtai Biopharm.

[0199] Example 2, detection of the affinity of the molecule to PD-L1 and Integrin αVβ6

[0200] The affinities of 156RGD-T54-Fc, 156RGD-N74-Fc, and control sample KN035 to hPDL1-His were detected based on Bio-Layer Interferometry (BLI) technology. The diluent was PBST20. First, the antibodies 156RGD-T54-Fc, 156RGD-N74-Fc, and control KN035 were diluted to 10 μg / mL and immobilized on a proA biosensor. Then, the hPDL1-His protein (ACRO Cat No. PD1-H52H3) was diluted to 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, and 0.78 nM, and different intensities of binding signals were detected. The results were fitted using a 1:1 model, and the equilibrium dissociation constant (KD) values of the samples were calculated. The results are shown in Table 1. The affinities of 156RGD-T54-Fc, 156RGD-N74-Fc, and control sample KN035 to hPDL1-His were 11.6 nM, 9.5 nM, and 8.8 nM, respectively, and the affinities were comparable.

[0201] Table 1

[0202] Using a similar method, the affinities of hu56RGD-T54-Fc, hu56RGD-N74-Fc, and control sample H2A2-hIgG1 to Integrin αVβ6 (Cat: IT6-H52E1, Lot: CG89P1-216KF1-1BH) in the presence or absence of metal ions were detected. The metal ions Ca 2+ Mg 2+ were added to the PBST diluent at a concentration of 100 mM. The results are shown in Tables 2 and 3. In the absence of metal ions Ca 2+ Mg 2+ , the affinities of hu56RGD-T54-Fc, hu56RGD-N74-Fc, and control sample H2A2-hIgG1 to Integrin αVβ6 were 32.1 nM, 24.5 nM, and 3.15 nM, respectively. In the presence of metal ions Ca 2+ Mg 2+ , the affinities of hu56RGD-T54-Fc, hu56RGD-N74-Fc, and control sample H2A2-hIgG1 to Integrin αVβ6 were 1.91 nM, 1.92 nM, and 50.70 nM, respectively. The affinities of hu56RGD-T54-Fc and hu56RGD-N74-Fc to Integrin αVβ6 were increased by about 20 times in the presence of metal ions compared to the absence of metal ions.

[0203] Table 2

[0204] Table 3

[0205] Using a similar method, the affinity of hu56RGDN74FM-FcagYTE, hu56RGDT54FM-Fc for hPDL1 and IntegrinαVβ6 was tested. The results showed that the KD values ​​of the affinity of hu56RGDN74FM-FcagYTE, hu56RGDT54FM-Fc, hu56RGDN74-Fc, and the control hu56-FC for hPDL1-His were 1.57 nM, 2.57 nM, 1.87 nM, and 2.28 nM, respectively, indicating that the affinity levels after antibody modification were not affected (Table 4). Under conditions where no metal ions are present, the KD values ​​of the affinity of hu56RGDN74FM-FcagYTE, hu56RGDT54FM-Fc, hu56RGDN74 Fc, and control h2A2-IgG to IntegrinαVβ6 were 49.65 nM, 29.80 nM, 38.96 nM, and 25.87 nM, respectively (Table 5).

[0206] Table 4

[0207] Table 5

[0208] Example 3: ELISA binding activity of the molecule with PD-L1

[0209] Dilute PD-L1-muFc (prepared by fusing the 19F-239T fragment of the PD-L1 sequence (uniprot ID.Q9NZQ7) and the 112P-324K fragment of the mouse IgG1 sequence (uniprot ID.P01868) into a eukaryotic expression vector and express it using CB buffer at pH 9.6 to a concentration of 3 μg / ml. Dilute 100 μl / well and incubate overnight at 2–8°C. Discard the liquid from the plate and use PBST. 20 After washing (0.05%, v / v), add PBST containing 3% BSA. 20 Block the ELISA plate (0.05%, v / v) with 250 μl / well and incubate at 25°C for 2 h. Discard the liquid from the plate and wash. hu56RGDT54-Fcag, hu56RGDN74-Fc, and KN035 are serially diluted 4-fold from 15 μg / ml, resulting in 11 dilutions. Use PBS containing 1% BSA. 20Dilute (0.05%, v / v) at 100 μl / well and incubate at 25°C for 2 h. A blank control is also included. After discarding the liquid from the plate and washing, rinse with PBST containing 1% BSA. 20 Anti-Human IgG (Fc-specific)-Peroxidase Antibody Produced in goat (Sigma, Cat: A0170, Lot: 0000154644) was diluted 1:8000 at 100 μl / well and incubated at 25°C for 2 h. After washing the plate again, soluble single-component TMB substrate solution (TIANGEN, Cat: PA107-01, Lot: X0307) was added at 100 μl / well, and the plate was incubated at room temperature in the dark. Then, 100 μl of 1M H2SO4 was added to stop the colorimetric reaction. The absorbance values ​​were then read at 450 nm and 650 nm using a microplate reader, and a concentration-OD value curve was plotted. The results are shown in Figure 1, indicating that the PD-L1 binding activity of the molecule fused with the RGD peptide was preserved.

