Bifunctional antibodies against pd-l1 and tgfp

Abstract

A polypeptide is provided comprising, from N- to C-terminus, a) at least one heavy chain variable region of a heavy chain antibody that binds Programmed Death-Ligand 1 (PD-L1) (VHH domain) operably linked to an IgG Fc binding domain; and b) human TGFβRII or a fragment thereof capable of binding TGFβ. Also provided are antibodies comprising the polypeptide, amino acid sequences of the antibodies, cloning or expression vectors, cells, and methods for expressing or isolating the antibodies. Also provided are therapeutic compositions comprising the antibodies, and methods of treating cancer and other diseases with the bispecific antibodies.

Description

TECHNICAL FIELD

[0001] The present invention relates to a bifunctional antibody. Furthermore, the present invention provides polynucleotides encoding said antibody, vectors comprising said polynucleotides, host cells, methods of producing said antibody, and immunotherapy using said bifunctional antibody for the treatment of cancer, infections or other human diseases. BACKGROUND

[0002] Bifunctional antibodies can bind to two different targets or two different epitopes on a target, resulting in additive or synergistic effects superior to individual monospecific antibodies or target binding modules. The variable domain of heavy chain antibodies found in Camelidae, VHH, is considered the smallest naturally derived antigen binding fragment (12-15 kDa) that can be isolated from full-size immunoglobulins. Due to its small size and high antigen binding affinity, VHH is expected to have good penetration into tumors. In addition, VHH has high stability and simplicity in recombinant expression. Therefore, VHH shows great advantages over conventional antibodies in the application of bifunctional antibodies.

[0003] Programmed cell death ligand 1 (PD-L1, B7-H1, CD274) is a member of the immunoglobulin superfamily induced on a variety of cell types in lymphoid and peripheral tissues. Programmed cell death-1 protein (PD-1, CD279) is a member of the CD28 family expressed on activated T cells and other immune cells. The main role of the PD-1 pathway is to regulate immune responses in tissues and organs. It has been found that cancer cells can escape immune killing by upregulating the PD-1 / PD-L1 pathway in the tumor microenvironment [Boussiotis 2016 N Engl J Med].

[0004] TGF-β can promote tumor progression and facilitate immune evasion of tumors through its effects on the innate and adaptive immune system. The three TGF-β isoforms, TGF-β1, TGF-β2 and TGF-β3, are highly expressed in many tumor types and their serum concentrations are associated with poor clinical outcomes. TGF-β acts as an autocrine or paracrine signal in the tumor microenvironment, which promotes tumor progression in the tumor microenvironment by matrix modification, angiogenesis and induction of epithelial-mesenchymal transition (EMT). TGF-β signaling in bone marrow cells is also crucial in driving metastasis. In addition, TGF-β1 can directly inhibit T cell division as well as acquire effector function and natural killer (NK) function.

[0005] Antibodies targeting immune checkpoints are emerging as effective and feasible cancer treatment options, but not all patients respond to these as single agents. Simultaneous targeting of another immunosuppressive pathway is a promising approach to enhance immune checkpoint blockade, and bifunctional molecules designed to target both pathways simultaneously can offer strategic advantages over two single-agent combinations.

[0006] The present invention aims to develop a new and effective anti-tumor therapeutic agent consisting of a VHH directed against PD-L1, without light chain, and fused to a TGFβ Receptor II ECD (extracellular domain). SUMMARY

[0007] The present invention is based on the finding that a bifunctional polypeptide containing at least a portion of a TGFβ Receptor II (TGFβRII) capable of binding TGFβ and a VHH domain that binds to an immune checkpoint protein, such as human, monkey or mouse Programmed Death Ligand 1 (PD-L1), is an effective anti-tumor and anticancer therapeutic agent. The polypeptide can exhibit synergistic effects in cancer treatment compared to the effects of administering the two agents separately.

[0008] Thus, in a first aspect, the present invention provides a polypeptide comprising, from N- to C-terminus, at least one heavy chain variable region of a heavy chain antibody that binds to Programmed Death Ligand 1 (PD-L1) (VHH domain) operably linked to an IgG Fc binding domain; and a human TGFβRII (e.g. human TGFβRII extracellular domain (ECD), SEQ ID NO: 6) or a fragment thereof capable of binding TGFβ.

[0009] In one embodiment, the TGFβ is selected from the group consisting of TGFβ1, TGFβ2 and TGFβ3.

[0010] In one embodiment, the VHH domain comprises one or more of at least one heavy chain CDR (CDRH) selected from the group consisting of:

[0011] (a) a CDRH1 having at least 70% sequence identity to the CDRH1 set forth in SEQ ID NO: 1;

[0012] (b) a CDRH2 having at least 70% sequence identity to the CDRH2 set forth in SEQ ID NO: 2; and

[0013] (c) a CDRH3 having at least 70% sequence identity to the CDRH3 set forth in SEQ ID NO: 3.

[0014] In one particular embodiment, the VHH domain comprises one or more of at least one heavy chain CDR (CDRH) selected from the group consisting of:

[0015] (a) CDRH1 as set forth in SEQ ID NO: 1 or an amino acid addition, deletion, or substitution of no more than 2 amino acids in the amino acid sequence from said CDRH1 ;

[0016] (b) CDRH2 as set forth in SEQ ID NO: 2 or an amino acid addition, deletion, or substitution of no more than 2 amino acids in the amino acid sequence from said CDRH2; and

[0017] (c) CDRH3 as set forth in SEQ ID NO: 3 or an amino acid addition, deletion, or substitution of no more than 2 amino acids in the amino acid sequence from said CDRH3.

[0018] In one embodiment, the VHH domain comprises one or more of at least one heavy chain CDR (CDRH) selected from:

[0019] (a) CDRH1 comprising or consisting of SEQ ID NO: 1 ;

[0020] (b) CDRH2 comprising or consisting of SEQ ID NO: 2; and

[0021] (c) CDRH3 comprising or consisting of SEQ ID NO: 3.

[0022] In one embodiment, the VHH domain comprises:

[0023] (a) the amino acid sequence of SEQ ID NO: 4;

[0024] (b) an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 4; or

[0025] (c) an amino acid sequence that has one or more amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 4.

[0026] The polypeptide can further comprise an amino acid linker connecting the C-terminus of the VHH domain or IgG Fc binding domain to the N-terminus of human TGFβRII or a fragment thereof; the linker comprising:

[0027] (a) the amino acid sequence of SEQ ID NO: 5;

[0028] (b) an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 5; or

[0029] (c) an amino acid sequence having one or more additions, deletions, and / or substitutions of amino acids compared to SEQ ID NO: 5.

