A bifunctional fusion protein and its use

Abstract

The present disclosure provides a fusion protein comprising at least a portion of TGFβRII and an anti-PD-L1 antibody or an antigen-binding portion thereof, a method for producing the fusion protein, a method for treating a disease or condition using the fusion protein, and uses thereof.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of International Patent Application PCT / CN2020 / 076658, filed on February 25, 2020, the entire content of which is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing in electronic form, the entire content of which is incorporated herein by reference. Technical Field

[0005] The present disclosure generally relates to bifunctional fusion proteins, methods for their preparation, and uses. Background Art

[0006] PD - 1 is one of the immune checkpoint proteins, an inhibitory member of the CD28 family, and is expressed on activated CD4+ T cells, CD8+ T cells, and B cells. Its ligand, PD - L1, is a type I transmembrane protein that is presumed to play a major role in suppressing the adaptive immune system. The binding of PD - L1 to PD - 1 transmits an inhibitory signal through interaction with phosphatases (SHP - 1 or SHP - 2) via immunoreceptor tyrosine - based switch motifs (ITSMs). As a result, this pathway inhibits T - cell proliferation and T - cell functions, such as cytokine production and cytotoxic activity. The PD - 1 / PD - L1 axis plays a major role in down - regulating the immune system [1, 2].

[0007] Monoclonal antibodies targeting PD - 1 or PD - L1 can block the binding of PD - 1 / PD - L1 and enhance the immune response against cancer cells. These drugs have shown great potential in treating certain cancers. Several pharmaceutical companies have developed multiple approved therapeutic antibodies targeting PD - 1 / PD - L1, including Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), and Durvalumab (Imfinzi). These drugs have shown efficacy in treating various types of cancer, including cutaneous melanoma, non - small cell lung cancer, renal cancer, bladder cancer, head and neck cancer, and Hodgkin's lymphoma. The use of these drugs in treating many other types of cancer is also under investigation [3].

[0008] Transforming growth factor-β (TGF-β) is a family of structurally related proteins that includes TGF-β, activin / inhibin, and bone morphogenetic protein (BMP). Members of the TGF-β family control many cellular functions, including proliferation, apoptosis, differentiation, epithelial-mesenchymal transition (EMT), and migration. TGF-β dysregulation is associated with carcinogenesis. In the early stages of cancer, TGF-β exhibits tumor-suppressive effects by inhibiting cell cycle progression and promoting apoptosis. However, in the late stage, TGF-β exerts tumor-promoting effects, increasing tumor invasiveness and metastasis. In addition, the TGF-β signaling pathway communicates with other signaling pathways in a synergistic or antagonistic manner and regulates cellular functions. Given the critical role of TGF-β in tumor progression, this pathway is an attractive target for cancer therapy [4]. Several therapeutic tools have shown great potential in inhibiting TGF-β signaling, such as TGF-β antibodies, antisense oligonucleotides, and small molecule inhibitors of TGF-β receptor 1 (TGF-βR1). Finally, to develop future therapeutic methods, further research is necessary to identify new convergence points of TGF-β with other signaling pathways and carcinogens in the tumor microenvironment.

[0009] Recently, therapeutic agents that simultaneously target the PD1 / PD-L1 and TGF-β pathways have been reported, such as bifunctional proteins containing the extracellular domain (ECD) of TGFβRII and an anti-PD-L1 antibody. However, the development of such fusion antibodies still has room for improvement and clinical needs. In the present disclosure, a novel bifunctional protein containing the TGFβRII ECD and an anti-PD-L1 antibody is described. This fusion antibody protein exhibits excellent protein stability and in vivo antitumor activity. These results demonstrate the great potential of this novel fusion antibody as a candidate drug for further preclinical studies. Summary of the Invention

[0010] Broadly speaking, the present disclosure relates to providing compounds, methods, compositions, and articles of manufacture having antibodies and antibody-like proteins (such as fusion protein molecules) with improved efficacy. The benefits provided by the present disclosure are widely applicable to the fields of antibody therapy and diagnosis and can be used in combination with antibodies capable of reacting with various targets.

[0011] In one aspect, the present disclosure provides a fusion protein comprising an antibody or an antigen-binding portion thereof that specifically binds to PD-L1 fused to a human transforming growth factor-β receptor (TGFβR) or a portion thereof that can bind TGF-β, wherein the antibody or the antigen-binding portion thereof comprises:

[0012] Heavy chain CDR1 (HCDR1), which comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:1 by no more than 2 amino acids;

[0013] HCDR2, which comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:2 by no more than 2 amino acids;

[0014] HCDR3, which comprises the amino acid sequence of SEQ ID NO:3 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:3 by no more than 2 amino acids;

[0015] Light chain CDR1 (LCDR1), which comprises the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:4 by no more than 2 amino acids;

[0016] LCDR2, which comprises the amino acid sequence of SEQ ID NO:5 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:5 by no more than 2 amino acids; and

[0017] LCDR3, which comprises the amino acid sequence of SEQ ID NO:6 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:6 by no more than 2 amino acids.

[0018] In some embodiments, the antibody or antigen-binding portion thereof as disclosed herein comprises:

[0019] HCDR1 comprising SEQ ID NO:1; HCDR2 comprising SEQ ID NO:2; and HCDR3 comprising SEQ ID NO:3; and

[0020] LCDR1 comprising SEQ ID NO:4; LCDR2 comprising SEQ ID NO:5; and LCDR3 of SEQ ID NO:6.

[0021] In some embodiments, the antibody or antigen-binding portion thereof as disclosed herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises:

[0022] (A) The amino acid sequence as shown in SEQ ID NO:7;

[0023] (B) An amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:7; or

[0024] (C) An amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO:7;

[0025] and / or VL comprises:

[0026] (A) an amino acid sequence as set forth in SEQ ID NO:8;

[0027] (B) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:8; or

[0028] (C) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO:8.

[0029] In some embodiments, the human TGFβR is selected from TGFβRII or TGFβRIII, preferably TGFβRII. In some preferred embodiments, the fusion protein comprises a portion of TGFβRII rather than full-length TGFβRII, and this portion is the extracellular domain of TGFβRII.

[0030] In some embodiments, the human TGFβR or a portion thereof that can bind TGFβ as disclosed herein comprises:

[0031] (A) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of wild-type human TGFβRII;

[0032] (B) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of the extracellular domain of wild-type human TGFβRII; or

[0033] (C) a portion of wild-type human TGFβRII that retains at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the ability to bind TGFβ.

[0034] In some embodiments, the TGFβR or a portion thereof that can bind TGFβ comprises or consists of the amino acid sequence of the extracellular domain of wild-type human TGFβRII, i.e., the amino acid sequence of SEQ ID NO:9.

[0035] In some embodiments, the antibody or an antigen-binding portion thereof included in the fusion protein is a whole antibody, ScFv, Fab, F(ab’)2, or Fv fragment, e.g., a whole antibody.

[0036] In some embodiments, the antibody or an antigen-binding portion thereof comprises a VH region operably linked to a heavy-chain Fc region. For example, the antibody or an antigen-binding portion thereof can be a whole antibody and comprises VH-CH1-hinge-Fc in the heavy chain and VL-CL in the light chain.

[0037] In some embodiments, the antibody or antigen-binding portion thereof is an IgG1, IgG2, IgG3 or IgG4 isotype, preferably the IgG1 isotype.

[0038] In some embodiments, the Fc region of the antibody or antigen-binding portion thereof is operably linked to the N-terminus of human TGFβR or a portion thereof, optionally via a linker. The linker can be a peptide linker. In some embodiments, the linker comprises (G4S)n, where n = 1-4, for example n can be 1, 2, 3 or 4.

[0039] In some embodiments, the antibody or antigen-binding portion thereof is a humanized or fully human antibody, such as a fully human antibody. In some embodiments, the heavy and light chains of the fusion protein comprise SEQ ID NO:10 and 11, respectively.

[0040] In one aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or antigen-binding portion thereof and / or human TGFβR or a portion thereof of the fusion protein as defined above.

[0041] In one aspect, the present disclosure provides a vector comprising a nucleic acid molecule as defined herein. In one aspect, the present disclosure provides a host cell comprising a nucleic acid molecule or vector as disclosed herein.

[0042] In one aspect, the present disclosure provides a pharmaceutical composition comprising a fusion protein as disclosed herein and a pharmaceutically acceptable carrier.

[0043] In one aspect, the present disclosure provides a method for producing a fusion protein as disclosed herein, comprising the steps of:

[0044] - expressing the fusion protein in a host cell as disclosed herein; and

[0045] - isolating the fusion protein from the host cell.

[0046] In one aspect, the present disclosure provides a method for modulating an immune response in a subject, comprising administering to the subject a fusion protein or pharmaceutical composition as disclosed herein.

[0047] In one aspect, the present disclosure provides a method for inhibiting the growth of tumor cells related to PD-1 / PD-L1 in a subject, comprising administering to the subject an effective amount of a fusion protein or pharmaceutical composition as disclosed herein.

[0048] In one aspect, the present disclosure provides a method for preventing or treating PD-1 / PD-L1-related cancer in a subject, comprising administering to the subject an effective amount of a fusion protein or pharmaceutical composition as disclosed herein. The cancer may be selected from colon cancer, lymphoma, lung cancer, liver cancer, cervical cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer or gastric cancer. In certain embodiments, the cancer is colon cancer or lung cancer, such as NSCLC.

[0049] In some embodiments, the fusion protein as disclosed herein is administered in combination with a chemotherapeutic agent, radiotherapy, and / or other agents for cancer immunotherapy.

[0050] In one aspect, the present disclosure provides a fusion protein as disclosed herein for treating or preventing PD-1 / PD-L1-related cancer.

[0051] In one aspect, the present disclosure provides the use of a fusion protein as disclosed herein in the preparation of a medicament for regulating an immune response related to PD-1 / PD-L1 or inhibiting the growth of tumor cells related to PD-1 / PD-L1 in a subject.

[0052] In one aspect, the present disclosure provides the use of a fusion protein as disclosed herein in the preparation of a medicament for treating or preventing PD-1 / PD-L1-related cancer.

[0053] In one aspect, the present disclosure provides a kit for treating or diagnosing cancer, which comprises a fusion protein as disclosed herein in a container.

[0054] The above content is an overview and thus includes simplifications, generalizations and omissions of details where necessary; thus, those skilled in the art will recognize that this overview is merely illustrative and not intended to limit in any way. Other aspects, features and advantages of the methods, compositions and / or devices and / or other subject matter described herein will become apparent in the teachings shown herein. The overview is provided to introduce some selected concepts in a simplified manner, which will be further described in the detailed description below. This overview is not intended to identify the key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. In addition, the contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 shows the binding results of the antibody to human TGF-β1, TGF-β2 and TGF-β3 as determined by ELISA when TGF-β is immobilized (A) or when the antibody is immobilized (B). Human IgG1 is an isotype control.