[0210] Example 4: Blocking activity of the molecule against the binding of PD-1 and PD-L1

[0211] Dilute PD-L1-muFc to 3 μg / ml with CB buffer (pH 9.6), 100 μl / well, and incubate overnight at 2–8°C. Discard the liquid from the plate and use PBST. 20 After washing (0.05%, v / v), add PBST containing 3% BSA. 20 Block the microplate with 0.05% (v / v) solution, 250 μl / well, and incubate at 25°C for 2 h. Discard the liquid from the plate and wash thoroughly with PBST containing 1% BSA. 20 Dilute hu56RGDT54-Fcag, hu56RGDN74-Fc, and KN035 to 50 μg / ml with (0.05%, v / v) diluent, and simultaneously dilute PD1-muFc to 1 μg / ml to obtain a mixed sample. Then, use PBST containing 1% BSA with 1 μg / ml PD1-muFc. 20 (0.05%, v / v) diluent was serially diluted 4-fold, resulting in 11 dilutions. 100 μl / well was incubated at 25°C for 2 h. Simultaneously, PBST containing 1% BSA was prepared. 20 (0.05%, v / v) and PBST containing 1% BSA with 1 μg / ml PD1-muFc 20 (0.05%, v / v) dilution buffer was used as a control. After discarding the liquid from the plate and washing, it was rinsed with PBST containing 1% BSA. 20(0.05%, v / v) diluted Goat anti-Mouse IgGl Secondary Antibody, HRP (Thermo, Cat: PA1-74421, Lot: WD3238656) at 1:3000, 100 μΐ / well, 25°C for 2h. After washing the plate again, KPL SureBlue TMB Microwell Peroxidase Substrate (1-Component) (seracare, Cat: 5120-0074, Lot: 10583101) was added to develop color at room temperature in dark. Then 1M H2SO4 100 μΐ / well was added to stop the color reaction. After that, the microplate was put on a microplate reader to read the absorbance value at 450nm and 650nm wavelength. The concentration-OD value curve was drawn. The results were shown in Figure 2. The molecules with RGD peptide retained the binding activity to PD-1 and PD-L1. TM TMB Microwell Peroxidase Substrate (1-Component) (seracare, Cat: 5120-0074, Lot: 10583101) was added to develop color at room temperature in dark. Then 1M H2SO4 100 μΐ / well was added to stop the color reaction. After that, the microplate was put on a microplate reader to read the absorbance value at 450nm and 650nm wavelength. The concentration-OD value curve was drawn. The results were shown in Figure 2. The molecules with RGD peptide retained the binding activity to PD-1 and PD-L1.

[0212] Example 5, Blocking activity of the molecules to CD80 and PD-L1 binding

[0213] The blocking activity of hu56RGDT54-Fcag, hu56RGDN74-Fc, KN035 to CD80 and PD-L1 binding was investigated using similar method as in Example 4, except that the concentration of CD80-muFc (35V-242N fragment of human CD80 sequence (uniprot ID. P33681) and 112P-324K fragment of mouse IgGl sequence (uniprot ID. P01868) fused, integrated into eukaryotic expression vector for expression and preparation) in the mixed sample was 100 μg / ml, and then 1% BSA PBST containing 100 μg / ml CD80-muFc 20 (0.05%, v / v) diluted Goat anti-Mouse IgGl Secondary Antibody, HRP (Thermo, Cat: PA1-74421, Lot: WD3238656) at 1:3000, 100 μΐ / well, 25°C for 2h. After washing the plate again, KPL SureBlue TMB Microwell Peroxidase Substrate (1-Component) (seracare, Cat: 5120-0074, Lot: 10583101) was added to develop color at room temperature in dark. Then 1M H2SO4 100 μΐ / well was added to stop the color reaction. After that, the microplate was put on a microplate reader to read the absorbance value at 450nm and 650nm wavelength. The concentration-OD value curve was drawn. The results were shown in Figure 2. The molecules with RGD peptide retained the binding activity to PD-1 and PD-L1.