[0030] In one embodiment, the human TGFβRII or fragment thereof comprises:

[0031] (a) the amino acid sequence of SEQ ID NO: 6;

[0032] (b) an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6; or

[0033] (c) an amino acid sequence having one or more additions, deletions, and / or substitutions of amino acids compared to SEQ ID NO: 6.

[0034] The TGFβRII or fragment thereof 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.

[0035] In one embodiment, the polypeptide can comprise

[0036] (a) the amino acid sequence of SEQ ID NO: 7;

[0037] (b) an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 7; or

[0038] (c) an amino acid sequence having one or more additions, deletions, and / or substitutions of amino acids compared to SEQ ID NO: 7.

[0039] The present application also provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises two of the above polypeptides.

[0040] The sequences of the polypeptides or antibodies are shown in Table 1:

[0041] Table 1. Antibody sequences

[0042]

[0043]

[0044] In another aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the above polypeptide or the above antibody or antigen-binding fragment thereof.

[0045] The present invention provides a cloning or expression vector comprising a nucleic acid molecule encoding the above-mentioned polypeptide or its antibody or its antigen-binding fragment.

[0046] The present invention also provides cells comprising the above-described nucleic acids or one or more of the above-described clones or expression vectors.

[0047] In another aspect, the present invention provides a method comprising culturing the cells of the present invention and isolating polypeptides or antibodies.

[0048] In another aspect, the present invention provides pharmaceutical compositions comprising the polypeptide, or antibody, or an antigen-binding fragment of said antibody, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0049] The present invention also provides the above-described polypeptide or antibody for use in treating tumors or inhibiting the growth of tumor cells in a subject.

[0050] The present invention also provides a method for treating a tumor or inhibiting tumor cell growth in a subject, comprising administering a therapeutically effective amount of the aforementioned polypeptide or antibody to the subject.

[0051] The present invention also provides the use of the above-described polypeptides or antibodies in the preparation of medicaments for treating tumors or inhibiting tumor cell growth in subjects.

[0052] In this invention, the tumor is selected from colorectal tumors, breast tumors, ovarian tumors, pancreatic tumors, gastric tumors, prostate tumors, kidney tumors, cervical tumors, myeloma, lymphoma, leukemia, thyroid tumors, endometrial tumors, uterine tumors, bladder tumors, neuroendocrine tumors, head and neck tumors, liver tumors, nasopharyngeal tumors, testicular tumors, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

[0053] Features and advantages of the present invention

[0054] Bifunctional molecules targeting both the PD-L1 and TGFβ pathways can offer several benefits in cancer treatment. Compared to anti-PD-L1 therapy, bifunctional antibodies can improve response rates. Attached Figure Description

[0055] Figure 1 A schematic diagram of WT1126-U15T1.G1-1.uIgG1 is shown.

[0056] Figure 2 The results of ELISA with fixed human TGF-β1, TGF-β2 and TGF-β3 are shown.

[0057] Figure 3 ELISA results showing binding to soluble TGF-β2.

[0058] Figure 4 ELISA results showing binding to cell surface human PD-L1.

[0059] Figure 5 FACS results showing binding to cell surface cynomolgus PD-L1.

[0060] Figure 6 FACS results showing binding to cell surface mouse PD-L1.

[0061] Figure 7 Results showing binding to immobilized TGF-β1 in a dual target ELISA.

[0062] Figure 8 Results showing binding to immobilized human PD-L1 in a dual target ELISA.

[0063] Figure 9 FACS results showing blocking of PD-1 / PD-L1 by competition.

[0064] Figure 10 TGF-β1 signaling blockade in RGA assay.

[0065] Figure 11 PD-1 / PD-L1 signaling blockade in RGA assay.

[0066] Figure 12 A shows allo-MRL assay results for IL-2 secretion in human CD4+ T cells. Figure 12 B shows allo-MRL assay results for IFN-γ secretion in human CD4+ T cells.

[0067] Figure 13 Serum stability test results.

[0068] Figure 14 A shows body weight growth curve for CT26 model; Figure 14 B shows tumor growth curve for CT26 model. DETAILED DESCRIPTION

[0069] The following description of the disclosure is merely exemplary in nature of the various implementations of the disclosure. Thus, specific modifications to the illustrative embodiments described herein will be apparent to those skilled in the art, and, once known, can be implemented by those skilled in the art without departing from the scope of the disclosure. It is therefore intended that the disclosure be considered only by way of the illustrative embodiments described herein. All references, including publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0070] As used herein, a noun without a quantifier will mean one or more than one (i.e., at least one). For example, “a polypeptide complex” means one polypeptide complex or more than one polypeptide complex.

[0071] As used herein, the term “about” or “approximately” means a quantity, level, value, number, frequency, percentage, scale, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight or length. In some particular embodiments, the term “about” or “approximately” preceding a value indicates a range of that value plus or minus 15%, 10%, 5%, or 1%.

[0072] Throughout this disclosure, the word “comprise” or “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. “Consisting of’ is intended to include and be limited to whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory and that no other elements can be present. “Consisting essentially of’ is intended to include the elements listed after the phrase “consisting essentially of” and to exclude other elements of any kind, whether or not further restricted by the phrase “consisting essentially of.” Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and can or can not be present depending upon whether or not they affect the activity or action of the listed elements.

[0073] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, or an assembly of multiple polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. The term "amino acid" refers to both naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, such as those listed above, as well as modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon that is bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but otherwise function in a manner similar to naturally occurring amino acids. The α-carbon refers to the first carbon atom of an amino acid, indicated by a symbol "α" that is bonded to a hydrogen, a carboxyl group, an amino group, and an R group. The β-carbon refers to the second carbon atom of an amino acid, indicated by the symbol "β" that is bonded to one of the substituents of the α-carbon and a hydrogen. The system of nomenclature continues in a similar fashion for substituents of the α- and β-carbons. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The term "protein" generally refers to large polypeptides. The term "peptide" generally refers to short polypeptides. Polypeptide sequences are generally described with the left-hand end of the sequence as the amino terminus (N-terminus) and the right-hand end of the sequence as the carboxy terminus (C-terminus). As used herein, "polypeptide complex" refers to a complex comprising one or more polypeptides that are associated with performing some function. In certain embodiments, the polypeptides are immunologically related.

[0074] The term "fragment of TGFβRII" refers to a fragment of a sequence that is substantially identical to SEQ ID NO: 6, that is at least 20 (e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 175, or 200) amino acids in length, that retains at least some TGFβ binding activity (e.g., at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99%) of a wild-type receptor or corresponding wild-type fragment. Typically such fragments are soluble fragments. An exemplary such fragment is the TGFβRII extracellular domain having the sequence of SEQ ID NO: 6.