[0056] Figure 2 shows the binding results of the antibody to cells expressing human PD-L1 (A), cynomolgus monkey PD-L1 (B), and mouse PD-L1 (C) as determined by FACS.

[0057] Figure 3 shows that when TGF-β1 (A) or human PD-L1 (B) is immobilized, the antibody binds both PD-L1 and TGF-β1 simultaneously as determined by ELISA.

[0058] Figure 4 Shows the results of the antibody blocking the binding of PD-1 to cell surface PD-L1 as determined by FACS.

[0059] Figure 5A Shows the results of the antibody blocking TGF-β1 signaling in the RGA assay. Figure 5B Shows the results of the antibody blocking human PD-1 / PD-L1 signaling in the RGA assay. Data are presented as mean ± SEM.

[0060] Figure 6 shows the results of IL-2 (A) and IFN-γ (B) production by the antibody in the allogeneic mixed lymphocyte reaction.

[0061] Figure 7 Shows the serum stability results of the antibody by dual-binding ELISA test and PD-L1-binding FACS test.

[0062] Figure 8 shows the body weight changes (A) and anti-tumor efficacy (B) induced by antibody administration in the mouse HCC827 PBMC model.

[0063] Figure 9 Shows the results of the in vivo pharmacokinetic study of the WT1122 antibody. Detailed Description

[0064] Although the present invention may be embodied in many different forms, specific illustrative embodiments thereof that verify the principles of the present invention are disclosed herein. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. Additionally, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0065] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings as commonly understood by one of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include plural forms and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “a protein” includes a plurality of proteins; reference to “a cell” includes a mixture of cells, etc. In this application, unless otherwise stated, the use of “or” means “and / or.” In addition, the use of the term “comprising” and other forms (such as “includes” and “containing”) is not limiting. Further, the ranges provided in the specification and the appended claims include the endpoints and all values between the endpoints.

[0066] Generally, the terms associated with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, as well as the techniques thereof, are well-known and commonly used terms in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6th Edition, W.B. Saunders Company (2010); Sambrook J. & Russell D.M. Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The terms associated with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as laboratory procedures and techniques, are those well-known and commonly used in the art.

[0067] Definitions

[0068] For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0069] The term "antibody" or "Ab" is used herein in the broadest sense and encompasses a variety of antibody structures, including polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies). A native intact antibody generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be divided into κ and λ light chains. The heavy chains can be divided into μ, δ, γ, α, and ε, which define the isotypes of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable region is joined to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (termed complementarity determining regions (CDRs)) separated by relatively conserved regions (termed framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the N-terminus to the C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site. The distribution of amino acids in the various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-883. Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies. Fusion proteins comprising an anti-PD-L1 antibody or antigen-binding portion thereof, as disclosed herein, also fall within the scope of antibodies.

[0070] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, which may be used interchangeably in the context of this application, refer to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind an antigen with the same antigen specificity as the full-length antibody, and / or that competes with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W. ed., 2nd ed., Raven Press, N.Y. (1989)), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be derived, e.g., from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the variable domains and optionally the constant domains of the antibody. Such DNA is known and / or can be readily obtained from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biological techniques, e.g., to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, generate cysteine residues, modify, add or delete amino acids, etc.

[0071] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units composed of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides) or restricted FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the expression "antigen-binding fragment" as used herein. In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. The variable domain and the constant domain can be directly linked to each other or can be linked by a complete or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule.

[0072] As used herein, the term "variable domain / domain of an antibody" or "variable region" refers to the variable region of an antibody or a fragment thereof that contains one or more CDRs. Although the variable domain may contain the complete variable region (such as HCVR or LCVR), it may also contain less than the complete variable region and still retain the ability to bind antigen or form an antigen-binding site.

[0073] The "Fc" of an antibody refers to the following part of the antibody, which contains the second (CH2) and third (CH3) constant regions of the first heavy chain joined to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc region may also contain all or part of the hinge region. The Fc portion of an antibody is responsible for various effector functions such as ADCC and CDC, but does not function in antigen binding. The ability of an antibody to initiate and regulate effector functions via its Fc domain is a key part of its protective activity in vivo. Although it was previously thought that the neutralizing activity of an antibody was solely the result of Fab-antigen interactions, increasing evidence indicates that its in vivo activity also highly depends on the interaction between the IgG Fc domain and its associated receptors, Fc gamma receptors (FcγR), which are expressed on the surface of effector lymphocytes.

[0074] The term "PD-L1", also known as programmed death ligand 1, is a 40 kDa type I transmembrane protein that is presumed to play a major role in suppressing the adaptive immune system. PD-L1 is the major ligand of programmed death 1 (PD-1). As used herein, the term "PD-L1", when referring to the amino acid sequence of the PD-L1 protein (such as provided by NCBI GenBank ID: NP_054862.1), includes the full-length PD-L1 protein, or the extracellular domain of PD-L1 (PD-L1 ECD) or a fragment containing PD-L1 ECD; it also includes fusion proteins of PD-L1 ECD, such as fragments fused with murine or human IgG Fc (mFc or hFc). In addition, as understood by those skilled in the art, the PD-L1 protein will also include those PD-L1 proteins with mutations (including but not limited to substitutions, deletions, and / or additions) in the amino acid sequence introduced naturally or artificially without affecting the biological function.

[0075] As used herein, the term "antibody that binds to PD-L1" or "anti-PD-L1 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize or bind to PD-L1. As used herein, the expression "anti-PD-L1 antibody" includes monovalent antibodies with single specificity, as well as bispecific antibodies that contain a first antigen-binding site that binds to PD-L1 and a second antigen-binding site that binds to a second (target) antigen.

[0076] The term "TGFβ", which is transforming growth factor beta (TGF-β), is a multifunctional cytokine belonging to the transforming growth factor superfamily, which includes three different mammalian isoforms (TGF-β1 to 3, HGNC symbols TGFB1, TGFB2, TGFB3) and many other signaling proteins. TGFβ is involved in paracrine signaling and can be found in many different tissue types, including the brain, heart, kidney, liver, bone, and testis. TGF-β dysregulation is associated with carcinogenesis. For example, a potential association has been reported between elevated PD-L1 expression and activated TGF-β signaling in certain human tumor samples (Justin M. David et al. Oncoimmunology. 2017;6(10):e1349589).

[0077] The term "TGFβR", which is a receptor of the TGFβ family, can be divided into three types: type I, type II, and type III. For a total of 13 TGFβ superfamily receptors, there are 7 type I receptors, 5 type II receptors, and 1 type III receptor. As used herein, "TGFβRII" or "TGFβ receptor II" refers to a polypeptide having the wild-type human TGFβ receptor type 2 isoform A sequence (e.g., NCBI Reference Sequence (RefSeq) accession number NP-001020018), or having the wild-type human TGFβ receptor type 2 isoform B sequence (e.g., the amino acid sequence of NCBI RefSeq accession number NP-003233) or having a sequence substantially identical to the wild-type amino acid sequence. TGFβRII can retain at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99% of the TGFβ binding activity of the wild-type sequence. The expressed TGFβRII polypeptide may lack a signal sequence.

[0078] As used herein, the term "monoclonal antibody" or "mAb" refers to a preparation of antibody molecules of a single molecular component. Monoclonal antibodies exhibit a single binding specificity and affinity for a specific epitope.

[0079] As used herein, the term "human antibody" or "fully human antibody" is intended to include antibodies having variable regions in which both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, then the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, the term "human antibody" is not intended herein to include antibodies in which CDR sequences derived from another mammalian species (e.g., mouse) are grafted onto human framework region sequences.

[0080] The term "humanized antibody" means an antibody in which the CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Other framework region modifications can be made within the human framework sequences.

[0081] The term "fusion protein", as used herein, refers to a polypeptide having two (or more) moieties covalently linked together, where each moiety is a peptide with different properties. The property can be a biological property, such as in vitro or in vivo activity. The property can also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two moieties can be directly linked by a single peptide bond or linked by a peptide linker containing one or more amino acid residues. Generally, the two moieties and the linker will be linked in-frame. In certain embodiments, the two moieties of the fusion protein are respectively an antigen-binding moiety that specifically binds to PD-L1 and a human TGFβ receptor (TGFβR) or a portion thereof that is capable of binding TGFβ. Such a fusion protein containing an antibody can also be regarded as an antibody in the present disclosure (e.g., referred to as a "fusion antibody").

[0082] The term "operably linked" refers to the juxtaposition (with or without a spacer or linker) of two or more biological sequences of interest such that they are in a relationship that permits them to function in the desired manner. In the case of polypeptides, it means linking polypeptide sequences in a manner that permits the resulting product to have the desired biological function. For example, an antibody variable region can be operably linked to a constant region to provide a stable product with antigen-binding activity. The term can also be used with respect to polynucleotides. By way of example, when a polynucleotide encoding a polypeptide is operably linked to regulatory sequences (such as a promoter, enhancer, silencer sequence, etc.), it means that the polynucleotide sequence is linked in a manner that permits regulation of the expression of the polypeptide from that polynucleotide.

[0083] As used herein, the term "Ka" is intended to represent the association rate of a particular antibody-antigen interaction, and the term "Kd" as used herein is intended to represent the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using well-established methods in the art. As used herein, the term "K D " is intended to represent the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The preferred method for determining the antibody Kd is by using surface plasmon resonance, preferably using a biosensor system such as system.

[0084] As used herein, the term "high affinity" for an IgG antibody means having a 1×10 -7 M or lower, more preferably 5×10 -8 M or lower, even more preferably 1×10 -8M or lower, even more preferably 5×10 -9 M or lower, and even more preferably 1×10 -9 M or lower of K D antibody.

[0085] As used herein, the term "EC 50 " is also referred to as "half maximal effective concentration", and refers to the concentration of a drug, antibody, or agent that induces a 50% response between baseline and maximum after a specific exposure time. In the context of the present application, the unit of EC 50 is "nM".

[0086] As used herein, the ability of "inhibiting binding" refers to the ability of an antibody or fusion protein to inhibit or block the binding of two molecules (e.g., PD1 and PD-L1) to any detectable level. In certain embodiments, the binding of the two molecules can be inhibited by the antibody or its antigen-binding fragment by at least 50%. In certain embodiments, such inhibitory effects can be greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In certain embodiments, the fusion protein of the present invention blocks the binding of PD1 to cell surface PD-L1 with an IC 50 not exceeding 0.1 nM.