[0214] Example 6, ELISA binding activity of the molecules to integrin αvβ6

[0215] Dilute Human Integrin alpha V beta 6 (ITGAV & ITGB6) Heterodimer Protein, His Tag & Tag Free (MALS verified) (Cat: IT6-H52E1, Lot: CG89P1-216KF1-1BH) to 3 μg / ml, 100 μl / well, store at 2-8°C overnight. Discard the liquid in the plate, wash with PBST 20 (0.05%, v / v) and then add PBST containing 3% BSA 20 (0.05%, v / v) to block the plate, 250 μl / well, incubate at 25°C for 2 h. Discard the liquid in the plate and wash with PBST containing 1% BSA with 1 mM MgCl2 and 1 mM CaCl2 20 (0.05%, v / v) to block the plate, 250 μl / well, incubate at 25°C for 2 h. Discard the liquid in the plate and wash with PBST containing 1% BSA with 1 mM MgCl2 and 1 mM CaCl2 20 (0.05%, v / v) to block the plate, 250 μl / well, incubate at 25°C for 2 h. Discard the liquid in the plate and wash with PBST containing 1% BSA with 1 mM MgCl2 and 1 mM CaCl2 TM TMB Microwell Peroxidase Substrate (1-Component) (seracare, Cat: 5120-0074, Lot: 10583101), develop color at room temperature in the dark. Then add 1 M H2SO4 100 μl / well to stop the color developing reaction. Then place the plate on a microplate reader to read the absorbance value at 450 nm and 650 nm. Draw the concentration-OD value curve. The results are shown in Figure 4a. The molecules fused with RGD peptide have binding activity to integrin αvβ6.

[0216] Using a similar method, the binding activity of the modified molecules to integrin αvβ6 was investigated (but without adding MgCl2 and CaCl2). The results are shown in Figure 4b. The modified molecule hu56RGD-T54FM-Fc still has considerable binding activity.

[0217] Using a similar method, the binding activity of the modified molecules to integrin αvβ6 was investigated. As shown in Figure 4c, the modified molecules hu56-RGDN74FMV05-Fc and hu56-RGDN74FMV08-Fc have good binding activity.

[0218] Using a similar method, the binding activity of the modified molecules to integrin αvβ6 was further investigated. As shown in Figures 4d and 4e, the modified molecules have good αvβ6 binding activity.

[0219] Example 7, ELISA binding activity of molecules to integrin αvβ8

[0220] Dilute Human ITGAV & ITGB8 Heterodimer Protein, His Tag & Tag Free (Acro, Cat: IT8-H52W4, Lot: G24-233NF1-1DV) to 3 μg / ml, 100 μl / well, in CB buffer, pH 9.6, and store in a 2-8°C refrigerator overnight. Discard the liquid in the plate and wash with PBST 20 (0.05%, v / v) and then add PBST containing 3% BSA 20 (0.05%, v / v) and then add PBST containing 3% BSA 20 (0.05%, v / v) and then add PBST containing 3% BSA 20 (0.05%, v / v) and then add PBST containing 3% BSA TMTMB Microwell Peroxidase Substrate (1-Component) (seracare, Cat: 5120-0074, Lot: 10658733), color development at room temperature in the dark. Then add 100 μl / well of 1M H2SO4 to stop the color development reaction. Then place the enzyme-labeled plate on the enzyme-labeled instrument to read the absorbance value at 450 nm and 650 nm wavelength, and draw the concentration-OD value curve. The results are shown in Figure 5a. The modified molecules have binding activity with integrin αvβ8.

[0221] Using a similar method, the binding activity of the modified molecules to integrin αvβ8 was further investigated. As shown in Figures 5b and 5c, the modified molecules all have good αvβ8 binding activity.

