Multifunctional antibodies

Abstract

The present invention relates to molecules comprising a tumor antigen-binding protein and the extracellular domain (ECD) or ligand-binding fragment of a transformed growth factor β receptor (TGFβR), as well as compositions and uses thereof.

Description

Technical Field

[0001] The present invention relates to molecules, compositions, and methods for treating cancer.

[0002] Related Applications This application claims the priority of Australian Provisional Application No. 2023 / 900642, the entire content of which is incorporated herein by reference.

Background Art

[0003] Radiation and other treatments for cancer can destroy cells and trigger inflammation, which initiates an immune response. Compensatory mechanisms exist in the body to promote wound healing and limit inflammation after an assault such as radiation. These mechanisms can be important in normal tissues, but in cancer tissues, these mechanisms can prematurely block the anti-tumor immune response and also make the tumor resistant to subsequent rounds of radiation.

[0004] There remains a need for new or improved molecules and methods for treating cancer.

[0005] Any reference to prior art herein does not admit or suggest that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art is regarded as relevant and / or reasonably foreseeable to be combined with other prior art by a person skilled in the art.

Summary of the Invention

[0006] The present invention relates to a molecule comprising a first portion in the form of a tumor antigen-binding protein and a second portion in the form of an extracellular domain (ECD) of a transforming growth factor β receptor (TGFβR) or a ligand-binding fragment.

[0007] As described herein, the tumor antigen-binding protein is (i) single chain Fv fragment (scFv), (ii) Dimer scFv (di-scFv), (iii) It may be in one form of (i) or (ii) linked to the constant region, Fc, or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

[0008] Furthermore, as described herein, antigen-binding proteins are (i) Diabody, (ii) Triabody, (iii) Tetrabody, (iv)Fab, (v)F(ab')2, (vi)Fv, (vii) Bispecific antibodies or other forms of multispecific antibodies, (viii) The antibody may be in one of the forms of (i) to (vii) linked to the constant region, Fc, or heavy chain constant domain (CH)2 and / or CH3.

[0009] Therefore, in any embodiment, the molecule may include an immunoglobulin, an antibody, a bispecific or multispecific antibody, an antibody fragment, a single-strand variable fragment (scFv), a bivalent or polyvalent scFv, or a tumor antigen-binding protein in the form of an antigen-binding domain of an Fc-containing polypeptide.

[0010] In any embodiment, the tumor antigen-binding protein is an antibody or an antigen-binding fragment thereof, and the ECD or ligand-binding fragment of TGFβR is ligated to the C-terminus of the heavy chain of the antibody or the antigen-binding fragment.

[0011] Alternatively, the tumor antigen-binding protein may be an antibody or its antigen-binding fragment, and the ECD or ligand-binding fragment of TGFβR may be ligated to the C-terminus of the light chain of the antibody or its antigen-binding fragment.

[0012] Alternatively, the tumor antigen-binding protein may be an antibody or its antigen-binding fragment, and the ECD or ligand-binding fragment of TGFβR is ligated to the C-terminus of the heavy chain of the antibody or its antigen-binding fragment.

[0013] In alternative embodiments, the ECD or ligand-binding fragment of TGFβR may be linked to a non-antigen-binding region of any tumor antigen-binding protein. For example, in cases where the tumor antigen-binding protein is an antibody or Fab, the ECD or ligand-binding fragment of TGFβR may be linked to an amino acid in any region of either the constant region or a variable domain that is not directly involved in antigen binding of the protein.

[0014] In any embodiment, the ECD or ligand-binding fragment of TGFβR may be directly linked to a tumor antigen-binding protein, or it may be linked via a peptide linker, a carbohydrate (e.g., a polyethylene glycol-based linker or similar), or a chemical conjugation.