[0075] The term "substantially identical" means that a polypeptide exhibits at least 50%, desirably 60%, 70%, 75%, or 80%, more desirably 85%, 90%, or 95%, and most desirably 99% amino acid sequence identity to a reference amino acid sequence. The length of the compared sequences will typically be at least 10 amino acids, desirably at least 15 contiguous amino acids, more desirably at least 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids, and most desirably the full-length amino acid sequence.

[0076] The term "antibody" as used in the present disclosure refers to an immunoglobulin or a fragment or derivative thereof, and includes any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. The term "antibody" also includes antibody fragments, such as scFv, dAb, bispecific antibodies comprising a first VHH domain and a TGFβRII or fragment thereof fusion, and other antibody fragments that retain the ability to bind antigen, i.e., to bind PD-L1.

[0077] An antigen-binding fragment typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily have to comprise both. The VH region and the VL region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). The numbering scheme of all CDR definitions in the present invention is determined by the Kabat numbering system. So-called nanobody fragments consist only of a VHH domain, a CH2 and a CH3 domain, but still retain some antigen binding functionality of the intact antibody.

[0078] "Fc" with respect to an antibody refers to the portion of the antibody consisting of the second and third constant regions of a first heavy chain bound by a second heavy chain to a second constant region (CH2) and a third constant region (CH3). The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not play a role in antigen binding.

[0079] As used herein, "CH2 domain" refers to the portion of the heavy chain molecule of an IgG antibody that extends, using conventional numbering schemes, for example, from about amino acid position 244 to amino acid position 360 (positions 244-360 in the Kabat numbering system; and positions 231-340 in the EU numbering system; see Kabat, E., et al., U.S. Department of Health and Human Services, (1983)).

[0080] "CH3 domain" extends from the CH2 domain to the C-terminus of the IgG molecule and comprises about 108 amino acids. Certain immunoglobulin classes, such as IgM, also comprise a CH4 region.

[0081] As used herein, the term "antigen binding portion" refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure.

[0082] The terms "programmed death ligand 1," "PD ligand 1," "PD-L1," "PD L1," "B7 homolog 1," "B7-H1," "B7H1," "CD274" are used interchangeably and include variants, isoforms, species homologs of human PD-L1, and analogs having at least one common epitope with PD-L1.

[0083] As used herein, the terms "homologous" and "homology" are interchangeable and refer to a nucleic acid sequence (or the complementary strand thereof) or an amino acid sequence that has at least 70% (e.g., at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence when optimally aligned.

[0084] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as identity. Conservative substitution of an amino acid residue is not considered to be identical. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved in various ways, for example, using publicly available tools, such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also, Altschul S.F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also, Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tools, or can customize the parameters suitable for alignment, for example, by selecting a suitable algorithm.

[0085] The term "bind" as used herein refers to a non-random binding reaction between two molecules, for example, between an antibody and an antigen. In certain embodiments, the polypeptide complexes and bispecific polypeptide complexes provided herein specifically bind an antigen with a binding affinity (K D )≤10 -6 M (e.g., <5 x 10 -7 M, <2 x 10 -7 M, <10 -7 M, <5 x 10 -8 M, <2 x 10 - 8 M, <10 -8 M, <5 x 10 -9 M, <2 x 10 -9 M, <10 -9 M, or <10 -10M). As used herein, K D is the ratio of the dissociation rate to the association rate (Koff / Kon), which can be determined using a surface plasmon resonance method, for example, using a Biacore instrument.

[0086] The term "operably linked" means that two or more biological sequences of interest are juxtaposed in such a way that they are in a relationship permitting them to function in their intended manner. When used in reference to polypeptides, it is intended to mean that the polypeptide sequences are linked in a manner which permits the product of the linkage to have the intended biological function. For example, an antibody variable region can be operably linked to a constant region to provide a stable product having antigen binding activity. The term can also be used in reference to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to regulatory sequences (e.g., promoters, enhancers, silencer sequences, etc.), it is intended to mean that the polynucleotide sequences are linked in a manner which permits the polypeptide to be expressed from the polynucleotide.

[0087] As used herein, the term "mutation" or "mutated" with respect to an amino acid residue means a substitution, insertion, or addition of an amino acid residue.

[0088] Format

[0089] The present application provides a polypeptide comprising, from N- to C-terminus, at least one variable region of a heavy chain of a heavy chain antibody that binds Programmed Death-Ligand 1 protein (PD-L1) operably linked to an IgG Fc binding domain; and a human TGFβRII (e.g., a human TGFβRII extracellular domain (ECD)) or a fragment thereof capable of binding TGFβ. The TGFβ is selected from the group consisting of TGFβ1, TGFβ2, and TGFβ3. The polypeptide can further comprise an amino acid linker connecting the C-terminus of the VHH domain or the IgG Fc binding domain to the N-terminus of the human TGFβRII or the fragment thereof. The present application also provides an antibody or an antigen binding fragment thereof, wherein the antibody or the antigen binding fragment thereof comprises the above two polypeptides Figure 1 ) linked to each other.

[0090] In one embodiment, the VHH domain comprises one or more of at least one heavy chain CDR (CDRH) selected from the group consisting of:

[0091] (a) a CDRH1 having at least 70%, 80%, or 90% sequence identity to the CDRH1 set forth in SEQ ID NO: 1 (GHFSNLAVN);

[0092] (b) a CDRH2 having at least 70%, 80%, or 90% sequence identity to the CDRH2 set forth in SEQ ID NO: 2 (GILWSGGSTFYADSVKG); and

[0093] (c) a CDRH3 having at least 70%, 80%, or 90% sequence identity to the CDRH3 set forth in SEQ ID NO: 3 (GTN).

[0094] In one embodiment, the VHH domain comprises one or more heavy chain CDRs (CDRH) selected from:

[0095] (a) a CDRH1 set forth in SEQ ID NO: 1 or a CDRH1 that differs from said CDRH1 in amino acid sequence by an amino acid addition, deletion, or substitution of 1 or 2 amino acids;

[0096] (b) a CDRH2 set forth in SEQ ID NO: 2 or a CDRH2 that differs from said CDRH2 in amino acid sequence by an amino acid addition, deletion, or substitution of 1 or 2 amino acids; and

[0097] (c) a CDRH3 set forth in SEQ ID NO: 3 or a CDRH3 that differs from said CDRH3 in amino acid sequence by an amino acid addition, deletion, or substitution of 1 or 2 amino acids.