[0222] Example 8, binding activity of molecules to overexpressing cells

[0223] Plasmids encoding αv(Uniprot ID: P06756) and β6 Uniprot ID: P18564) were co-transfected into HEK293 cells, and 48 h after transfection, HEK293 cells overexpressing αvβ6 (i.e., 293-αvβ6) were obtained; plasmids encoding αv and β8 (Uniprot ID: P26012) were co-transfected into HEK293 cells, and 48 h after transfection, HEK293 cells overexpressing αvβ8 (i.e., 293-αvβ8) were obtained. The cells were harvested and adjusted to a cell density of 1 × 10 6 The cells were harvested and adjusted to a cell density of 1 × 10

[0224] The results are shown in Figure 6. The test molecule can bind to HEK293 cells that overexpress ανβ6 and HEK293 cells that overexpress ανβ8 (hu56RGD-T54 in the figure is hu56RGDT54-Fc, hu56RGD-N74 is hu56RGDN74-Fc, Positive Control 2A2 is H2A2-IgG1, and Iso-hIgG1 is a negative control antibody, which is obtained by fusing the variable region of the anti-HEL antibody with the constant region of human IgG1).

[0225] Example 9: Molecular-based cellular blocking activity against PD-1 and PD-L1

[0226] (1) Karpas299 cells (human degenerative large cell lymphoma cells, highly expressing PD-L1, not expressing ανβ6, purchased from Nanjing Kebai) were collected, counted, and the cell density was adjusted to 1×10⁻⁶. 6 Cells / ml, 100 μl / well added to a 96-well flow cytometry plate, centrifuged and ready for use. Antibody was diluted with PBS (containing 1 mM MgCl2 and 1 mM CaCl2), serially diluted 3-fold starting at 400 nM, for a total of 9 spots. The sample dilution buffer contained PD-1-muFc protein (prepared by fusing the extracellular domain of human PD-1 (uniprot ID. Q15116) and the 112P-324K fragment of mouse IgG1 sequence (uniprot ID. P01868) into a eukaryotic expression vector at a final concentration of 5 μg / ml). 100 μl / well of the diluted sample was added to the cell-containing 96-well flow cytometry plate and incubated at 4°C for 30 min, followed by washing twice with PBS. Add 100 μl / well of mouse anti-human IgG-Fc (purchased from Biolegend, catalog number 366906) fluorescent secondary antibody diluted 100-fold with PBS (containing 1 mM MgCl2 and 1 mM CaCl2), incubate at 4°C for 30 min, and wash twice with PBS. Resuspend cells in PBS with 100 μl / well, and perform flow cytometry on a CytoFlex (purchased from Bechman) instrument to calculate the corresponding median fluorescence intensity. The blocking effect of the antibody is evaluated based on the median fluorescence intensity. The results are shown in Figure 7a. The molecule fused with the RGD peptide can block the binding of PD-1 and PD-L1 (hu56RGD-T54 in the figure is hu56RGDT54-Fc, hu56RGD-N74 is hu56RGDN74-Fc, and hu56 is KN035).

[0227] (2) PD-L1 / TCR Activator-CHO cells (purchased from BPS Bioscience, Cat No. 60536) were digested with pancreatin, centrifuged at 1000 rpm for 5 min, resuspended with 10% FBS+F-12K medium (purchased from Gibco, Cat No. 21127-022) and adjusted to a cell density of 4*10 5 cells / ml, 50ul 10% FBS+F-12K medium and 50ul cell suspension were added to each well of the 96-well white plate, and the plate was placed in a 37°C incubator overnight. The test molecules were prepared using the culture medium, with a maximum preparation concentration of 10nM (working concentration 5nM), 2-fold gradient dilution, a total of 9 concentration points; the culture supernatant in the 96-well white plate was discarded, 50ul of drug diluent was added, and the plate was placed in the incubator for 60min. PD-1 / NFAT Reporter-Jurkat cells (purchased from BPS Bioscience, Cat No. 60535) were collected, centrifuged at 1000 rpm for 5 min, resuspended with 2% FBS+1640 medium and adjusted to a cell density of 8*10 5 cells / ml, 50ul of each well was added to the 96-well white plate containing PD-L1 / TCR Activator-CHO cells. After the 96-well white plate was placed in the incubator for 6h, 50ul of luciferase substrate (One-Lumi Firefly Luciferase Reporter Gene Assay Kit, Biyun Tian, CAT: RG055M) was added to each well, and after 5min at room temperature, the RLUs value was read by MD-ID5 enzyme label instrument. The concentration-RLUs value was fitted with a 4-parameter curve using GraphPad Prism software, and the EC50 value was calculated. The results are shown in Figures 7b and 7c, and the molecule fused with RGD peptide retains the blocking activity of PD-1 and PD-L1.