[0015] It is understood that the tumor antigen-binding protein of the molecule may bind to any antigen that is associated with or specific to tumor cells, as further described herein. Non-limiting examples of tumor cell-associated antigens (TAAs) or antigens specific to them (TSAs) include 17-IA antigen, alpha-fetoprotein (AFP), alpha-actinin 4, antigens specific to A3, A33 antibodies, ART-4, B7, Ba 733, BAGE, bcl-2, bcl-6, BCMA, BrE3 antigen, CA125, CAMEL, CAP-1, carbonate anhydrase IX (CAIX), CASP-8 / m, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, C D70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD138, CD147, CD154, CD1 71, CDC27, CDK-4 / m, CDKN2A, CEA, CEACAM5, CEACAM6, complement factors (e.g., C3, C3a, C3b, C5a, and C5), colon-specific antigen p (CSA p), c-Met, CTLA-4, CXCR4, CXCR7, DAM, Dickkopf-related protein (DKK), ED-B fibronectin, EGFR, EGFRvIII, EGP-1(T ROP-2), EGP-2, ELF2-M, Ep-CAM, EphA2, EphA3, fibroblast-activating protein (FAP), fibroblast growth factor (FGF), Flt-1, Flt-3, folate-binding protein, folate receptor, G250 antigen, ganglioside (e.g., GC2, GD3, and GM2), GAGE, GD2, gplOO, GPC3, GRO-13, HLA-DR, HM1.24. Human chorionic gonadotropin (HCG) and its subunits, HER2, HER3, HMGB-1, hypoxia-inducible factor (HIF-1), HIF-la, HSP70-2M, HST-2, la, IFN-gamma, IFN-alpha, IFN-beta, IFN-X, IL-4R, IL-6R, IL-13R, IL13R-alpha-2, IL-15R, IL-17R, IL-18R, ILGF, ILGF-1R, insulin IGF-1, IGF-1R, Integrin ανβ3, Integrin α5β1, KC4 antigen, Killer cell immunoglobulin-like receptor (KIR), Kras, KS-1 antigen, KS1-4, La / SSB, LDR / FUT, Le1, Macrophage migration inhibitor (MIF), MAGE, MAGE-3, MART-1, MART-2, mCRP, MCP-1, Melanoma glycoprotein, Mesothelin, MIP-1 Examples include A, MIP-1B, MIF, mucins (e.g., MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, and MUM-3), NCA66, NCA95, NCA90, NY-ESO-1, PAM4 antigen, pancreatic cancer mucin, PD-1, PD-L1, PD-1 receptor, placental growth factor, platelet-derived growth factor receptor alpha (PDGFRa), p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, RS5, RANTES, SAGE, 5100, Survivin, Survivin-2B, T101, TAC, TAG-72, tenascin, Thomson-Friedenreich antigen, Tn antigen, TNF-alpha, tumor necrosis antigen, TRAG-3, TRAIL receptor, VEGF, VEGFR, and WT-1. Therefore, it is understood that in any embodiment, the tumor antigen-binding protein may be one that is capable of specifically binding to any such antigen.

[0016] Optionally, the tumor antigen-binding protein may be selected from or may contain an antigen-binding domain, which is derived from any one of the following: APOMAB(DAB4), atezolizumab, avelumab, bevacizumab, semiprimab, cetuximab, dataumumab, dinutuximab, durvalumab, elotuzumab, girentuximab, ipilimumab, isatuximab, J591 or huJ591, mogamulizumab, nectimumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, pertizimab, ramucirumab, rituximab, or trastuzumab.

[0017] Preferably, the tumor antigen-binding protein is capable of binding to tumor antigens expressed by solid tumors.

[0018] In certain embodiments, the tumor antigen is not EGFR or EGFRvIII.

[0019] In certain embodiments, the tumor antigen is not PD-1 or PD-L1.