[0098] In one embodiment, the VHH domain comprises one or more heavy chain CDRs (CDRH) selected from:

[0099] (a) a CDRH1 comprising or consisting of SEQ ID NO: 1 ;

[0100] (b) a CDRH2 comprising or consisting of SEQ ID NO: 2; and

[0101] (c) a CDRH3 comprising or consisting of SEQ ID NO: 3.

[0102] In one embodiment, the VHH domain comprises:

[0103] (a) a CDRH1 comprising or consisting of SEQ ID NO: 1 ;

[0104] (EVQLVESGGGLVQPGGSLRLSCAASGHFSNLAVNWFRQAPGKERELVAGILWSGGSTFYADSVKGRFTISRGNAENMLYLQMNSLRAEDTAVYYCNTGTNWGQGTLVTVSS) set forth in SEQ ID NO: 4;

[0105] (b) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; or

[0106] (c) an amino acid sequence that has one or more additions, deletions and / or substitutions of amino acids compared to SEQ ID NO: 4.

[0107] The polypeptide can further comprise an amino acid linker connecting the C-terminus of the VHH domain or IgG Fc binding domain to the N-terminus of the human TGFβRII or fragment thereof; the linker comprising:

[0108] (a) an amino acid sequence set forth in SEQ ID NO: 5 (GGGGGSGGGGSGGGGSGGGGS);

[0109] (b) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5; or

[0110] (c) an amino acid sequence that has one or more additions, deletions and / or substitutions of amino acids compared to SEQ ID NO: 5.

[0111] In one embodiment, the human TGFβRII or fragment thereof comprises:

[0112] (a) an amino acid sequence set forth in SEQ ID NO: 6

[0113] (IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD);

[0114] (b) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6; or

[0115] (c) an amino acid sequence that has one or more additions, deletions and / or substitutions of amino acids compared to SEQ ID NO: 6.

[0116] The TGFβRII or fragment thereof 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.

[0117] In one embodiment, the polypeptide can comprise

[0118] (a) an amino acid sequence set forth in SEQ ID NO: 7;

[0119] (b) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7; or

[0120] (c) an amino acid sequence that has one or more additions, deletions, and / or substitutions of amino acids compared to SEQ ID NO: 7.

[0121]

[0122] Method of preparation

[0123] The present disclosure provides isolated nucleic acids or polynucleotides encoding the polypeptide complexes, as well as the bispecific polypeptide complexes provided herein.​

[0124] The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single- or double-stranded form, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of nucleotides and / or deoxy nucleotides (see, Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0125] Nucleic acids or polynucleotides encoding the polypeptide complexes and bispecific polypeptide complexes provided herein can be constructed using recombinant techniques. To this end, DNA encoding the antigen binding portions (e.g., CDRs or variable regions) of the parent antibodies can be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are specific to the heavy and light chains of the antibodies). Likewise, DNA encoding the TCR constant regions can also be obtained. For example, a polynucleotide sequence encoding the heavy chain variable domain (VH) and a polynucleotide sequence encoding the first TCR constant region are obtained and operably linked to permit transcription and expression in a host cell to produce a first polypeptide. Similarly, a polynucleotide sequence encoding the VL is operably linked to a polynucleotide sequence encoding the second TCR constant region, permitting expression of the second polypeptide in the host cell. If desired, a polynucleotide sequence encoding one or more spacers is also operably linked to the other coding sequences to permit expression of the desired product.

[0126] The coding polynucleotide sequences can be further operably linked to one or more regulatory sequences, optionally in an expression vector, such that expression or production of the first and second polypeptides is possible and under appropriate control.

[0127] Recombinant techniques known in the art can be used to insert the polynucleotide sequence encoding into a vector for further cloning (amplification of DNA) or expression. In another embodiment, the polypeptide complexes and bispecific polypeptide complexes provided herein can be produced by homologous recombination known in the art. A number of vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1a), and a transcription termination sequence.

[0128] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. Typically, the construct also includes appropriate regulatory sequences. For example, a polynucleotide molecule can include regulatory sequences located 5' to the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding the site-directed modification polypeptide that are operably linked to the coding sequence in a manner that enables the desired transcript / gene to be expressed in a host cell. Vectors can be used to transform, transduce, or transfect host cells to cause the genetic elements they carry to be expressed within the host cell. Examples of vectors include plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1 -derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage; and animal viruses. Classes of animal viruses that are used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors can comprise a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, vectors can comprise an origin of replication. Vectors can also comprise materials that aid in their entry into cells, including but not limited to viral particles, liposomes, or protein coats.

[0129] In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., and includes plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, and the like, as well as other laboratory and commercially available vectors. Suitable vectors can include plasmid or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0130] Vectors comprising the polynucleotide sequences provided herein can be introduced into host cells for cloning or gene expression. The phrase "host cell," as used herein, refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0131] Suitable host cells for cloning or expression of DNA in the vectors herein are prokaryotic, yeast, or higher eukaryotic cells described above. Suitable prokaryotes include gram-negative or gram-positive organisms, including eubacteria, such as Enterobacteriaceae, e.g., Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., S. typhimurium; Serratia, e.g., S. marcescans; and Shigella; and Bacilli, e.g., B. subtilis and B. licheniformis; Pseudomonas, e.g., P. aeruginosa; and Streptomyces.

[0132] In addition to prokaryotes, eukaryotic microbes (e.g., filamentous fungi or yeast) are suitable cloning or expression hosts for vectors encoding polypeptide complexes and bispecific polypeptide complexes. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and useful, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as S. occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium; and Aspergillus hosts, such as A. nidulans and A. niger.

[0133] Suitable host cells for the expression of glycosylated polypeptide complexes, bispecific polypeptide complexes herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the virus herein according to the application, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco, also can be used as hosts.

[0134] However, of greatest interest is the propagation of vertebrate cells in culture (tissue culture), and vertebrate cell propagation in culture has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS cells, ATCC CRL 1651); human embryonic kidney line 293 (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), e.g., Expi293; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0135] Host cells transformed or co-transformed with the above-described expression or cloning vectors are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the subject cloning vectors.