[0228] Example 10, Inhibitory activity of molecules in TGFβ reporter gene system

[0229] Preparation of 293T-pro-TGF-β1 cells:

[0230] 1. Lentivirus supernatant preparation: HEK293T cells were placed in 10 cm culture dishes, and the culture medium was 10% FBS + DMEM, which was placed in a 37°C incubator overnight; prepare the virus packaging plasmid and target gene plasmid transfection mixture: mix pSPAX2 (purchased from Youbao Biology, VT1444), pMD2.G (a gift from Nanjing University) and the target gene plasmid containing pro-TGFβ1 (target gene sequence uniprot ID P01137), then add PEI (polyethylenimine, purchased from Polysciences, Inc. Cat# 24765-1) and mix, equilibrate at room temperature for 20 min; add the transfection mixture to the cells in the changed liquid, incubate in the incubator for 6 h, then add 1 mL of cell culture medium (DMEM + 10% FBS), and incubate overnight; remove the old culture medium, add 8 mL of preheated virus packaging medium (DMEM + 10% FBS + 1% Pen / Strep + 1% NEAA + 1% Sodium Pyruvate); start 36 h after changing the liquid, observe the cell state in time, and collect the virus supernatant in time, when collecting, place the culture plate at -80°C for 5 min, lyse the cells, aspirate the cell culture medium, and filter it with a 0.45 μm filter to remove cell debris.

[0231] 2. Virus supernatant transfection of cells: collect 293T cells, dilute the cells with DMEM medium containing 10% FBS, and plate the cells in a 6-well plate, and incubate in the incubator overnight; after removing the supernatant, add the virus supernatant in step 1, and then add Polybrene (purchased from Yixing Biology, Cat# 40804ES76) and mix well, and incubate overnight; then add pressure passage, and add 5 μg / mL doxycycline hydrochloride (Solarbio, D8960) before the experiment to induce for 72 h.

[0232] 293T-SBE-luc cells were prepared in a similar manner, except that the target gene plasmid containing the SBE response element sequence (see Promega plasmid pGL4.48 [luc2P / SBE / Hygro] Vector for sequence information) was used instead of the aforementioned target gene plasmid containing pro-TGFβ1.

[0233] The test sample was prepared using 10% FBS + DMEM medium, the highest concentration was 200 μg / mL (working concentration 50 μg / mL), 5-fold gradient dilution, a total of 4 concentration points. The logarithmic phase cultured Capan 2 cells (human pancreatic cancer cells, medium expression of αvβ6, low expression of PD-L1, purchased from Beina Biotechnology) were centrifuged at 1000 rpm for 5 minutes, resuspended with 10% FBS + DMEM medium and adjusted to a cell density of 4×10^5 cells / ml, 25 μL of Capan 2 cell suspension was added to each well of a 96-well white transparent bottom plate. 2 μg / mL puromycin + 5 μg / mL DOX (doxorubicin hydrochloride, purchased from Solarbio, catalog number D8960) + 10% FBS + DMEM medium for 72 hours 293T-pro-TGF-β1 cells were trypsinized, centrifuged at 1000 rpm for 5 minutes, and after the supernatant was discarded, resuspended with 10% FBS + DMEM medium and adjusted to a cell density of 8×10^5 cells / ml. The 293T-SBE-luc cells were trypsinized, centrifuged at 1000 rpm for 5 minutes, resuspended with 10% FBS + DMEM medium and adjusted to a cell density of 1×10^6 cells / ml. In a 96-well white plate, 25 μl of each sample diluent, 25 μl of Capan2 cells, 25 μl of 293T-pro-TGF-β1 cells and 25 μl of 293T-SBE-luc cells were added to each well, two replicate wells for each group, and incubated in the incubator for a total of 24 hours. Then, 50 μL of luciferase substrate (One-Lumi Fierfly Luciferase Reporter Gene Assay Kit, Biyun Tian, CAT: RG055M) was added to each well, and after 5 minutes at room temperature, the RLUS value was detected by MD-ID5 enzyme label instrument. Taking the RLU value of the cell well without adding sample as the control, according to the RLU value of each sample treatment well, the inhibition activity percentage of different samples under different concentration conditions was calculated, and the GraphPad Prism software was used for plotting.

[0234] The results are shown in Figure 8, and each sample showed concentration-dependent inhibition activity.