[0087] As used herein, the term "epitope" refers to the antigenic portion to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to as "antigenic determinant". An epitope or antigenic determinant typically consists of chemically active surface groups of a molecule such as amino acids, carbohydrates, or sugar side chains, and usually has a specific three-dimensional structure and specific charge characteristics. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, ed. (1996).

[0088] As used herein, the term "isolated" refers to a state obtained by artificial means from its natural state. If a certain "isolated" substance or component exists naturally, it may be because its natural environment has changed, or the substance has been separated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide exists naturally in a living body, and the same highly purified polynucleotide or polypeptide separated from this natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude a mixture of artificial or synthetic substances, nor other impure substances that do not affect the activity of the separated substance.

[0089] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to the PD-L1 protein is substantially free of antibodies that specifically bind to antigens other than the PD-L1 protein). However, an isolated antibody that specifically binds to the human PD-L1 protein may have cross-reactivity to other antigens such as the PD-L1 protein from other species. In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0090] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector permits the expression of a protein encoded by a polynucleotide inserted therein, the vector is called an expression vector. The vector can cause the genetic material elements carried therein to be expressed in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known to those of skill in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector can contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain an origin of replication.

[0091] As used herein, the term "host cell" refers to a cell system that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissues or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular subject cell but also the progeny of such a cell. Such progeny may differ from the parental cell due to mutations or environmental influences that may occur in succeeding generations, but such progeny are still included within the scope of the term "host cell".

[0092] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by aligning and comparing the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps (if any) in the alignment are preferably addressed by specific mathematical models or computer programs (i.e., "algorithms"). Methods that can be used to calculate the identity of nucleic acids or polypeptides for alignment include those described in Computational Molecular Biology, (Lesk, A.M. ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W. ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M. and Griffin, H.G. eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J. eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0093] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the formation of a specific immune response of antibodies or sensitized T lymphocytes in the immune system of an organism after stimulating the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the means of immunization.

[0094] As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. In a specific embodiment of the invention, the human PD-L1 gene is transfected into 293F cells.

[0095] As used herein, the term "SPR" or "surface plasmon resonance" refers to and encompasses the optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, e.g., using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For a detailed description, see U., et al. (1993) Ann. Biol. Clin. 51:19-26; U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0096] As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). Instruments for performing FACS are known to those of skill in the art and are commercially available to the public. Examples of such instruments include the FACSStar Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the Epics C from Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).

[0097] The term "subject" includes any human or non-human animal, preferably a human.

[0098] As used herein, the term "cancer" refers to any tumor or malignant cell growth, proliferation, or metastasis-mediated solid and non-solid tumors such as leukemia that give rise to a medical condition.

[0099] As used herein, the terms "treat", "treatment", and "therapy" when used in the context of treating a condition generally refer to the treatment and therapy of a human or animal in which some desired therapeutic effect is achieved, e.g., inhibition of the progression of the condition, including a decrease in the rate of progression, a halt in the rate of progression, regression of the condition, improvement of the condition, and cure of the condition. Treatment also includes treatment as a prophylactic measure (i.e., prevention). For cancer, "treatment" may refer to inhibiting or slowing tumor or malignant cell growth, proliferation, or metastasis or some combination thereof. For a tumor, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

[0100] As used herein, the term "effective amount" refers to the amount of an active compound or the amount of a material, composition, or dosage containing the active compound that is effective, when administered according to the desired treatment regimen, to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, when used in combination with treating a PD-1 / PD-L1-related disease or disorder, an "effective amount" refers to the amount or concentration of an antibody or its antigen-binding portion effective to treat the disease or disorder.

[0101] As used herein, the terms "prevent", "prevention", or "prophylaxis" with respect to a disease condition in a mammal refer to preventing or delaying the onset of the disease or preventing the manifestation of its clinical or subclinical symptoms.

[0102] As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient, and / or its salts are chemically and / or physically compatible with the other ingredients in the formulation and are physiologically compatible with the recipient.

[0103] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, which are well-known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th Edition, Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic, or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.

[0104] As used herein, the term "adjuvant" refers to a non-specific immune enhancer that can enhance the immune response against an antigen in an organism or change the type of immune response when delivered to the organism together with the antigen or delivered to the organism in advance. There are various adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0105] A fusion protein comprising an anti-PD-L1 antibody and a TGFβR or a portion thereof

[0106] This application provides a fusion protein that can specifically bind to both PD-L1 and TGFβ (such as TGFβ1 and TGFβ3), thus not only targeting the PD-L1 antigen or cells expressing PD-L1, but also promoting the local consumption of TGFβ in the microenvironment. Broadly speaking, such a fusion protein can also be regarded as an antibody because it can specifically bind to PD-L1, antagonize the activity of PD-L1, and block the PD-1 / PD-L1 signaling pathway. This fusion protein can also be referred to as a "bifunctional protein" or a "TGFβ-capture - PD-L1-targeted antibody fusion" herein.

[0107] The fusion proteins herein can comprise (a) an antibody or an antigen-binding portion thereof that specifically binds to PD-L1, and (b) a human transforming growth factor-β receptor (TGFβR) or a portion thereof (also referred to as a TGFβ capturer) that is capable of binding TGFβ, wherein (a) and (b) can be fused via a linker. The antibody or its antigen-binding portion can have various forms, such as a whole antibody, a monospecific antibody, a bispecific antibody, a ScFv, a domain antibody (SdAb), a VHH, a Fab, an F(ab')2 or an Fv fragment, etc., as long as it has a specific binding affinity for PD-L1. The human transforming growth factor-β receptor can be selected from TGFβRII and TGFβRIII, preferably TGFβRII. The fusion protein can comprise a portion of wild-type TGFβRII that retains some or all of the TGFβ binding ability, for example, the fusion protein can comprise the extracellular domain (ECD) of TGFβRII.

[0108] The linker between (a) and (b) connects the C-terminus of the antibody or its antigen-binding portion that specifically binds to PD-L1 to the N-terminus or C-terminus of the human transforming growth factor-β receptor (TGFβR) or a portion thereof that can bind TGFβ. In some embodiments, in the absence of an Fc region, the linker can be connected to the C-terminus of the variable domain of the antibody (such as Fab, domain Ab or ScFv); alternatively, in the case where the antibody is a whole antibody or a heavy chain antibody, the linker can be connected to the C-terminus of the Fc region.

[0109] Combining anti-PD-L1 and TGFβ capturer in a single agent elicits a synergistic anti-tumor effect due to simultaneously blocking the interaction between PD-L1 on tumor cells and PD-1 on immune cells, as well as neutralizing TGFβ in the tumor microenvironment. As confirmed by the examples, compared with M7824, the fusion proteins of the present disclosure exhibit better blocking of the PD-1 / PD-L1 signaling pathway and more effectively enhance the production of IFNγ.

[0110] Specifically, the fusion proteins of the present invention provide one or more of the following properties:

[0111] (a) being able to bind to human PD-L1 with a KD of no more than 7×10 -10 M and to bind to TGFβ1 with a KD of no more than 2×10 -12 M, as determined by SPR;

[0112] (b) being able to bind to cynomolgus monkey PD-L1 with an EC50 of no more than 2 nM, as determined by FACS;

[0113] (c) being able to bind to PD-L1 and TGFβ1 simultaneously;

[0114] (d) capable of achieving PD-1 / PD-L1 signal transduction blockade and TGF-β1 blockade;

[0115] (e) potently enhancing IL-2 and IFNγ production in the allogeneic mixed lymphocyte reaction;

[0116] (f) stable in an accelerated stability study; and

[0117] (g) stable in human serum for at least 14 days.

[0118] An antibody or an antigen-binding portion thereof that specifically binds to PD-L1

[0119] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1 comprises one or more heavy chain CDRs (HCDRs), and the heavy chain CDRs are selected from at least one group consisting of:

[0120] (i) HCDR1, which comprises SEQ ID NO:1 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:1 by no more than 2 amino acids;

[0121] (ii) HCDR2, which comprises SEQ ID NO:2 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:2 by no more than 2 amino acids; and

[0122] (iii) HCDR3, which comprises SEQ ID NO:3 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:3 by no more than 2 amino acids; and / or

[0123] comprises one or more light chain CDRs (LCDRs), and the light chain CDRs are selected from at least one group consisting of:

[0124] (i) LCDR1, which comprises SEQ ID NO:4 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:4 by no more than 2 amino acids;

[0125] (ii) LCDR2, which comprises SEQ ID NO:5 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:5 by no more than 2 amino acids; and

[0126] (iii) LCDR3, which comprises SEQ ID NO:6 or an amino acid sequence with amino acid additions, deletions, and / or substitutions that differ from SEQ ID NO:6 by no more than 2 amino acids.

[0127] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises (i) an HCDR1 comprising or consisting of SEQ ID NO:1; (ii) an HCDR2 comprising or consisting of SEQ ID NO:2; and (iii) an HCDR3 comprising or consisting of SEQ ID NO:3; and / or the light chain variable region comprises: (i) an LCDR1 comprising or consisting of SEQ ID NO:4; (ii) an LCDR2 comprising or consisting of SEQ ID NO:5; and (iii) an LCDR3 comprising or consisting of SEQ ID NO:6.

[0128] In some embodiments, the heavy chain variable region comprises: (i) the amino acid sequence of SEQ ID NO:7; (ii) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:7; or (iii) an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions compared to SEQ ID NO:7; and / or

[0129] the light chain variable region comprises: (i) the amino acid sequence of SEQ ID NO:8; (ii) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:8; or (iii) an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions compared to SEQ ID NO:8.

[0130] The fusion proteins of the present disclosure comprising the above-described antibody or antigen-binding portion thereof can bind to human PD-L1 with high affinity. The binding of the antibodies of the present disclosure to PD-L1 can be evaluated using one or more techniques established in the art (e.g., ELISA). The binding specificity of the antibodies of the present disclosure can also be determined by monitoring the binding of the antibody to cells expressing the PD-L1 protein (e.g., flow cytometry). For example, the antibody can be tested by flow cytometry assays, in which the antibody is reacted with a cell line expressing human PD-L1, such as CHO-K1 or 293F cells transfected to express PD-L1 on their cell surface. Additionally or alternatively, the binding of the antibody can be tested in a BIAcore binding assay, including binding kinetics (e.g., K D values). Other suitable binding assays include ELISA or FACS assays, e.g., using recombinant PD-L1 protein.