[0136] To produce the polypeptide complexes and bispecific polypeptide complexes provided herein, host cells transformed with expression vectors can be cultured in a variety of media. Commercially available media, such as Ham’s F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Re. 30,985 can be used as culture media for the host cells. Any of these media can be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamycin™ drug), trace elements (defined as inorganic compounds generally present at final concentrations in the range of micromolar to millimolar), and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations that would be known to those of skill in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0137] In certain embodiments, the polypeptide complex or bispecific polypeptide complex can be indirectly linked to a conjugate, or indirectly linked, e.g., through another conjugate or through a linker. For example, a polypeptide complex or bispecific polypeptide complex having a reactive residue, e.g., a cysteine, can be linked to a thiol-reactive reagent, where the reactive group is, e.g., a maleimide, iodoacetamide, pyridyl disulfide, or other thiol-reactive conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0138] For another example, the polypeptide complex or bispecific polypeptide complex can be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. For yet another example, the polypeptide complex or bispecific polypeptide complex can be linked to a linker, which is further linked to a conjugate. Examples of linkers include bifunctional coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl esters), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidophenyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and his-active fluorides (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP) to provide disulfide linkages.

[0139] The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable labels can include a fluorescent label (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas red), an enzyme-substrate label (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, phospholipase, lysozyme, saccharide oxidase, or beta-D-galactosidase), a radioisotope (e.g., 123I, 124I, 125I, 131I, 35S, 3H, 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi, and 32P, other lanthanides, luminescent labels), a chromophoric moiety, digoxigenin, biotin / avidin, a DNA molecule for detection, or gold. In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety, such as PEG, which helps to increase the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, copolymers of ethylene glycol / propylene glycol, and the like. In certain embodiments, the conjugate can be a purification moiety, such as a magnetic bead. A “cytotoxic moiety” can be any agent that is harmful or can damage or kill a cell. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, de-carbazine, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (actinomycin D) and bleomycin), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0140] Methods of conjugating a conjugate to a protein, such as an antibody, immunoglobulin, or fragment thereof, can be found, for example, in U.S. Patent No. 5,208,020; U.S. Patent No. 6,441,163; WO2005037992; WO2005081711; and WO2006 / 034488, which are incorporated herein by reference in their entirety.

[0141] Pharmaceutical composition

[0142] The present disclosure also provides pharmaceutical compositions comprising the polypeptide complex or bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier.

[0143] The term "pharmaceutically acceptable" means that the carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients constituting a formulation and physiologically acceptable to the recipient thereof.

[0144] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is biologically acceptable and non-toxic to the subject. Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, aqueous vehicle, non-aqueous vehicle, antimicrobial agent, isotonic agent, buffering agent, antioxidant, anesthetic, suspending / dispersing agent, masking or chelating agent, diluent, adjuvant, excipient, or non-toxic auxiliary substance, other components known in the art, or a combination thereof.

[0145] Method of treatment

[0146] The present disclosure also provides methods of treatment comprising: administering to a subject in need thereof a therapeutically effective amount of the polypeptide complex or bispecific polypeptide complex provided herein, thereby treating or preventing a condition or disease. In certain embodiments, the subject has been identified as having a condition that can be responsive to the polypeptide complex or bispecific polypeptide complex provided herein.

[0147] The term "subject" as used herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The term "patient" or "subject" can be used interchangeably unless otherwise indicated.

[0148] The terms "treatment" and "method of treatment" refer to both therapeutic treatment and prophylactic / preventative measures. Those in need of treatment can include those already with the particular medical disease as well as those in which the disease is to be prevented.

[0149] In certain embodiments, the disorders and diseases include tumors and cancers, e.g., non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, e.g., classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and EBV-negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, e.g., primary CNS lymphoma, spinal axis tumors, brain stem glioma.

[0150] Examples

[0151] Example 1: Preparation of Experimental Materials

[0152] 1. Preparation of PD-L1 and TGFβ antigens

[0153] His-tagged human PD-L1 extracellular domain (ECD) antigen was purchased from Sino Biological (Cat# 10084-H08H). His-tagged mouse PD-L1 ECD antigen was purchased from Sino Biological (Cat# 50010-M08H). His-tagged cyno PD-L1 ECD antigen was purchased from Sino Biological (Cat# 90251-C08H). Human TGFβ1, TGFβ2, and TGFβ3 antigens were purchased from R&D Systems (Cat# 7754-BH, 7754-BH / CF; Cat# 302-B2, 302-B2 / CF; Cat# 8420-B3, 8420-B3 / CF).

[0154] 2. Establishment of PD-L1 -expressing cell lines

[0155] Human PD-L1 expressing cell line (W315-CHO-K1.hPro1.C11), mouse PD-L1 expressing cell line (W315-293F.mPro1.C1) and cynomolgus monkey PD-L1 expressing cell line (W315-293F.cynoPro1.2A2) were inhouse. CHO-K1 or 293F cells were transfected with expression vectors containing the gene encoding full length human PD-L1 or cynomolgus monkey PD-L1 or mouse PD-L1 using Lipofectamine 2000 (ThermoFisher-11668027). Cells were cultured in medium containing appropriate selection pressure and stable cell lines were obtained by limiting dilution.

[0156] 3. Preparation of Benchmark Antibody (BMK) and Control Antibody (cAb)

[0157] The anti-PD-L1 BMK antibody fused to TGFβRII ECD was named WT112-BMK2-IgG1 and was constructed based on the M7824 sequence in Merck Patent GmbH patent US9676863B2. The plasmid containing the heavy chain gene and the plasmid containing the light chain gene were co-transfected into Expi293 cells using Expi293 expression kit (ThermoFisher-A14524).

[0158] The control antibody WT112-cAb2 fused to TGFβRII ECD was constructed in the same way as WT112-BMK2-IgG1. The antibody part of WT112-cAb2 is a human IgG1 isotype control antibody; the linker between the Fc and the TGFβRII ECD is (G4S)4; the TGFβRII ECD sequence is the same as in WT112-BMK2-IgG1.

[0159] 4. Production of WT1126-U15T1.G1-1.uIgG1

[0160] WT1126-U15T1.G1-1.uIgG1 is an anti-PD-L1 VHH Fc fusion antibody fused to TGFβRII ECD. The DNA encoding the anti-PD-L1 VHH variable region was inserted into a modified pcDNA3.3 expression vector containing the Fc of human IgG1. The C-terminus of the Fc is the sequence encoding the TGFβRII ECD with a (G4S)4 linker in between, the same as in WT112-BMK2-IgG1. The schematic of WT1126-U15T1.G1-1.uIgG1 is shown in Figure 1

[0161] Example 2: In vitro characterization

[0162] ​1. ELISA detection of binding to human TGF-β

[0163] Binding of antibodies to human TGF-β1, TGF-β2 and TGF-β3 was determined by ELISA. Plates were coated with human TGF-β1, TGF-β2 or TGF-β3, respectively, overnight at 4°C. After blocking and washing, different concentrations of the lead antibodies were added to the plates and incubated for 1 hour at room temperature. Plates were then washed and incubated with HRP-labeled goat anti-human IgG antibody (Bethyl) for 1 hour. After washing, TMB substrate was added and the color development reaction was stopped with 2 M HC1. Absorbance at 450 nm and 540 nm was read using a microplate reader (SpectraMax M5e).