[0235] Example 11, binding experiment of molecules and human tumor cells

[0236] Capan2 cells (human pancreatic cancer cells, medium expression of ανβ6, low expression of PD-L1, purchased from North Naibio) were digested with 0.25% Trypsin-EDTA (1X) Solution (purchased from Gibco, item number 25200056) and Karpas299 cells (human anaplastic large cell lymphoma cells, high expression of PD-L1, no expression of ανβ6, purchased from Nanjing Kebai) were harvested, counted and adjusted to a cell density of 1x10 6 The cells were centrifuged and resuspended in PBS (containing 1 mM MgCl2and 1 mM CaCl2) at a density of 1x10

[0237] The results are shown in Figure 9. The test molecules can bind to human tumor cells Karpas299 and Capan2 (hu56RGD-T54 in the figure is hu56RGDT54-Fc, hu56RGD-N74 is hu56RGDN74-Fc, and hu56 is KN035).

[0238] Using a similar method, the binding activity of the variant molecules to Capan2 cells and HCC70 cells (human breast cancer cells, high expression of ανβ6, low expression of PD-L1, purchased from Nanjing Kebai) was tested. The results are shown in Figure 10. The variant molecules hu56RGDT54B1-Fc (shown as hu56RGD-T54B1 in the figure) and hu56RGDT54FM-Fc (shown as hu56RGD-T54M in the figure) can bind to human tumor cells Capan2 and HCC70.

[0239] Example 12, Serum stability test

[0240] The hu56-RGDN74FMV08-Fc, hu56-RGDN74FMV05-Fc, hu56RGD-T54FM-Fc, hu56-RGDN74-Fcag and hu56-RGDT54-Fcag were prepared into stability samples with a concentration of 100 μg / ml using filtered bacteria-free cynomolgus monkey blank serum (purchased from Blue Island Biological, batch number 20181214), and the prepared samples were divided and placed at 37°C for 0, 1, 3, 5, 7, 14 days, respectively. The samples at the corresponding time points were taken out and frozen at -80°C for final detection. After all the stability samples were taken out, the sample concentration was detected by ELISA method to investigate the serum stability of the candidate molecules. The ELISA method is as follows:

[0241] Dilute Human Integrin alpha V beta 6 (ITGAV & ITGB6) Heterodimer Protein, His Tag & Tag Free (MALS verified) (Cat: IT6-H52E1, Lot: CG89P1-216KF1-1BH) to 3 μg / ml, 100 μl / well, and place in a 2-8°C refrigerator overnight. After washing the plate with PBST (0.05%, v / v), add 3% BSA-containing PBST 20 (0.05%, v / v) to block the enzyme-labeled plate, 250 μl / well, 25°C incubation for 2h. Discard the liquid in the plate and wash with PBST containing 5% cynomolgus monkey blank serum, 1 mM MgCl2, 1 mM CaCl2, 1% BSA 20 (0.05%, v / v) to block the enzyme-labeled plate, 250 μl / well, 25°C incubation for 2h. Discard the liquid in the plate and wash with PBST containing 5% cynomolgus monkey blank serum, 1 mM MgCl2, 1 mM CaCl2, 1% BSA 20 (0.05%, v / v) to block the enzyme-labeled plate, 250 μl / well, 25°C incubation for 2h. Discard the liquid in the plate and wash with PBST containing 5% cynomolgus monkey blank serum, 1 mM MgCl2, 1 mM CaCl2, 1% BSA 20(0.05%, v / v) Dilute Goat Anti-Human IgG, Monkey ads-HRP (SouthernBiotech, Cat:2049-05, Lot:L3320-QD81C) 1:5000, 100 μl / well, incubate at 25℃±2℃ for 2 h. After washing the plate again, add KPL SureBlue. TM TMB Microwell Peroxidase Substrate (1-Component) (seracare, Cat: 5120-0074, Lot: 10662425, Lot: 10658733) was incubated at room temperature in the dark. 100 μl of 1M H₂SO₄ was added to each well to terminate the reaction. After termination, the absorbance values ​​were read at 450 nm and 650 nm using a microplate reader. Based on the standard curve for each sample, the concentration-OD value calculation formula was fitted. Using the OD colorimetric value of each sample and the sample dilution factor, the sample concentration was calculated, and finally, the percentage change in sample concentration was calculated. A graph showing the percentage change in concentration over time was then plotted.

[0242] As shown in Figure 11, the modified FM series molecules have improved serum stability, with FMV05 and FMV08 exhibiting even better stability.