[0020] In preferred embodiments of the present invention, the tumor antigen-binding protein may be selected from those that bind to prostate-specific membrane antigen (PSMA), carbonate anhydrase IX (CAIX), PDGFRa, or La / SSB. Exemplary amino acid sequences of tumor antigen-binding proteins for binding to PSMA, CAIX, PDGFRα, and La / SSB are further described herein and include those in Tables 1, 2, 5, and 6, respectively.

[0021] In any embodiment, the second portion of the molecule is in the form of the extracellular domain (ECD) or ligand-binding fragment of a transformed growth factor β receptor (TGFβR), where TGFβR is type I (TGFβRI), type II (TGFβRII), or type III (TGFβRIII)TGFβR. In a preferred embodiment, the second portion of the molecule comprises an amino acid sequence derived from or containing the ECD or ligand-binding domain of type II TGFβR (TGFβRII).

[0022] Exemplary amino acid sequences of TGFβR and its isoforms' ECDs and their fragments are further described herein and include those shown in Table 3.

[0023] In one embodiment, the tumor antigen-binding domain and the ECD or ligand-binding fragment of TGFβR are linked via a linker.

[0024] In any embodiment, any molecule of the present invention may be conjugated to a therapeutic agent. The therapeutic agent may be conjugated to the molecule directly or indirectly, for example, by halogenation of an amino acid residue. Preferably, the therapeutic agent is indirectly conjugated to the molecule via a linker or chelator moiety. In one example, the molecule is conjugated to a chelating moiety, which is selected from the group consisting of TMT (6,6''-bis[N,N'',N'''-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2''-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N''(N'''-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, Diamsar, DTPA, CHX-A''-DTPA, TETE, Te2A, HBED, DFO, DFOsq, and HOPO, or other chelating agents described herein. In a preferred embodiment, the therapeutic agent is a radionuclide.

[0025] In some embodiments, the radionuclide can be an alpha-emitting radionuclide or a beta-emitting radionuclide. As used herein, the terms radioactive isotope, radioisotope, radionuclide, and radioactive nuclide can be used interchangeably.

[0026] The radionuclide can be selected from the group consisting of actinium 225 (225Ac), astatine 211 (211At), bismuth 212 and bismuth 213 ( 212 Bi, 213 Bi), copper 67 ( 67 Cu), iodine 123, 124, 125, or 131 ( 123 I, 124 I, 125 I, 131 I)( 123 I), lead 212 ( 212 Pb), lutetium 177 ( 177 Lu), radium 223 and radium 224 ( 223 Ra, 224 Ra), samarium 153 ( 153 Sm), scandium 47 ( 47 Sc), strontium 90 ( 90 Sr), and yttrium 90 ( 90 Y). In some embodiments, the radionuclide conjugated to the molecule is lutetium 177 .

[0027] In some embodiments, the therapeutic agent, preferably the radioactive isotope, can be conjugated to the tumor antigen-binding protein portion of the molecule. In alternative or additional embodiments, the therapeutic agent, preferably the radioactive isotope, can be conjugated to a portion of a molecule that includes the ECD or ligand-binding fragment of TGFβR. In alternative embodiments, the therapeutic agent, preferably the radioactive isotope, can be conjugated to the molecule in a non-site-specific manner.

[0028] The present invention also provides a bioconjugate molecule comprising a first portion in the form of a tumor antigen-binding protein and a second portion in the form of the extracellular domain (ECD) or ligand-binding fragment of a transforming growth factor β receptor (TGFβR), which is suitable for radiolabeling of therapeutic agents, preferably therapeutic radionuclides.

[0029] In one embodiment, the present invention provides a bioconjugate comprising a molecule of the present invention conjugated to any chelate moiety or linker group suitable for further conjugation to a radionuclide. In particular, the chelating agent or linker group can indirectly conjugate the radionuclide to the molecule.