[0131] For example, the antibodies of the present disclosure have a K -7 of 1×10 D M or lower, a K -8 of 5×10 D M or lower, a K -8K of M or lower D 、1×10 -8 K of M or lower D 、5×10 -9 K of M or lower D 、4×10 -9 K of M or lower D 、3×10 -9 K of M or lower D 、2×10 -9 K of M or lower D 、1×10 -9 K of M or lower D 、5×10 -10 K of M or lower D 、1×10 -10 K of M or lower D Bind to human PD-L1 protein, as measured by surface plasmon resonance. Alternatively, the antibodies of the present disclosure can bind to cell lines expressing human or cynomolgus monkey PD-L1 with an EC50 of less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM or even less than 0.5 nM, as determined by FACS.

[0132] Unless otherwise specified, the assignment of amino acids to each CDR can be according to one of the numbering schemes provided below: Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Edition), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, ed. (2007) Handbook of Therapeutic Antibodies, 3rd Edition, Wiley-VCH VerVEGF GmbH and Co.

[0133] The variable regions and CDRs in an antibody sequence can be identified according to general rules developed in the art (such as the Kabat numbering system as described above) or by aligning the sequence with a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in and available from the "Abysis" website at www.bioinf.org.uk / abs (maintained by A.C. Martin of the Department of Biochemistry & Molecular Biology, University College London, London, England) and the VBASE2 website at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). The Abysis database is preferably used to analyze sequences, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with structural data from the PDB, see Protein Sequence and Structure Analysis of Antibody Variable Domains in the book by Dr. Andrew C.R. Martin. In: Antibody Engineering Lab Manual (eds: Duebel, S. and Kontermann, R., Springer-VerVEGF, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg.uk / abs). The Abysis database website also includes general rules that have been developed for identifying CDRs that can be used according to the teachings herein. Unless otherwise specified, all CDRs described herein are obtained according to the Abysis database website of Kabat.

[0134] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0135] Additionally or alternatively, the protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such a search can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. The BLAST protein search can be carried out with the XBLAST program with a score = 50 and wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the present invention. To obtain a gapped alignment for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0136] In other embodiments, the CDR amino acid sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the specific CDR amino acid sequences contained in the corresponding sequences described above. In other embodiments, the amino acid sequences of the variable regions can be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequences described above.

[0137] Preferably, the CDRs of the isolated antibody or antigen-binding portion thereof contain no more than 2 amino acid or no more than 1 amino acid conservative substitutions. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the basic properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a substitution of a residue with a physically or functionally similar residue (e.g., having a similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), amino acids having acidic side chains (e.g., aspartic acid and glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, an amino acid residue is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997), which are incorporated herein by reference).

[0138] A TGFβ capturer comprising a human transforming growth factor β receptor (TGFβR) or a portion thereof

[0139] TGFβ has three ligand isotypes, TGFβ1, 2, and 3, which all exist as homodimers. There are also three TGFβR receptors (TGFβR), called type I, II, and III TGFβR, respectively. TGFβRI is the signaling chain and cannot bind ligands. TGFβRII binds ligands TGFβ1 and 3 with high affinity but does not bind TGFβ2. TGFβRIII is a positive regulator of the binding of TGFβ to its signaling receptors and binds all three TGFβ isotypes with high affinity. TGFβ1 and TGFβ2 are reported to play major roles in the tumor microenvironment and cardiac physiology, respectively. Thus, therapeutic agents that neutralize TGFβ1 but not TGFβ2 can provide an optimal therapeutic index by minimizing cardiac toxicity without compromising anti-tumor activity. Accordingly, TGFβRII was selected, and the extracellular domain of TGFβRII has a length of only 136 amino acid residues (SEQ ID No: 9), which makes it very suitable for constructing antibody-captor fusion proteins.

[0140] In the fusion protein, the human transforming growth factor β receptor (TGFβR) or a portion thereof can comprise:

[0141] (A) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of wild-type human TGFβRII;

[0142] (B) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of the extracellular domain of wild-type human TGFβRII; or

[0143] (C) a portion of wild-type human TGFβRII that retains at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the binding ability to TGFβ.

[0144] In some embodiments, the TGFβ captor included in the fusion protein herein is the extracellular domain of TGFβRII or a portion thereof. In some specific embodiments, the TGFβ captor comprises or consists of the sequence shown in SEQ ID NO:9.

[0145] A nucleic acid molecule encoding the fusion protein of the present disclosure

[0146] In some aspects, the present disclosure relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding one or more of the following:

[0147] (i) an antibody or an antigen-binding portion thereof, or the VH or VL domain of an antibody;

[0148] (ii) human TGFβRII or a portion thereof of the fusion protein; and

[0149] (iii) The heavy or light chain of the fusion protein.

[0150] In some aspects, the present disclosure relates to vectors comprising nucleic acid sequences encoding as disclosed herein. In the context of the present invention, the vector can be any suitable vector, including chromosomal, extrachromosomal, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, bacteriophage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and bacteriophage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the PD-L1 antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, including for example linear expression elements (described, e.g., in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), compact nucleic acid vectors (described, e.g., in US 6,077,835 and / or WO 00 / 70087), plasmid vectors such as pBR322, pUC 19 / 18 or pUC 118 / 119, "midge" minimal-size nucleic acid vectors (described, e.g., in Schakowski et al., Mol Ther 3, 793-800 (2001)), or nucleic acid vector constructs as precipitates, such as CaPO4-precipitated constructs (described, e.g., in WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, SomaticCell Genetics 7, 603 (1981)). Such nucleic acid vectors and their uses are well known in the art (see, e.g., US 5,589,466 and US 5,973,972).

[0151] In one embodiment, the vector is suitable for expressing a fusion protein as disclosed herein in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503 - 5509 (1989)), pET vectors (Novagen, Madison WI), etc. The vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (reviewed in: F. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516 - 544 (1987)).

[0152] The vector may also or alternatively be a vector suitable for expression in a mammalian cell, for example, a vector containing glutamine synthetase as a selectable marker, such as the vector described in Bebbington (1992) Biotechnology (NY) 10:169 - 175.

[0153] The nucleic acid and / or vector may also contain a nucleic acid sequence encoding a secretion / localization sequence that can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or the cell culture medium. Such sequences are known in the art and include secretion leaders or signal peptides.

[0154] The expression vector may contain or be associated with any suitable promoter, enhancer, and other expression - facilitating elements. Examples of such elements include strong expression promoters (such as the human CMV IE promoter / enhancer and RSV, SV40, SL3 - 3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, an origin of replication for plasmid production in Escherichia coli, an antibiotic resistance gene as a selectable marker, and / or convenient cloning sites (such as polylinkers). The nucleic acid may also contain an inducible promoter, such as CMV IE, opposite to a constitutive promoter.

[0155] In yet another aspect, the present disclosure relates to a host cell containing the vector described herein. Thus, the present invention also relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfected tumor cell, that produces the fusion protein of the present invention.

[0156] The fusion protein can be expressed in recombinant eukaryotic or prokaryotic host cells such as transfected tumor cells, which produce the fusion protein as defined herein.

[0157] Examples of host cells include yeast, bacteria, plant, and mammalian cells such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NS0 cells or lymphocytes. For example, in one embodiment, the host cell may comprise a first and a second nucleic acid construct stably integrated into the cell genome. In another embodiment, the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises the first and second nucleic acid constructs as described above.

[0158] In another aspect, the present invention relates to a transgenic non-human animal or plant which comprises a nucleic acid encoding one or both of the heavy and light chains of a fusion protein as disclosed herein, wherein the animal or plant produces the fusion protein.

[0159] In yet another aspect, the present disclosure relates to a hybridoma which produces an antibody for a fusion protein as defined herein.

[0160] In one aspect, the present invention relates to an expression vector which comprises

[0161] (i) a nucleic acid sequence which encodes an antibody or an antigen-binding portion thereof, or a VH or VL domain of an antibody, according to any one of the embodiments disclosed herein;

[0162] (ii) a nucleic acid sequence which encodes a human TGFβRII or a portion thereof of a fusion protein according to any one of the embodiments disclosed herein;

[0163] (iii) a nucleic acid sequence which encodes a heavy or light chain of a fusion protein; or

[0164] (iv) a combination of two or more of the above.

[0165] In one aspect, the present disclosure relates to a nucleic acid construct encoding one or more of the amino acid sequences listed in the sequence listing.

[0166] In one aspect, the present disclosure relates to a method for producing a fusion protein according to any one of the embodiments disclosed herein, comprising culturing a host cell as disclosed herein which comprises one or more expression vectors for expressing the fusion protein, and purifying the fusion protein from the culture medium. In one aspect, the present invention relates to a host cell comprising the expression vector as defined above. In one embodiment, the host cell is a recombinant eukaryotic, recombinant prokaryotic or recombinant microbial host cell.

[0167] A pharmaceutical composition

[0168] In some aspects, the present invention relates to pharmaceutical compositions comprising at least one fusion protein as disclosed herein and a pharmaceutically acceptable carrier.

[0169] Components of the composition

[0170] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present invention may also be administered in combination with, for example, another immunostimulant, anti-cancer agent, antiviral agent or vaccine such that the fusion protein as disclosed herein enhances the immune response against the antigen. The pharmaceutically acceptable carrier may include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, aqueous medium, non-aqueous medium, antimicrobial agent, isotonic agent, buffer, antioxidant, anesthetic, suspending / dispersing agent, chelating agent, diluent, adjuvant, excipient or non-toxic auxiliary substances, combinations of various components known in the art or more.

[0171] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifying agents or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoethanol, mercaptoacetic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene and / or propyl gallate. As disclosed in the present invention, in a solvent of an antibody or antigen-binding fragment of a composition of the present invention containing one or more antioxidants such as methionine of a reduced antibody or its antigen-binding fragment, it may be oxidized. Oxidation-reduction can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending the shelf life. Thus, in some embodiments, the present invention provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present invention further provides various methods in which an antibody or antigen-binding fragment thereof is mixed with one or more antioxidants such as methionine. Thus, the antibody or antigen-binding fragment thereof can be prevented from being oxidized to extend its shelf life and / or increase its activity.

[0172] For further illustration, pharmaceutically acceptable carriers can include, for example, aqueous carriers such as sodium chloride injection, Ringer's injection, isosmotic dextrose injection, sterile water injection or dextrose and lactated Ringer's injection, non-aqueous carriers such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antibacterial or antifungal concentrations of antimicrobial agents, isotonic agents such as sodium chloride or dextrose, buffering agents such as phosphate or citrate buffers, antioxidants such as sodium bisulfite, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethylene glycol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antimicrobial agent used as a carrier can be added to the pharmaceutical composition in a multi-dose container containing phenol or cresol, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0173] Administration, formulations and dosages

[0174] The pharmaceutical compositions of the present invention can be administered in vivo to a subject in need thereof by various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. Suitable formulations and administration routes can be selected according to the intended application and treatment protocol.