[0243] sequence list

[0244] SEQ ID NO: 32(156RGDT54-Fc) (T54 means that the amino acid T54 is inserted after the 54th amino acid Thr from the N-terminus, the same below) (Gray highlights indicate CDR regions (defined by Kabat), yellow highlights indicate RGD peptides, underlines indicate spacer peptides, and bold text indicates mutated amino acids, the same applies below.)

Claims

1. A fusion protein comprising an immunoglobulin single variable domain capable of binding PD-L1, said immunoglobulin single variable domain comprising a CDR1, a CDR2 and a CDR3, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 1; the CDR2 comprises the amino acid sequence of SEQ ID NO: 2 and the CDR3 comprises the amino acid sequence of SEQ ID NO: 3; or, the CDR1 consists of the amino acid sequence of SEQ ID NO: 1, the CDR2 consists of the amino acid sequence of SEQ ID NO: 2 and the CDR3 consists of the amino acid sequence of SEQ ID NO: 3; the fusion protein further comprises a polypeptide inserted in the CDR2 region or the FR3 region of the immunoglobulin single variable domain, said polypeptide comprising a motif capable of binding a second antigen or receptor.

2. The fusion protein according to claim 1, the immunoglobulin single variable domain being a VHH.

3. The fusion protein according to claim 1 or 2, the immunoglobulin single variable domain comprising the amino acid sequence of SEQ ID NO: 4; or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity thereto; or consisting of the amino acid sequence of SEQ ID NO:

4.

4. The fusion protein according to any one of claims 1 to 3, the immunoglobulin single variable domain being a humanized VHH.

5. The fusion protein according to any one of claims 1 to 4, the immunoglobulin single variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 5 to 10; or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5 to 10; or consisting of the amino acid sequence of any one of SEQ ID NOs: 5 to 10.

6. The fusion protein according to any one of claims 1 to 5, the polypeptide being inserted after amino acid position 50, 51, 52, 52A, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 in the CDR2 region; preferably, the polypeptide is inserted after amino acid position 50, 51, 52, 52A, 53, 54 or 55 in the CDR2 region; more preferably, the polypeptide is inserted after amino acid position 53 or 54 in the CDR2 region; the CDR2 region is numbered according to the Kabat numbering system. ​ ​ ​ 7. The fusion protein according to any one of claims 1-6, wherein the polypeptide is inserted after amino acid position 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 82A, 82B, 82C, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 or 94 of the FR3 region; preferably, the polypeptide is inserted after amino acid position 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or 76 of the FR3 region; more preferably, the polypeptide is inserted after amino acid position 70, 71, 72, 73 or 74 of the FR3 region; even more preferably, the polypeptide is inserted after amino acid position 71 or 73 of the FR3 region; the FR3 region is numbered according to the Kabat numbering system.

8. The fusion protein according to any one of claims 1-7, wherein the N-terminus and / or the C-terminus of the polypeptide each independently further comprises a spacer peptide consisting of 1-4 amino acids, for example, the N-terminus and the C-terminus of the polypeptide each independently comprises the spacer peptide.

9. The fusion protein according to claim 8, wherein the spacer peptide is selected from one or more of A, G, S, P, AA, AS, GG, GS, SS, PP, ASGS, GSAS and GGGS; preferably, the polypeptide comprises the following combination of spacer peptides at the N-terminus and the C-terminus, respectively: (a) N-terminus: A, C-terminus: AA; (b) N-terminus: A, C-terminus: AS; (c) N-terminus: A, C-terminus: A; (d) N-terminus: PP, C-terminus: AS; (e) N-terminus: PP, C-terminus: ASGS; (f) N-terminus: PP, C-terminus: A; and (g) N-terminus: PP, C-terminus: AA.

10. The fusion protein according to any one of claims 1-9, wherein the second antigen or receptor is an integrin, preferably avb6 and / or avb8.

11. The fusion protein according to any one of claims 1-10, wherein the polypeptide comprises an Arg-Gly-Asp (RGD) motif.

12. The fusion protein according to claim 11, wherein the polypeptide comprising the RGD motif is derived from Pro-TGF-b, preferably, the polypeptide is derived from Pro-TGF- b1, Pro-TGF-b2 or Pro-TGF-b3, for example, is a fragment of 11 contiguous amino acids of the protein comprising the RGD.