[0164] Binding curves of antibodies to human TGF-β1, TGF-β2 and TGF-β3 coated on plates are shown in Figure 2 WT1126-U15T1.G1-1.uIgG1 showed similar affinity to WT112-BMK2-IgG1. They bound strongly to immobilized TGF-β1 (EC 50 = 0.67 nM) and TGF-β3 (EC 50 = 0.89 nM), but not to immobilized TGF-β2.

[0165] Binding of antibodies to human TGF-β2 was determined by ELISA with the test antibodies immobilized. After blocking and washing, different concentrations of TGF-β2 were added to the plates and incubated for 1 hour at room temperature. Plates were then washed and incubated with biotinylated TGF-β2 detection antibody (R&D, DY240) for 1 hour, followed by incubation with streptavidin-HRP for 1 hour. After washing, TMB substrate was added and the color development reaction was stopped with 2 M HC1. Absorbance at 450 nm and 540 nm was read using a microplate reader (SpectraMax M5e). Binding curves of antibodies to soluble human TGF-β2 are shown in Figure 3 Immobilized WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 could bind to soluble TGF-β2 with comparable EC 50 values of 0.11 nM and 0.05 nM, respectively.

[0166] 2. FACS detection of binding to human PD-L1

[0167] Different concentrations of test antibodies were incubated with W315-CHO-K1.hPro1.C11 cells expressing hPD-L1 for 1 hour at 4°C. After washing, cells were incubated with PE-labeled goat anti-human IgG-Fc antibody (Jackson ImmunoResearch). Finally, MFI of cells was measured by flow cytometry and analyzed by FlowJo. Binding curves to human PD-L1 transfected cells are shown in Figure 4 Figure 6. WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 strongly bind to cell surface human PD-L1 with EC 50 of 0.14 nM and 0.35 nM, respectively.

[0168] 3. Cross-species binding by FACS

[0169] Binding of test antibodies to cynomolgus or mouse PD-L1 was determined by FACS. Different concentrations of test antibodies were incubated with W315-293F.cynoPro1.2A2 cells expressing cynomolgus PD-L1 or W315-293F.mPro1.C1 cells expressing mouse PD-L1 for 1 hour at 4°C, and then binding of antibodies to cell surface was detected by PE-labeled goat anti-human IgG-Fc antibody (Jackson ImmunoResearch). MFI of cells was measured by flow cytometry and analyzed by FlowJo. Binding to cynomolgus PD-L1 transfected cells is shown in Figure 5 Figure 7, and binding to mouse PD-L1 transfected cells is shown in Figure 6 Figure 8. WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 can strongly bind to cell surface cynomolgus and mouse PD-L1. EC 50 for cynomolgus PD-L1 binding of WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 were 0.63 nM and 0.96 nM, respectively. EC 50 for mouse PD-L1 binding were 0.71 nM and 1.3 nM, respectively.

[0170] 4. Simultaneous binding to human PD-L1 and human TGF-β1

[0171] Simultaneous binding of the test antibody to human TGF-β1 and human PD-L1 was determined by ELISA. Plates were coated with human TGF-β1 overnight at 4°C. After blocking and washing, different concentrations of test antibody were added to the plates and incubated at room temperature for 1 hour. The plates were then washed and incubated with biotinylated human PD-L1 ECD protein, followed by incubation with streptavidin-HRP (Invitrogen) for 1 hour. After washing, TMB substrate was added, and the colorimetric reaction was terminated with 2M HCl. Absorbance was read at 450 nm and 540 nm using a ELISA reader (SpectraMax M5e). Similarly, dual-target binding was also tested by coating the plates with human PD-L1. After incubation with different concentrations of test antibody and then with TGF-β1 antigen, biotinylated human TGF-β1 detection antibody (R&D, Cat 840117) and subsequently streptavidin-HRP (Invitrogen) were added to the plates. Finally, add TMB substrate and terminate the colorimetric reaction with 2M HCl. Read the absorbance at 450 nm and 540 nm using a microplate reader (SpectraMax M5e).

[0172] Figure 7 and Figure 8 The results showed that when TGF-β1 was immobilized, WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 simultaneously bound to PD-L1 and TGF-β1, EC2 50 The concentrations were 0.24 and 0.17 nM, respectively; when the antibody was immobilized, EC... 50 The values ​​are 0.03 nM and 0.06 nM, respectively.

[0173] 5. Detection of PD-1 / PD-L1 blocking via competitive FACS

[0174] Different concentrations of lead 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 hour. The binding of human PD-1 to human PD-L1-expressing cells was detected using PE-labeled anti-mouse IgG Fc antibody (Abcam). The mFc binding factor (MFI) of the cells was measured by flow cytometry and analyzed using FlowJo. Figure 9 As shown, WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 block the binding of PD-1 to PD-L1 on the cell surface, IC50 50 The values ​​are 0.03 nM and 0.11 nM, respectively.

[0175] 6. Reporter gene assay (RGA)

[0176] Blockade of TGF-β1 signaling was tested by RGA. RGA cell line was constructed by stable expression of full-length human activin receptor II B and stably integrated SBE luciferase reporter. To test TGF-β1 signaling blockade activity of the test antibodies, human TGF-β1 and different concentrations of antibodies were pre-mixed and added to RGA cells, incubated at 37°C, 5% CO2 overnight. After incubation, reconstituted luciferase substrate (Promega, Cat E6130) was added and luciferase intensity was measured by microplate spectrophotometer. WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 showed strong TGF-β1 signaling blockade activity in RGA assay. IC 50 50% of WT112-BMK2 (IC 50 = 0.7 nM) was 0.7 nM, as Figure 10 shown.

[0177] Blockade of PD-1 / PD-L1 signaling was tested by RGA. PD-1 RGA cell line was constructed by stable expression of full-length PD-1 and NFAT luciferase reporter in Jurkat E6-1 cells. PD-1 RGA 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 different concentrations of test antibodies at 37°C, 5% CO2 for 4 to 6 hours. After incubation, reconstituted luciferase substrate was added and luciferase intensity was measured by microplate spectrophotometer. As Figure 11 shown, WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 showed strong hPD-1 / PD-L1 signaling blockade activity in RGA assay. IC 50 of WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 were 0.28 and 0.59 nM, respectively.