[0175] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and their controlled release forms.

[0176] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant body fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific dosage regimen (including dose, timing, and repetition) will depend on the specific individual and the individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0177] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining such reduction of tumor cells, reducing the proliferation of tumor cells, or delaying the development of metastasis. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, sustained continuous release formulations of the therapeutic compositions of the invention may be appropriate.

[0178] Those skilled in the art will understand that the appropriate dose can vary from patient to patient. Determining the optimal dose generally involves balancing the level of therapeutic benefit against any risks or adverse side effects. The dose level selected will depend on a variety of factors, including but not limited to the activity of the particular compound, the route of administration, the time of administration, the rate of clearance of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, and the species, sex, age, weight, medical condition, general health, and previous medical history of the patient. The amount of the compound and the route of administration are ultimately determined by a physician, veterinarian, or clinician, but the dose is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0179] Generally, the fusion proteins of the invention can be administered in a variety of ranges. These include from about 5 μg / kg body weight to about 100 mg / kg body weight per dose; from about 50 μg / kg body weight to about 5 mg / kg body weight per dose; from about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include from about 100 μg / kg body weight to about 20 mg / kg body weight and from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the dose per dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight.

[0180] In any case, the fusion protein of the present invention is preferably administered to a subject in need thereof as needed. Those skilled in the art can determine the frequency of administration, for example, based on considerations such as the medical condition being treated, the age of the subject being treated, the severity of the medical condition being treated, the general health of the subject being treated, and the like by the attending physician.

[0181] In certain preferred embodiments, the treatment process involving the fusion protein of the present invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the fusion protein of the present invention can be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dose can be changed or the interval adjusted based on patient response and clinical practice.

[0182] The dose and regimen of the disclosed therapeutic composition can also be determined empirically in an individual to whom one or more administrations have been given. For example, an individual can be given incremental doses of the therapeutic composition produced as described herein. In selected embodiments, the dose can be gradually increased or decreased or reduced based on empirically determined or observed side effects or toxicity. To evaluate the efficacy of the selected composition, markers of a particular disease, disorder, or condition can be followed as described above. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement evaluated by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or oncoantigens identified according to the methods described herein, reduction in pain or paralysis; improvement in speech, vision, respiration, or other disabilities associated with the tumor; increased appetite; or improvement in quality of life or extension of survival measured by accepted tests. Those skilled in the art will appreciate that the dose will vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations in the individual, and past and concurrent treatments used.

[0183] A compatible formulation for parenteral administration (e.g., intravenous injection) will contain the fusion protein disclosed herein at a concentration of from about 10 μg / ml to about 100 mg / ml. In certain selected embodiments, the concentration of the fusion protein will include 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml or 1 mg / ml. In other preferred embodiments, the concentration of the fusion protein will include 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml.

[0184] The use of the present invention

[0185] In some aspects, the present invention provides a method of treating a disorder in a subject, which comprises administering to a patient in need thereof (e.g., a human) a therapeutically effective amount of a fusion protein as disclosed herein. For example, such a disorder is a cancer.

[0186] The methods provided by the present disclosure can be used to treat or prevent a variety of cancers involving PD-1 / PD-L1, whether malignant or benign, and whether primary or secondary. These cancers can be solid cancers or hematological malignancies. Examples of these cancers include lung cancers such as bronchial carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondroid hamartoma (non-cancerous), and sarcoma (cancerous); heart cancers such as myxoma, fibroma, and rhabdomyoma; bone cancers such as osteochondroma, chondroma, chondroblastoma, chondroid chondroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; brain cancers such as glioma (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ cell tumor, teratoma, dermoid cyst, and hemangioma; cancers in the digestive system such as colon cancer, leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colorectal polyp, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, hypernephroma, and transitional cell carcinoma of the renal pelvis; bladder cancer; hematological cancers such as acute lymphoblastic leukemia (acute lymphocytic leukemia), acute myeloid (myelocytic, myeloid, myeloblastoid, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myeloid (myeloid, myelogenous, granulocytic) lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative diseases such as polycythemia vera, myelofibrosis, thrombocytosis, and chronic myeloid leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi sarcoma, and Paget's disease; head and neck cancers; eye-related cancers such as retinoblastoma and intraocular melanoma; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial cancer), cervical cancer (cervical tumor), ovarian cancer (ovarian tumor), vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal); non-cancerous growths of the parathyroid gland; pancreatic cancer; and hematological cancers such as leukemia, myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma. In a specific embodiment, the cancer is colon cancer.In some other embodiments, the cancer is lung cancer, such as non-small cell lung cancer (NSCLC).

[0187] In some embodiments, examples of cancers include, but are not limited to, B-cell cancers, including B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD)), as well as abnormal blood vessel proliferation associated with nevus flammeus, edema (e.g., associated with brain tumors), B-cell proliferative disorders, and Meigs' syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, frontline low-grade NHL, grade III / IV NHL, chemotherapy-tolerant NHL, precursor B-lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicular center lymphoma (including aggressive frontline NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky disease NHL, Burkitt lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sézary syndrome, cutaneous (cutaneous) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

[0188] In some embodiments, examples of cancers further include, but are not limited to, B cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B cell non-Hodgkin lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-cleaved cell lymphoma / Burkitt lymphoma (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma, and non-Burkitt lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B cell lymphoma, and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B cell diffuse large cell lymphoma (DLCL) cell lymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma, angiocentric lymphoma - pulmonary B cell lymphoma), f) hairy cell leukemia, g) lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, h) acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, i) plasmacytoma, plasma cell myeloma, multiple myeloma, plasmacytoma, and / or j) Hodgkin's disease.

[0189] In some other embodiments, the disorder is an autoimmune disease. Examples of autoimmune diseases that can be treated with an antibody or an antigen-binding portion thereof include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. The antibody or an antigen-binding portion thereof can also be used to treat or prevent infectious diseases, inflammatory diseases (e.g., allergic asthma), and chronic graft-versus-host disease.

[0190] Used in combination with chemotherapy

[0191] The fusion proteins disclosed herein can be used in combination with anti-cancer agents, cytotoxic agents, or chemotherapeutic agents.

[0192] The term "anti-cancer agent" or "anti-proliferative agent" means any agent that can be used to treat cell proliferative disorders such as cancer and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents and immunotherapeutic agents. It should be understood that in the selected embodiments as described above, such anti-cancer agents can include conjugates and can be associated with the disclosed fusion proteins prior to administration. More specifically, in some embodiments, a selected anti-cancer agent is linked to an unpaired cysteine of an antibody to provide an engineered conjugate. Thus, such engineered conjugates are expressly contemplated within the scope of the present invention. In other embodiments, the disclosed anti-cancer agents will be administered in combination with a site-specific conjugate comprising different therapeutic agents as described above.

[0193] As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell destruction. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxins, Staphylococcal enterotoxin A), fungi (e.g., α-sarcin, restrictocin), plants (abrin, ricin, momordin, viscotoxin, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, hordotoxin, Aleurites fordii protein, caryophyllin, Phytolacca mericana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, croton toxin, Saponaria officinalis inhibitor, gelonin, mitegellin, restrictocin, phenomycin, neomycin, and trichothecenes) or animals (e.g., cytotoxic ribonucleases such as extracellular pancreatic ribonucleases; DNase I, including fragments and / or variants thereof).

[0194] For the purposes of the present invention, "chemotherapeutic agent" includes chemical compounds (e.g., cytotoxic agents or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. These chemical agents typically target intracellular processes required for cell growth or division and are thus particularly effective against cancer cells, which typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. In general, chemotherapeutic agents can include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become neoplastic or produce neoplastic progeny (e.g., TICs). These agents are typically used in combination and are generally most effective, for example, in regimens such as CHOP or FOLFIRI.

[0195] Examples of anti-cancer agents (either as components of site-specific conjugates or in unconjugated form) that can be used in combination with the fusion proteins of the present invention include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines and methylmelamines, polyacetylenes (acetogenins), camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, docarimycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carcinomycin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, triferric doxorubicin, rhodomycin, streptozocin, streptozotocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinie, enzalutamide, folic acid analogs, purine analogs, androgens, anti-adrenals, folic acid supplements such as frolinic acid, acetoglucuronide, aldophosphamide glycoside, aminolevulinic acid, eniluracil, aclarubicin, bestrabucil, bisantrene, edatrexate, defofamine, colchicine amide, diaziquone, elfornithine, elidronic acid, epochlorohydrin, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, pipobroman, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazine, procarbazine, Polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; enmerphalan; sizofiran; spirogermanium; tinuzole acid; triaziquone; 2,2',2”-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, baccharmin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes; chlorambucil, Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine, Vinorelbine; nogalamycin; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine; ibandronate; irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; inhibitors of PKC-α, Raf, H-Ras, EGFR and VEGF-A (which reduce cell proliferation), and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents for modulating or inhibiting the hormonal action on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase which regulates estrogen production in the adrenal gland, and anti-androgens; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitor and HER2 expression inhibitor; vaccines, rIL-2; Topoisomerase 1 inhibitor; rmRH; vinorelbine and esperamicin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0196] Used in combination with radiotherapy

[0197] The present invention also provides combinations of the fusion protein with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as γ-irradiation, X-rays, UV-irradiation, microwaves, electron emission, etc.). Combinatorial therapies using targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed antibodies can be used in combination with targeted anti-cancer agents or other targeting means. Generally, radiotherapy is administered in a pulsed manner over a period of about 1 week to about 2 weeks. Radiotherapy can be administered to a subject with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple sequential doses.

[0198] A pharmaceutical package and a kit

[0199] Also provided are pharmaceutical packages and kits comprising one or more containers containing one or more doses of an antibody or antigen-binding portion thereof. In some embodiments, unit doses are provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or antigen-binding portion thereof, with or without one or more other reagents. For other embodiments, such unit doses are supplied in single-use prefilled syringes for injection. In other embodiments, the composition contained in the unit dose can comprise saline, sucrose, or the like; buffers such as phosphate, etc.; and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition can be provided as a lyophilized powder, which can be reconstituted after addition of a suitable liquid (such as sterile water or salt solution). In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the one or more containers indicates that the encapsulated composition is for the treatment of a selected tumor disease condition.