13. The fusion protein according to claim 11, wherein the polypeptide comprising the RGD motif is derived from foot-and-mouth disease virus (FMDV), for example, is a fragment of 11 contiguous amino acids of the protein comprising the RGD.

14. The fusion protein according to any one of claims 10-13, wherein the polypeptide comprising the RGD sequence is selected from the amino acid sequence set forth in any one of SEQ ID NOs: 11-14 or the polypeptide comprising the RGD sequence has 1, 2 or 3 substitutions, deletions or additions of amino acids compared to the amino acid sequence set forth in any one of SEQ ID NOs: 11-14. ​ 15. The fusion protein of any one of claims 1-14, wherein the intervening polypeptide in combination with the spacer peptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 17-22 or the intervening polypeptide has 1, 2, or 3 amino acid substitutions, deletions, or additions compared to the amino acid sequence of any one of SEQ ID NOs: 17-22.

16. The fusion protein of any one of claims 1-15, wherein the fusion protein comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 23-31; or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 23-31.

17. The fusion protein of any one of claims 1-16, further comprising an immunoglobulin Fc region.

18. The fusion protein of claim 17, wherein the immunoglobulin Fc region is a human immunoglobulin Fc region, preferably an Fc region of human IgGl, human IgG2, human IgG3, or human IgG4, or a variant thereof.

19. The fusion protein of claim 18, wherein the amino acid sequence of the immunoglobulin Fc region is set forth in any one of SEQ ID NOs: 15, 16, or 53-61.

20. The fusion protein of any one of claims 17-19, wherein the fusion protein comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 32-51; or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 32-51.

21. The fusion protein of any one of claims 1-20, wherein the fusion protein has at least one of the following characteristics: (2) blocks the interaction of PD-L1 and PD-1; (3) blocks the interaction of PD-L1 and CD80; (4) is capable of binding to tumor cells that overexpress PD-L1.

22. The fusion protein of any one of claims 1-21, wherein the fusion protein has at least one of the following characteristics: (1) a KD value of less than 1 ╳ 10 -7 M, preferably less than 1 ╳ 10 -8 M, more preferably less than 1 ╳ 10 -9 M, even more preferably less than 1 ╳ 10 -10 M; (c) is capable of binding to cells that overexpress ανβ6 and / or ανβ8; (d) is capable of inhibiting the activation of TGF-β, preferably TGF-β1 and / or TGF-β3.

23. The fusion protein of any one of claims 1-22, wherein the fusion protein is a homodimer.

24. A nucleic acid molecule encoding the fusion protein of any one of claims 1-23. (a) a KD value of less than 1 ╳ 10 -6 M, preferably less than 1 ╳ 10 -7 M, more preferably less than 1 ╳ 10 -8 M, even more preferably less than 1 ╳ 10 -9 M; (b) a KD value of less than 1 ╳ 10 -6 M, preferably less than 1 ╳ 10 -7 M, more preferably less than 1 ╳ 10 -8 M, even more preferably less than 1 ╳ 10 -9 M; 25. An expression vector comprising the nucleic acid molecule of claim 24 operably linked to an expression control element. ​ ​ ​ ​ 26. A recombinant cell comprising the nucleic acid molecule of claim 24 and / or transformed with the expression vector of claim 25, and capable of expressing the fusion protein.

27. A pharmaceutical composition comprising the fusion protein of any one of claims 1-23 and / or the nucleic acid molecule of claim 24 and / or the expression vector of claim 25 and / or the recombinant cell of claim 27, and a pharmaceutically acceptable carrier.

28. A kit comprising the fusion protein of any one of claims 1-23 and / or the pharmaceutical composition of claim 27.

29. A method of treating and / or preventing cancer, comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-23, the nucleic acid molecule of claim 24, the expression vector of claim 25 and / or the pharmaceutical composition of claim 27.

30. The method of claim 29, wherein the cancer is selected from anaplastic large cell lymphoma, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer, and osteosarcoma.

31. A method of treating and / or preventing a fibrotic disease or disorder, comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-23, the nucleic acid molecule of claim 24, the expression vector of claim 25 and / or the pharmaceutical composition of claim 27.

32. The method of claim 31, wherein the fibrotic disease or disorder is selected from pulmonary fibrosis, liver fibrosis, cardiac or vasculature fibrosis, renal fibrosis, skin fibrosis, gastrointestinal tract fibrosis, bone marrow or hematopoietic tissue fibrosis, nervous system fibrosis, joint fibrosis, or a combination thereof.

Images