[0178] 7. Allogeneic mixed lymphocyte reaction (allo-MLR)

[0179] Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from healthy donors using 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). Purified CD4 + T cells were isolated from human CD4 + T cells using the 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 various lead antibodies, positive and negative control antibodies. Plates were incubated at 37°C, 5% CO2. Supernatants were harvested at day 3 and day 5 for IL-2 and IFN-g assays, respectively. Human IL-2 and IFN-g release was measured by ELISA using matched antibodies. Recombinant human IL-2 (R&D) and IFN-g (PeproTech) were used as standards, respectively. Plates were pre-coated with capture antibodies specific for human IL-2 (R&D) or IFN-g (Pierce), respectively. After blocking, 50 pL of standards or samples were pipetted into each well and incubated for 2 hours at ambient temperature. After removal of unbound material, biotin-conjugated detection antibodies specific for the respective cytokine were added to the wells and incubated for one hour. HRP-labeled streptavidin was then added to the wells, incubated for 30 minutes at ambient temperature. Color development was achieved by dispensing 50 pL of TMB substrate, followed by termination of the reaction with 50 pL of 2N HC1. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. Figure 12 The results shown indicate that WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 can enhance IL-2 production + in a dose-dependent manner in the human CD4 Figure 12 A) and IFN-g production Figure 12 B) in the human CD4 + T cell allo-MLR assay (by two-way ANOVA analysis with isotype control. *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001)

[0180] 8. Serum stability

[0181] WT1126-U15T1.G1-1.uIgG1 was incubated with freshly isolated human serum (serum content >95%) at 37°C in a 5% CO2 incubator. At the indicated time points, aliquots of the serum-treated samples were removed from the incubator and snap-frozen in liquid nitrogen and stored at 20°C until ready for testing. Samples were quickly thawed prior to stability testing. The operation of the simultaneous binding ELISA, the ELISA for binding to human TGF-β1 and the FACS for binding to human PD-L1 were tested as described above. As shown in Figure 8, the WT1126-U15T1.G1-1.uIgG1 samples showed normal binding to the targets, indicating that the antibody was stable in human serum for at least 14 days. Figure 13

[0182] 9. Accelerated stability study of antibody proteins

[0183] 9.1 Sample handling and accelerated stability study

[0184] WT1126-U15T1.G1-1.uIgG1 was dialyzed in PBS buffer through dialysis bags (Spectrum-888-10987, MWCO 3.5 kDa) and then diluted to 2 pg / ml. The accelerated stability study was performed by incubating the test antibody at 4°C, 25°C and 40°C for 1 day, 4 days and 7 days, respectively, and at -80°C for 3 cycles of freeze-thaw (Table 2). Immediately after incubation under each test condition, a visual inspection of the samples was performed to carefully detect the presence of any particles. All samples appeared as clear solutions free of particles. The antibody stability of each treated sample was analyzed by SDS-PAGE, analytical SEC-HPLC, DSF and DLS determination. The samples after different treatments were stable in DSF and DLS analysis compared to the sample freshly thawed from -80°C (TO in Table 2). SEC-HPLC analysis showed an increase in the percentage of low molecular weight (LMW%) after incubation in PBS buffer at 40°C for 4 days and 7 days (Table 2). After formulation buffer optimization, the antibody was stable in sodium acetate buffer at 40°C (Table 3).

[0185] 9.2 DSF detection of thermal stability

[0186] ​DSF assays were performed using real-time quantitative PCR (QuantStudio 7 Flex, Thermo Fisher Scientific). Briefly, 19 pL of antibody solution was mixed with 1 pL of 62.5x SYPRO Orange solution (Invitrogen) and 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 was collected. The negative derivative of the fluorescence change at different temperatures was calculated and the maximum was defined as the melting temperature. Data acquisition and Tm calculation were automatically performed by the operating software (QuantStudio™ Real-Time PCR Software vl.3). The Tm of WT1126-U15T1.G1-1.uIgG1 in PBS buffer was about 65°C (Table 2).

[0187] 9.3DLS measures molecular radius

[0188] Molecular radius measurements were performed using a DynaPro Plate Reader III dynamic light scattering (DLS) instrument (Wyatt Dynapro™). Five acquisitions were collected for each protein sample and each acquisition time was 5 seconds. In a 1536 plate (Aurora microplate), each well contained 7.5 pL of solution. 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 25.9 nM to 33.5 nM, which is comparable to the sample freshly thawed from -80°C (T0 radius of 28 nM).

[0189] Table 2. Accelerated stability results in PBS

[0190]

[0191]

[0192] T0: has been thawed once (from -80°C).

[0193] 3X: sample has been thawed 3 times more than T0.

[0194] Table 3. Accelerated stability test at 40°C in sodium acetate buffer

[0195]

[0196] 10. SPR detects full kinetic binding affinity to PD-L1

[0197] The binding affinity of WT1126-U15T1.G1-1.uIgG1 to human, mouse and cynomolgus PD-L1 was determined by SPR, using a Biacore 8K for detection. The antibodies were captured on an anti-human IgG Fc antibody immobilized CM5 sensor chip (GE). Different concentrations of human or cynomolgus PD-L1 were injected over the sensor chip at a flow rate of 30 uL / min, with an association phase of 180 seconds, followed by a dissociation phase of 3600 seconds. Different concentrations of mouse PD-L1 were injected over the sensor chip at a flow rate of 30 uL / min, with an association phase of 120 seconds, followed by a dissociation phase of 1200 seconds. After each binding cycle, the chip was regenerated with 10 mM glycine, pH 1.5.

[0198] The blank surface and buffer channel sensorgrams were subtracted from the test sensorgrams. For the binding of WT1126-U15T1.G1-1.uIgG1 to mouse PD-L1, the 0 to 300 seconds curve was used in the fitting process. The experimental data were fitted by a 1 : 1 model using Langmiur analysis. The molar concentration of human, mouse and cynomolgus PD-L1 was calculated using a molecular weight of 40 kDa. As shown in Table 4, WT1126-U15T1.G1-1.uIgG1 has similar affinity to human and cynomolgus PD-L1.