[0200] The present invention also provides a kit for generating a single-dose or multi-dose administration unit of a fusion protein and optionally one or more anti-cancer agents. The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials, such as glass or plastic, and contains a pharmaceutically effective amount of the disclosed conjugated or unconjugated form of the antibody. In other preferred embodiments, one or more containers include a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a subcutaneous injection needle). Such kits typically contain a pharmaceutically acceptable formulation of the antibody in a suitable container and optionally one or more anti-cancer agents in the same or different containers. The kit can also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, in addition to the antibody or its antigen-binding portion of the present invention, such kits can contain any one or more anti-cancer agents, such as chemotherapeutic agents or radiotherapeutic agents; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and / or other anti-cancer agents.

[0201] More specifically, the kit can have a single container containing the disclosed antibody or its antigen-binding portion, with or without additional components, or they can have separate containers for each desired reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution can be pre-mixed in a molar equivalent combination or with one component in excess of the other. Alternatively, the antibody of the kit and any optional anti-cancer agents can be stored separately in different containers prior to administration to a patient. The kit can also include a second / third container device for holding a sterile pharmaceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0202] When the components of the kit are provided as one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or saline solution. However, the components of the kit can be provided as dry powders. When the reagent or component is provided as a dry powder, the powder can be reconstituted by adding a suitable solvent. It is contemplated that the solvent can also be provided in another container.

[0203] As briefly described above, the kit can also contain means for administering the antibody or its antigen-binding portion and any optional components to a patient, such as one or more needles, I.V. bags or syringes, or even eye droppers, pipettes, or other similar devices through which the formulation can be injected or introduced into an animal or administered to an affected area of the body. The kit of the present invention typically also includes means for holding vials or the like and other tightly sealed components for commercial sale, such as injection or blow-molded plastic containers in which the desired vials and other devices are placed and held.

[0204] Summary of the Sequence Listing

[0205] This application is accompanied by a sequence listing containing many amino acid sequences. Table A below provides an overview of the sequences included.

[0206] An exemplary fusion protein disclosed herein is designated WT1122-U14T1.G15-1.uIgG1 (abbreviated as WT1122).

[0207] Table A

[0208]

[0209] Examples

[0210] The present invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. These examples are not intended to represent that the following experiments are all or the only experiments conducted.

[0211] Example 1

[0212] Preparation of Antigens, Benchmark Antibodies, and Cell Lines

[0213] 1.1 Preparation of Antigens

[0214] The human PD-L1 extracellular domain (ECD) antigen with a His tag was purchased from Sino Biological (Catalog No. 10084-H08H). The cynomolgus monkey (cyno) PD-L1 ECD antigen with a His tag was purchased from Sino Biological (Catalog No. 90251-C08H). The human TGFβ1, TGFβ2, and TGFβ3 antigens were purchased from R&D Systems (Catalog No. 7754-BH, 7754-BH / CF; Catalog No. 302-B2, 302-B2 / CF; Catalog No. 8420-B3, 8420-B3 / CF).

[0215] 1.2 Establishment of a Cell Line Expressing PD-L1

[0216] Cell lines expressing human PD-L1 (W315-CHO-K1.hPro1.C11), mouse PD-L1 (W315-293F.mPro1.C1), and cynomolgus monkey PD-L1 (W315-293F.cynoPro1.2A2) were generated as follows. Briefly, CHO-K1 or 293F cells were transfected with an expression vector containing the gene encoding full-length human PD-L1 or cynomolgus monkey PD-L1 or mouse PD-L1 using Lipofectamine 2000 (ThermoFisher-11668027). The cells were cultured in a medium containing an appropriate selection pressure. Stable cell lines were obtained by limiting dilution.

[0217] 1.3 Generation of benchmark antibody (BMK)

[0218] The anti-PD-L1 BMK antibody fused with TGFβRII ECD is designated WT112-BMK2-IgG1 and was constructed based on the sequence of M7824 in US9676863B2 of Merck Patent GmbH. Plasmids containing the heavy chain gene and the light chain gene were co-transfected into Expi293 cells using the Expi293 Expression Kit (ThermoFisher-A14524). The cells were cultured for several days, and the supernatant was collected for protein purification.

[0219] Example 2

[0220] Generation of anti-PD-L1 antibody fused with TGFβRII ECD

[0221] WT1122-U14T1.G15-1.uIgG1 is a monoclonal anti-PD-L1 antibody fused with TGFβRII ECD. The sequence of the anti-PD-L1 antibody is from clone W3152-r11.135.5-zAb17-m6 in PCT / CN2020 / 110494 (incorporated herein by reference in its entirety). The C-terminus of the Fc is linked to the sequence of TGFβRII ECD, which is the same as that in WT112-BMK2-IgG1 (SEQ ID NO: 9). The linker between the Fc and TGFβRII ECD is (G4S) 4 . The CDR sequences of WT1122-U14T1.G15-1.uIgG1 (referred to herein as "WT1122" for short) are listed in Table 1 below.

[0222] Table 1

[0223]

[0224] Example 3

[0225] In vitro characterization of WT1122

[0226] 3.1 Human TGF-β binding ELISA

[0227] The binding of the antibody to human TGF-β1, TGF-β2, and TGF-β3 was determined by ELISA. Plates were coated overnight at 4 °C with human TGF-β1, TGF-β2, or TGF-β3. After blocking and washing, various concentrations of the test antibody were added to the plates and incubated for 1 h at room temperature. The plates were then washed and incubated for 1 h with an HRP-labeled goat anti-human IgG antibody (Bethyl). After washing, TMB substrate was added, and the color development reaction was terminated with 2 M HCl. Absorbance at 450 nm and 540 nm was read using a microplate reader (SpectraMax M5e).

[0228] The binding curves of the antibody to immobilized human TGF-β1, TGF-β2, and TGF-β3 are shown as Figure 1A follows. WT1122 showed similar affinity to WT112-BMK2-IgG1. They bound tightly to immobilized TGF-β1 (EC50 = 0.5 nM) and TGF-β3 (EC50 = 0.8 nM), but not to immobilized TGF-β2.

[0229] The binding of the antibody to human TGF-β2 was also determined by ELISA with immobilized test antibody. After blocking and washing, various concentrations of TGF-β2 were added to the plates and incubated for 1 h at room temperature. The plates were then washed and incubated for 1 h with a biotinylated TGF-β2 detection antibody (R&D, DY240), followed by incubation with streptavidin-HRP for 1 h. After washing, TMB substrate was added, and the color development reaction was terminated with 2 M HCl. Absorbance at 450 nm and 540 nm was read using a microplate reader (SpectraMax M5e).

[0230] The binding curves of the antibody to soluble human TGF-β2 are shown as Figure 1B follows. Immobilized WT1122 and WT112-BMK2-IgG1 were able to bind soluble TGF-β2 with comparable EC50 values of 0.07 nM and 0.05 nM, respectively.

[0231] 3.2 Human PD-L1 binding FACS

[0232] Test antibodies at various concentrations were incubated with W315-CHO-K1.hPro1.C11 expressing human PD-L1 at 4°C for 1 hour. After washing, the cells were incubated with a PE-labeled goat anti-human IgG-Fc antibody (Jackson Immuno Research). Finally, the MFI of the cells was measured by flow cytometry and analyzed by FlowJo.

[0233] The binding curves to human PD-L1 transfected cells are shown in Figure 2A Figure. WT1122 and WT112-BMK2-IgG1 bound tightly to human PD-L1 on the cell surface with EC50 values of 0.7 nM and 1.21 nM, respectively.

[0234] 3.3 Cross-species binding FACS

[0235] The binding of the test antibodies to cynomolgus monkey or mouse PD-L1 was determined by FACS. Test antibodies at various concentrations were incubated with W315-293F.cynoPro1.2A2 cells expressing cynomolgus monkey PD-L1 or W315-293F.mPro1.C1 cells expressing mouse PD-L1 at 4°C for 1 hour, and then the binding of the antibodies to the cell surface was detected with a PE-labeled goat anti-human IgG-Fc antibody (Jackson Immuno Research). The MFI of the cells was measured by flow cytometry and analyzed by FlowJo.

[0236] Figure 2B The binding to cynomolgus monkey PD-L1 transfected cells is shown in Figure 2C Figure, and the binding to mouse PD-L1 transfected cells is shown in Figure. WT1122 and WT112-BMK2-IgG1 were able to bind tightly to cynomolgus monkey and mouse PD-L1 on the cell surface. The EC50 values of WT1122 and WT112-BMK2-IgG1 for binding to cynomolgus monkey PD-L1 were 1.08 nM and 1.59 nM, respectively, and the EC50 values for binding to mouse PD-L1 were 1.2 nM and 1.5 nM, respectively.

[0237] 3.4 Simultaneous binding to human PD-L1 and human TGF-β1

[0238] The simultaneous binding of the test antibody to human TGF-β1 and human PD-L1 was assayed by ELISA. Plates were coated with human TGF-β1 overnight at 4 °C. After blocking and washing, various concentrations of the test antibody were added to the plates and incubated for 1 h at room temperature. The plates were then washed and incubated with biotinylated human PD-L1 ECD protein and then streptavidin-HRP (Invitrogen) for 1 h. After washing, TMB substrate was added and the color development reaction was terminated with 2 M HCl. Absorbance at 450 nm and 540 nm was read using a microplate reader (SpectraMax M5e).

[0239] Similarly, simultaneous dual-target binding was also tested by coating plates with human PD-L1. After incubation with various concentrations of the test antibody and then the TGF-β1 antigen, biotinylated human TGF-β1 detection antibody (R&D, catalog number 840117) and then streptavidin-HRP (Invitrogen) were added to the plates. Finally, TMB substrate was added and the color development reaction was terminated with 2 M HCl. Absorbance at 450 nm and 540 nm was read using a microplate reader (SpectraMax M5e).

[0240] Figure 3A and Figure 3B The results shown indicate that when TGF-β1 was fixed, WT1122 and WT112-BMK2-IgG1 simultaneously bound PD-L1 and TGF-β1 with EC50 values of 0.29 and 0.17 nM, respectively ( Figure 3A ); when human PD-L1 was fixed, the EC50 values were 0.01 nM and 0.02 nM, respectively ( Figure 3B ).

[0241] 3.5 PD-1 / PD-L1 blockade determined by competitive FACS

[0242] Various concentrations of the test antibody, positive and negative control antibodies were mixed with mFc-labeled human PD-1 and then incubated with transfected cells expressing human PD-L1 at 4 °C for 1 h. Binding of human PD-1 to human PD-L1-expressing cells was detected by a PE-labeled anti-mouse IgG Fc antibody (Abcam). The MFI of the cells was measured by flow cytometry and analyzed by FlowJo.