[0030] Optional chelating agents include TMT (6,6''-bis[N,N'',N'''-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2''-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N'' (N'''-tetraacetic acid, also known as tetraxetan), TCMC (tetraprimary amide of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide), CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), NOTA (1,4,7-triazacyclononane-triacetic acid)Diamsar(3,6,10,13,16 Selected from the group consisting of ,19-hexaazabicyclo[6.6.6]icosane-1,8-diamine), DTPA (pentetic acid or diethylenetriaminepentaacetic acid), CHX-A''-DTPA ([(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diaminepentaacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8), 11-tetraacetic acid, Te2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), HBED, DFO (desferrioxamine), DFOsq (DFO-squalamide), and HOPO (3,4,3-(LI-1,2-HOPO)), or any other chelating agent described herein.

[0031] In any embodiment, the bioconjugate comprises the molecule of the present invention conjugated to the chelated portion, the chelated portion being selected from the group consisting of TMT(6,6''-bis[N,N'',N'''-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2''-terpyridine), DOTA(1,4,7,10-tetraazacyclododecane-NN',N''(N'''-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, Diamsar, DTPA, CHX-A''-DTPA, TETE, Te2A, HBED, DFO, DFOsq, and HOPO).

[0032] In any embodiment, the bioconjugate includes the molecule of the present invention conjugated to a bifunctional linker, such as bromoacetyl, thiol, succinimide ester, TFP ester, or maleimide, or the molecule of the present invention conjugated using any amine or thiol modification chemistry known in the art.

[0033] In any embodiment, the chelating agent or linker group may be conjugated to the tumor antigen-binding protein portion of the molecule. In alternative or additional embodiments, the chelating agent or linker group may be conjugated to the portion of the molecule containing the ECD or ligand-binding fragment of TGFβR.

[0034] The present invention also provides nucleic acids encoding molecules or bioconjugates, or components thereof, such as tumor antigen-binding proteins, immunoglobulin variable domains, antibodies, dab, discFv, scFv, Fab, Fab', F(ab')2, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, or multispecific antibodies, fusion proteins, or conjugates as described herein.

[0035] For example, such a nucleic acid is contained in an expression construct in which the nucleic acid is operably ligated to a promoter. Such an expression construct may be contained within a vector, such as a plasmid.

[0036] In the example of the present invention involving a single polypeptide chain antigen-binding protein, the expression construct may include a promoter linked to the nucleic acid encoding the polypeptide chain.

[0037] In examples involving multiple polypeptide chains forming an antigen-binding protein, the expression construct includes, for example, a nucleic acid encoding a polypeptide containing VH operably linked to a promoter, and for example, a nucleic acid encoding a polypeptide containing VL operably linked to a promoter.

[0038] In another example, an expression construct is, for instance, a operably linked component such as: (i) promoter (ii) Nucleic acid encoding the first polypeptide, (iii) Internal ribosome entry site, and (iv) Nucleic acid encoding a second polypeptide, This is a bisistron expression construct containing the elements in the order from 5' to 3', The first polypeptide comprises VH (and optionally, the extracellular domain (ECD) or ligand-binding fragment of TGFβR), and the second polypeptide comprises VL (and optionally, the extracellular domain (ECD) or ligand-binding fragment of TGFβR), or vice versa.

[0039] The present invention also envisions separate expression constructs, one of which encodes a first polypeptide comprising VH, and the other of which encodes a second polypeptide comprising VL. For example, the present invention also envisions (i) A first expression construct comprising a nucleic acid that encodes a polypeptide containing VH operably linked to a promoter, and optionally further encodes a polypeptide containing the extracellular domain (ECD) or ligand-binding fragment of TGFβR, (ii) A composition comprising a second expression construct comprising a nucleic acid encoding a polypeptide containing a VL operably linked to a promoter (optionally further encoding a polypeptide containing the extracellular domain (ECD) or ligand-binding fragment of TGFβR).