[0199] Table 4. Binding affinity of WT1126-U15T1.G1-1.uIgG1 to human, cynomolgus and mouse PD-L1

[0200] Analyte Ligand ka (1 / Ms) kd (1 / s) KD (M) Human PD-L1 WT1126-U15T1.G1-1.uIgG1 1.15E+06 3.07E-04 2.67E-10 Cynomolgus PD-L1 WT1126-U15T1.G1-1.uIgG1 1.01E+06 4.65E-04 4.61E-10 Mouse PD-L1 WT1126-U15T1.G1-1.uIgG1 2.58E+05 1.08E-02 4.19E-08

[0201] 11. SPR detection of full kinetic binding affinity to FcRn

[0202] The antibody binding affinity to 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 over the sensor chip at a flow rate of 30 uL / min, with an association phase of 60 seconds, followed by a dissociation phase of 90 seconds. The chip was then regenerated after each binding cycle with 10 mM glycine, pH 1.5. The blank surface and buffer channel sensorgrams were subtracted from the test sensorgrams. The experimental data were fitted by a steady state affinity model. The molar concentration of the analyte FcRn was calculated using a molecular weight of 45 KDa. The running buffer was PBST, pH 6.0.

[0203] WT1126-U15T1.G1-1.uIgG1 and WT112-BMK2-IgG1 have similar affinity to FcRn (Table 5).

[0204] Table 5. Affinity to FcRn by SPR

[0205]

[0206] Example 3: In vivo anti-tumor efficacy study

[0207] WT1126-U15T1.G1-1.uIgG1 anti-tumor efficacy study was tested in CT26 model of BALB / C female mice. 8-week-old female BALB / C mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were used in this study. CT26 cells were cultured in vitro as monolayer cultures in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C, 5% CO2. Routine subculture of tumor cells was performed twice a week with 0.25% trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.

[0208] For the therapeutic model, CT26 tumor cells (100 μL RPMI, 5.0 x 10 5 When the average tumor volume reached about 50 mm 3 When the average tumor volume reached about 50 mm 2) were calculated. Results are expressed as mean and standard error (mean ± SEM). Data were analyzed using Graphpad Prism 6.0 and p values were analyzed using T test. p < 0.05 was considered statistically significant.

[0209] As shown in Figure 2A, all mice were normal and gained weight slowly during the experiment, indicating that the antibody was not toxic. As shown in Figure 2B, the average tumor volume of the vehicle group was 2550 mm3 17 days after the first dose, which indicated that the CT26 model was well established. Figure 14 Figure 14 As shown in Figure 2B, the average tumor volume of the vehicle group was 2550 mm3 17 days after the first dose, which indicated that the CT26 model was well established. 3 This indicated that the CT26 model was well established. Compared with the vehicle group, WT1126-U15T1.G1-1.uIgG1 showed potent anti-tumor effect and significantly inhibited tumor growth. The TGI (tumor growth inhibition) of each group was calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]x100. Ti is the average tumor volume of the treatment group on a given date. T0 is the average tumor volume of the treatment group on the first day of treatment. Vi is the average tumor volume of the vehicle control group on the same day as Ti, and V0 is the average tumor volume of the vehicle group on the first day of treatment. The TGI of WT112-BMK2-IgG1 on day 17 was 3.67%, 54.26% and 66.46% at the dose levels of 2 mg / kg, 6.5 mg / kg and 20 mg / kg, respectively. The TGI of WT1126-U15T1.G1-1.uIgG1 was 42.21%, 48.69% and 56.05% at 1.2 mg / kg, 4 mg / kg and 12 mg / kg, respectively. WT1126-U15T1.G1-1.uIgG1 showed superior anti-tumor activity to BMK2 at low doses (p < 0.01 by T test of G2 vs. G5), and comparable anti-tumor activity at medium and high doses (p was not significant (ns) by T test of G3 vs. G6 and G4 vs. G7). SEQUENCE LISTING <110> Shanghai Magway Biotechnology Co., Ltd. <120> Bispecific antibody against PD-L1 and TGFβ <130> MP22032889P <160> 7 <170> PatentIn version 3.3 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VHH CDRH1 ​<400> 1 Gly His Phe Ser Asn Leu Ala Val Asn 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VHH CDRH2 <400> 2 Gly Ile Leu Trp Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> VHH CDRH3 <400> 3 Gly Thr Asn 1 <210> 4 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VHH domain <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Phe Ser Asn Leu Ala 20 25 30 Val Asn Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Gly Ile Leu Trp Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Gly Asn Ala Glu Asn Met Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Gly Thr Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 5 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 5 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser 20 <210> 6 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> TGFβRII <400> 6 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> 7 <211> 498 <212> PRT <213> Artificial Sequence <220> <223> Full-length Polypeptide <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Phe Ser Asn Leu Ala 20 25 30 Val Asn Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Gly lie Leu Trp Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Gly Asn Ala Glu Asn Met Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Gly Thr Asn Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Glu 100 105 110 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 115 120 125 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 130 135 140 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 145 150 155 160 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 165 170 175 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr 180 185 190 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 195 200 205 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 210 215 220 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg 225 230 235 240 Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 245 250 255 Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 260 265 270 Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 275 280 285 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 290 295 300 Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 305 310 315 320 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 325 330 335 Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 340 345 350 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser lie Pro Pro His Val Gin 355 360 365 Lys Ser Val Asn Asn Asp Met lie Val Thr Asp Asn Asn Gly Ala Val 370 375 380 Lys Phe Pro Gin Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys 385 390 395 400 Asp Asn Gin Lys Ser Cys Met Ser Asn Cys Ser lie Thr Ser lie Cys 405 410 415 Glu Lys Pro Gin Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu 420 425 430 Asn lie Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His 435 440 445 Asp Phe lie Leu Glu Asp Ala Ala Ser Pro Lys Cys lie Met Lys Glu 450 455 460 Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp 465 470 475 480 Glu Cys Asn Asp Asn lie lie Phe Ser Glu Glu Tyr Asn Thr Ser Asn 485 490 495 Pro Asp

Claims

1. A polypeptide comprising, from N- to C-terminus, a) at least one heavy chain variable region of a heavy chain antibody (VHH domain) that binds to human, cynomolgus or mouse Programmed Death Ligand 1 protein (PD-L1) operably linked to an IgG Fc binding domain; and b) human TGFβRII or a fragment thereof capable of binding to TGFβ1 ; the amino acid sequence of the polypeptide is SEQ ID NO:

7.

2. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of claim 1.

3. A cloning or expression vector comprising the nucleic acid molecule of claim 2.

4. A cell comprising the nucleic acid molecule of claim 2 or one or more cloning or expression vectors of claim 3.

5. A method of producing the polypeptide of claim 1, comprising culturing the cell of claim 4 and isolating the polypeptide.

6. A pharmaceutical composition comprising the polypeptide of claim 1, and one or more of a pharmaceutically acceptable excipient, diluent, and carrier.

7. Use of the polypeptide of claim 1 in the manufacture of a medicament for treating a tumor or inhibiting the growth of tumor cells, the tumor being a colorectal tumor, the tumor cells being colorectal tumor cells.

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