[0243] As Figure 4 shown, WT1122 and WT112-BMK2-IgG1 blocked the binding of PD-1 to cell surface PD-L1 with IC50 values of 0.04 nM and 0.32 nM, respectively.

[0244] 3.6 Reporter gene assay (RGA)

[0245] The blockade of TGF-β1 signaling was tested by RGA assay. The RGA cell line was generated by stable expression of full-length human activin receptor II B and a stably integrated SBE luciferase reporter gene. To determine the TGF-β1 signaling blockade activity of the test antibody, human TGF-β1 and various concentrations of the antibody were pre-mixed and added to the RGA cells, and incubated overnight at 37 °C in 5% CO2. After incubation, the reconstituted luciferase substrate (Promega, catalog number E6130) was added, and the luciferase intensity was measured by a microplate spectrophotometer.

[0246] The blockade of PD-1 / PD-L1 signaling was tested by RGA assay. The PD-1RGA cell line was prepared by stable expression of the full-length of PD-1 and the NFAT luciferase reporter gene in Jurkat E6-1 cells. The PD-1RGA cells were incubated with artificial APCs expressing human PD-L1 (CHO-K1 cells expressing human PD-L1 and OKT3 sc-Fv) in the presence of various concentrations of the test antibody at 37 °C in 5% CO 2 for 4 - 6 hours. After incubation, the reconstituted luciferase substrate was added, and the luciferase intensity was measured by a microplate spectrophotometer.

[0247] As Figure 5A shown, WT1122 showed comparable TGF-β1 blockade to WT112-BMK2 with an IC50 of 1.2 nM, and the IC50 of WT112-BMK2 was 0.7 nM. As Figure 5B shown, in the RGA assay, WT1122 and WT112-BMK2-IgG1 showed strong hPD-1 / PD-L1 signal blockade activity. The IC50s of WT1122 and WT112-BMK2-IgG1 were 0.31 nM and 0.59 nM, respectively.

[0248] 3.7 Allogeneic mixed lymphocyte reaction (allo-MLR)

[0249] Fresh human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Paque PLUS (Stem Cell) gradient centrifugation. Monocytes were isolated using CD14 MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. Cells were cultured in medium containing GM-CSF (Amoytop Biotech) and IL-4 (R&D) for 5 to 7 days to generate dendritic cells (DCs). Human CD4+ T cells were isolated using a human CD4+ T cell enrichment kit (Stem Cell) according to the manufacturer's protocol. Purified CD4+ T cells were co-cultured with allogeneic immature DCs (iDCs) in 96-well plates in the presence of test antibodies, positive and negative control antibodies. The plates were incubated at 37 °C and 5% CO2. Supernatants were harvested on days 3 and 5 respectively for IL-2 and IFN-γ assays.

[0250] Human IL-2 and IFN-γ release were measured by ELISA using matched antibody pairs. Recombinant human IL-2 (R&D) and IFN-γ (PeproTech) were used as standards respectively. Capture antibodies specific for human IL-2 (R&D) or IFN-γ (Pierce) were pre-coated on the plates respectively. After blocking, 50 μL of standards or samples were pipetted into each well and incubated for 2 hours at ambient temperature. After removing unbound substances, biotin-conjugated detection antibodies specific for the respective cytokines were added to the wells and incubated for one hour. Then HRP-labeled streptavidin was added to the wells and incubated for 30 minutes at room temperature. Color was developed by adding 50 μL of TMB substrate and then stopped with 50 μL of 2N HCl. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer.

[0251] Figures 6A-6B The results shown in Figure 6A ) demonstrated that WT1122 and WT112-BMK2-IgG1 were able to enhance IL-2 production in the human CD4+ T cell allogeneic MLR assay in a dose-dependent manner ( Figure 6B ).

[0252] 3.8 Serum stability

[0253] WT1122 was cultured in freshly isolated human serum (serum content > 95%) at 37 °C in a 5% CO2 incubator. At the indicated time points, aliquots of serum-treated samples were removed from the incubator, snap-frozen in liquid nitrogen, and stored at -20 °C until ready for testing. Samples were rapidly thawed immediately before the stability test. The procedures combining ELISA, human TGF-β1-binding ELISA, and human PD-L1-binding FACS were as described above.

[0254] As Figure 7 shown, these WT1122 samples showed normal binding to the target, indicating that the antibody was stable in human serum for at least 14 days.

[0255] 3.9 Antibody Protein Accelerated Stability Study

[0256] 3.9.1 Sample Handling and Accelerated Stability Study

[0257] WT1122 was dialyzed into PBS buffer through a dialysis bag (Spectrum-888-10987, MWCO 3.5 kDa) and then diluted to 2 μg / ml. Accelerated stability studies were conducted by incubating the antibody at 4 °C, 25 °C, and 40 °C for 1 day, 4 days, and 7 days, respectively, and subjecting it to 3 freeze-thaw cycles at -80 °C (Table 2). After incubation under each test condition, the samples were immediately visually inspected for the presence of any particles. All samples were shown to be clear solutions without particles. Antibody stability of each treated sample was analyzed by SDS-PAGE, analytical SEC-HPLC, DSF, and DLS assays. The results shown in Table 2 indicate that WT1122 was stable in the accelerated stability study.

[0258] 3.9.2 Thermal Stability by DSF

[0259] DSF analysis was performed using real-time fluorescence quantitative PCR (QuantStudio 7Flex, Thermo Fisher Scientific). Briefly, 19 μL of the antibody solution was mixed with 1 μL of 62.5× SYPRO Orange solution (Invitrogen) and then added to a 96-well plate (Biosystems). The plate was heated from 26 °C to 95 °C at a rate of 0.9 °C / min, and the resulting fluorescence data were collected. The negative derivative of the fluorescence change relative to different temperatures was calculated, and the maximum value was defined as the melting temperature. Data collection and Tm calculation were automatically performed by the operating software (QuantStudioTM Real-Time PCR Software v1.3). The Tm of WT1122 in PBS buffer was approximately 65.6 °C (Table 2).

[0260] 3.9.3 Molecular Radius Measurement by DLS

[0261] Molecular radius measurements were studied using a DynaPro Plate Reader III dynamic light scattering (DLS) instrument (Wyatt DynaproTM). Five acquisitions were performed for each protein sample, with each acquisition lasting 5 s. Each well contained 7.5 μL of solution in a 1536-well plate (Aurora microplate). For each measurement, the diffusion coefficient was determined. The radius was automatically calculated by the operating software (DYNAMICS 7.8.1.3). The results in Table 2 show that the radius of the samples after different treatments ranged from 13.6 nm to 14.9 nm, which is comparable to that of the sample just melted from -80 °C (the radius of T0 was 13.7 nm).

[0262] Table 2. Accelerated stability results in PBS

[0263]

[0264]

[0265] T0: One freeze-thaw cycle (from -80 °C).

[0266] 3X: The sample was freeze-thawed 3 more times than T0.

[0267] 3.10 Complete kinetic binding affinity for PD-L1 (by SPR)

[0268] The binding affinity of WT1122 to human, mouse, and cynomolgus monkey PD-L1 was detected by SPR analysis using a Biacore 8K. Antibodies were captured on a CM5 sensor chip (GE) immobilized with anti-human IgG Fc antibody. Different concentrations of human or cynomolgus monkey PD-L1 were injected into the sensor chip at a flow rate of 30 μL / min for 180 s in the association phase, followed by dissociation for 3600 s. Different concentrations of mouse PD-L1 were injected into the sensor chip at a flow rate of 30 μL / min for 120 s in the association phase, followed by dissociation for 1200 s. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5).

[0269] The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgrams. For the binding of WT1122 to mouse PD-L1, the curve from 0 - 300 s was used in the fitting process. The experimental data were fitted using a 1:1 model by Langmiur analysis. A molecular weight of 40 kDa was used to calculate the molar concentration of human, mouse, and cynomolgus monkey PD-L1. As shown in Table 3, WT1122 has similar affinities for human and cynomolgus monkey PD-L1.

[0270] Table 3. Binding affinity of WT1122 to human, cynomolgus monkey, and mouse PD-L1

[0271]

[0272] 3.11 Complete kinetic binding affinity for TGFβ (by SPR)

[0273] The antibody binding affinity for TGFβ was detected by SPR assay using a Biacore8K. Each antibody was immobilized on a CM5 sensor chip (GE). Different concentrations of human TGFβ1 and TGFβ3 were injected into the sensor chip at a flow rate of 50 μL / min for 240 s in the association phase, followed by dissociation for 1200 s. Different concentrations of human TGFβ2 were injected into the sensor chip at a flow rate of 50 μL / min for 240 s in the association phase, followed by dissociation for 300 s. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5).

[0274] The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data were fitted by the 1:1 model using Langmiur analysis. The molar concentrations of the analytes TGFβ1, TGFβ2, and TGFβ3 were calculated using molecular weights of 22 kDa, 24 kDa, and 22 kDa, respectively. The results are listed in Table 4.

[0275] Table 4. Binding affinity of WT1122 for TGFβ determined by SPR.

[0276]

[0277] 3.12 Complete kinetic binding affinity for FcRn (by SPR)

[0278] The antibody binding affinity for human FcRn (ARCO, FCM-H5286) was detected using a Biacore 8K. Each antibody was immobilized on a CM5 sensor chip (GE). Different concentrations of human FcRn were injected into the sensor chip at a flow rate of 30 μL / min for 60 s in the association phase, followed by dissociation for 90 s. Then, after each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5). The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data were fitted by the steady-state affinity model. A molecular weight of 45 kDa was used to calculate the molar concentration of the analyte FcRn. The running buffer was PBST, pH 6.0.

[0279] The affinities of WT1122 and WT112-BMK2-IgG1 for FcRn were similar (Table 5).

[0280] Table 5. Affinity for FcRn determined by SPR

[0281]

[0282] Example 4

[0283] In vivo anti-tumor efficacy study

[0284] 4.1 In vivo anti-tumor efficacy study in HCC827 PBMC model

[0285] In the HCC827 model, the anti-tumor efficacy of WT1122 was studied in NCG female mice. Female NCG mice (Nanjing Galaxy Biopharmaceutical Co., Ltd.) at 13 - 14 weeks of age were used in the study. HCC827 cells were maintained in vitro in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in an atmosphere of 5% CO2 in air at 37°C in monolayer culture. The tumor cells were passaged twice a week routinely and treated with 0.25% trypsin-EDTA. Cells growing in the exponential growth phase were harvested and the tumor inoculum was counted.

[0286] For the treatment model, HCC827 tumor cells (5.0×10 6 cells, containing 50% Matrigel in 200 μL PBS) were subcutaneously inoculated into the right anterior side of each mouse. When the average tumor volume reached approximately 173 mm 3 , the animals were randomly divided into 5 groups, with 7 mice in each group. The mice were intravenously injected with human PBMC (5.0×10 6 , Hemocare, lot number 19057819). 1 - 2 h after PBMC implantation, the animals were treated with drugs by intraperitoneal injection on days 0, 3, 7, and 10, for a total of 4 injections. Four groups (G2 - G5) were injected with 20 mg / kg of WT112-BMK2-IgG1 or 0.2 mg / kg, 2 mg / kg, and 20 mg / kg of WT1122, respectively. The control group (G1) was injected with vehicle - PBS. The date of the first injection was considered day 0. For all tumor studies, the mice were weighed and tumor growth was measured twice a week using calipers.

[0287] All procedures related to animal handling, care, and treatment in this study were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai SIPPR-BK Laboratory Animal Co., Ltd. and followed the guidance of the American Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). The tumor volume was calculated using the formula (1 / 2 (length × width 2 ))). The results were expressed as mean and standard error (mean ± SEM). The data were analyzed by two-way ANOVA Tukey multiple comparison test using Graphpad Prism 6.0, and P < 0.05 was considered statistically significant.

[0288] As Figure 8A shown, all mice were normal during the experiment without obvious weight loss, indicating that the antibody was non-toxic. As Figure 8B shown, on the 16th day after the first administration, the average tumor volume in the vehicle group was 798 mm 3 , indicating that the HCC827 model had been well established. Compared with the vehicle group, WT1122 showed a potent anti-tumor effect and significantly inhibited tumor growth. The TGI on the 16th day for each group was 55.91% for WT112-BMK2-IgG1 (20 mg / kg), and 55.42%, 50.72% and 77.13% for WT1122 at 0.2 mg / kg, 2 mg / kg and 20 mg / kg, respectively. WT1122 showed better anti-tumor activity than BMK2 at high dose (p < 0.05), and comparable anti-tumor activity at low and medium doses (p > 0.05).

[0289] 4.2 In vivo pharmacokinetic study of WT1122 at 30 mg / kg in cynomolgus monkeys

[0290] Two adult cynomolgus monkeys (one male + one female) were administered 30 mg / Kg WT1122 by intravenous injection. Body weight, food consumption and clinical observations were made daily. Electrocardiogram (ECG), blood and serum samples were collected at different time points. Blood was collected into tubes containing EDTA-K2 for hematological examination, and 2.0 mL of blood was collected into tubes without additives for serum chemistry determination. Standard clinical chemistry and hematological analyses were performed. For PK and immunological analyses, approximately 1.2 mL of blood was collected, approximately 0.5 mL of serum was collected by centrifugation at 3500 rpm and 4 °C for 15 minutes, and frozen and stored at approximately -70 °C or lower. Daily clinical observations were made and recorded. All procedures related to animal handling, care and treatment were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Guangzhou University of Chinese Medicine Science and Technology Park Company following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The concentration of WT1122 in serum was determined by a bioanalytical ELISA method. Non-compartmental pharmacokinetic analysis of serum concentration was performed using Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA). The linear / log trapezoidal rule was applied to obtain PK parameters, and the data were expressed as mean ± SD.

[0291] Animals tolerated a single intravenous injection of WT1122 well at a dose of 30 mg / kg, and no obvious side effects were observed, including electrocardiogram, daily clinical observation, body weight, and major hematological parameters (ALT, AST, WBC, RBC, PLT, QTc, etc.). As shown in Table 6 and Figure 9 as follows, the terminal half-life was 92.1 h, and the AUC 0-inf was 34993 h*μg / mL, and the clearance rate was 20.9 ml / day / kg.

[0292] Table 6. PK profile of WT1122 after a single intravenous injection of 30 mg / kg

[0293]

[0294]

[0295] Those skilled in the art can recognize and understand the description of this patent. Without departing from its essence or basic characteristics, the present invention can be implemented in other specific forms. Since the foregoing description of the present invention only discloses its exemplary embodiments, other variations should be understood to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims to indicate the scope and content of the present invention.

[0296] References

[0297] [1] Alsaab HO, Sau S, Alzhrani R, et al. PD-1 and PD-L1 Checkpoint Signaling Inhibition for Cancer Immunotherapy: Mechanism, Combinations, and Clinical Outcome. Frontiers in Pharmacology 2017; 8:561.

[0298] [2] Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and autoimmunity. Immunological Reviews 2010; 236:219–42.

[0299] [3] Gong, Jun, Chehrazi-Raffle, Alexander et al. Development of PD-1 and PD-L1 inhibitors as a form of cancer immunotherapy: a comprehensive review of registration trials and future considerations. Journal for Immunotherapy of Cancer 2018; 6:8.

[0300] [4] Justin M. David et al. A novel bifunctional anti-PD-L1 / TGF-β Trap fusion protein (M7824) efficiently reverts mesenchymalization of human lung cancer cells. Oncoimmunology. 2017; 6(10):e1349589. Sequence Listing <110> Shanghai WuXi Biologics Inc. <120> A Bifunctional Fusion Protein and Its Use <130> IEC196146PCT <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 1 Gly Phe Ser Leu Thr Glu Asn Ser Val Ser 1 5 10 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 2 Ala Val Trp Ser Ser Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 3 Ser Thr Tyr Ser Asn Asp Phe Tyr Tyr Tyr Phe Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 4 Ser Gly Ser Glu Leu Pro Lys Arg Tyr Ala Tyr 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 5 Lys Asp Ser Glu Arg Pro Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 6 Ser Ser Thr Tyr Gly Asp Arg Lys Leu Pro Ile 1 5 10 <210> 7 <211> 121 <212> PRT <213> Artificial sequence <220> <223> VH <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Glu Asn 20 25 30 Ser Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ala Val Trp Ser Ser Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Arg Ser Thr Tyr Ser Asn Asp Phe Tyr Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 8 <211> 108 <212> PRT <213> Artificial sequence <220> <223> VL <400> 8 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Ser Glu Leu Pro Lys Arg Tyr Ala 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Ile Val Arg Val Ile Tyr 35 40 45 Lys Asp Ser Glu Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Thr Tyr Gly Asp Arg Lys Leu 85 90 95 Pro Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 9 <211> 136 <212> PRT <213> Human <400> 9 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 10 <211> 606 <212> PRT <213> Artificial Sequence <220> <223> HC <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Glu Asn 20 25 30 Ser Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ala Val Trp Ser Ser Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Arg Ser Thr Tyr Ser Asn Asp Phe Tyr Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val Asn 465 470 475 480 Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln 485 490 495 Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys 500 505 510 Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln 515 520 525 Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 530 535 540 Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu 545 550 555 560 Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro 565 570 575 Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp 580 585 590 Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 605 <210> 11 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> LC <400> 11 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Ser Glu Leu Pro Lys Arg Tyr Ala 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Ile Val Arg Val Ile Tyr 35 40 45 Lys Asp Ser Glu Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Thr Tyr Gly Asp Arg Lys Leu 85 90 95 Pro Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210

Claims

1. A fusion protein, which consists of an antibody or an antigen-binding portion thereof that specifically binds to PD-L1; a linker; and the extracellular domain of human TGFβRII, wherein, the C-terminus of the heavy chain of the antibody or its antigen-binding portion is linked to the extracellular domain of human TGFβRII through a linker, and the antibody or its antigen-binding portion comprises: Heavy chain CDR1 shown in SEQ ID NO:1; Heavy chain CDR2 shown in SEQ ID NO:2; Heavy chain CDR3 shown in SEQ ID NO:3; Light chain CDR1 shown in SEQ ID NO:4; Light chain CDR2 shown in SEQ ID NO:5; and Light chain CDR3 shown in SEQ ID NO:6; the extracellular domain of human TGFβRII is the extracellular domain of human TGFβRII shown in SEQ ID NO:

9.

2. The fusion protein of claim 1, wherein the antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region is the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:7; the light chain variable region is the light chain variable region of the amino acid sequence shown in SEQ ID NO:

8.

3. The fusion protein of claim 1, wherein the antibody or its antigen-binding portion is a whole antibody, ScFv, Fab, F(ab’)2, or Fv fragment.

4. The fusion protein of claim 2, wherein the heavy chain variable region of the antibody or its antigen-binding portion is operably linked to an Fc region.

5. The fusion protein of claim 4, wherein the Fc region is of IgG1 isotype.

6. The fusion protein of claim 4, wherein the Fc region is operably linked to the extracellular domain of human TGFβRII shown in SEQ ID NO:9 via a linker.

7. The fusion protein of claim 6, wherein the linker is a peptide linker.

8. The fusion protein of claim 7, wherein the linker is (G4S)n and n = 2 - 4.

9. The fusion protein of any one of claims 1 - 8, wherein the antibody or its antigen-binding portion is a humanized antibody or a fully human antibody.

10. The fusion protein of any one of claims 1 - 8, wherein the fusion protein comprises a heavy chain and a light chain, the heavy chain is the heavy chain of the amino acid sequence shown in SEQ ID NO:10, and the light chain is the light chain of the amino acid sequence shown in SEQ ID NO:

11.

11. An isolated nucleic acid molecule, which comprises a nucleic acid sequence encoding the fusion protein as defined in any one of claims 1 - 10.

12. A vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12.

14. A pharmaceutical composition, which comprises the fusion protein of any one of claims 1 - 10 and a pharmaceutically acceptable carrier.

15. A method for producing the fusion protein as defined in any one of claims 1 - 10, comprising the following steps: - expressing the fusion protein in a host cell comprising a nucleic acid sequence encoding the fusion protein; and - Isolate the fusion protein from the host cell.

16. Use of the fusion protein according to any one of claims 1-10 or the pharmaceutical composition according to claim 14 in the preparation of a medicament for inhibiting the growth of solid tumor cells related to PD-1 / PD-L1 in a subject.

17. The use according to claim 16, wherein the solid tumor is selected from colon cancer, lung cancer, liver cancer, cervical cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer and gastric cancer.

18. The use according to claim 16, wherein the solid tumor is lung cancer.

19. The use according to claim 18, wherein the lung cancer is NSCLC.

20. The use according to any one of claims 16-19, wherein the fusion protein or pharmaceutical composition is administered in combination with a chemotherapeutic agent, radiotherapy and / or other agents for cancer immunotherapy.

21. A kit for treating or diagnosing cancer, comprising the fusion protein defined in any one of claims 1-10 in a container.

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