[0040] The present invention provides cells comprising a vector or nucleic acid as described herein. Preferably, the cells are isolated, substantially purified, or recombinant. In one example, the cells comprise an expression construct of the present invention, or (i) A first expression construct comprising a nucleic acid encoding a polypeptide containing VH operably linked to a promoter (optionally further encoding a polypeptide containing the extracellular domain (ECD) or ligand-binding fragment of TGFβR), (ii) A second expression construct comprising a nucleic acid encoding a polypeptide containing a VL operably linked to a promoter (optionally further encoding a polypeptide containing the extracellular domain (ECD) or ligand-binding fragment of TGFβR), The first and second polypeptides associate to form the molecule of the present invention.

[0041] Examples of cells of the present invention include bacterial cells, yeast cells, insect cells, or mammalian cells.

[0042] The present invention also provides compositions comprising the above-mentioned molecules of the present invention. Optionally, the compositions may comprise one or more pharmaceutically acceptable carriers or excipients.

[0043] In one aspect, the present invention provides a composition comprising the molecule of the present invention and a treatment for cancer, wherein the treatment for cancer is suspected to cause, or is known to cause, an increase in TGFβ activity in the tumor microenvironment when administered to a subject.

[0044] Preferably, the treatment for cancer is an antibody or antibody fragment conjugated with a radionuclide, optionally, that binds to or specifically binds to an antigen expressed by cancer. In such embodiments, the antibody or antibody fragment may be for binding to the same cancer antigen as the molecule of the present invention.

[0045] For example, in one embodiment, the composition comprises i) a molecule comprising a first portion in the form of a tumor antigen-binding protein for binding to CAIX and a second portion in the form of the extracellular domain (ECD) of a transforming growth factor β receptor (TGFβR), and ii) an antibody or its antigen-binding fragment for binding to CAIX. Optionally, the molecule in i) contains a radionuclide, but the antibody in ii) does not. Alternatively, the molecule in i) does not contain a radionuclide, and the antibody in ii) contains a radionuclide. Alternatively, the molecule in i) and the antibody in ii) each contain a radionuclide.

[0046] In another example, in one embodiment, the composition comprises: i) a molecule comprising a first portion in the form of a tumor antigen-binding protein for binding to PSMA and a second portion in the form of the extracellular domain (ECD) of a transforming growth factor β receptor (TGFβR); and ii) an antibody or its antigen-binding fragment for binding to PSMA. Optionally, the molecule in i) contains a radionuclide, but the antibody in ii) does not. Alternatively, the molecule in i) does not contain a radionuclide, and the antibody in ii) contains a radionuclide. Alternatively, the molecule in i) and the antibody in ii) each contain a radionuclide.

[0047] In another example, in one embodiment, the composition comprises: i) a molecule comprising a first portion in the form of a tumor antigen-binding protein for binding to PDGFRα and a second portion in the form of the extracellular domain (ECD) of a transforming growth factor β receptor (TGFβR); and ii) an antibody or its antigen-binding fragment for binding to PDGFRα. Optionally, the molecule in i) contains a radionuclide, but the antibody in ii) does not. Alternatively, the molecule in i) does not contain a radionuclide, and the antibody in ii) contains a radionuclide. Alternatively, the molecule in i) and the antibody in ii) each contain a radionuclide.

[0048] In another example, in one embodiment, the composition comprises: i) a molecule comprising a first portion in the form of a tumor antigen-binding protein for binding to La / SSB and a second portion in the form of the extracellular domain (ECD) of a transforming growth factor β receptor (TGFβR); and ii) an antibody or its antigen-binding fragment for binding to LA / SSB. Optionally, the molecule in i) contains a radionuclide, but the antibody in ii) does not. Alternatively, the molecule in i) does not contain a radionuclide, and the antibody in ii) contains a radionuclide. Alternatively, the molecule in i) and the antibody in ii) each contain a radionuclide.

[0049] The present invention also provides various methods and uses of the molecules and compositions described herein.

[0050] In one embodiment, the present invention provides a method for treating, preventing, or minimizing the progression of cancer in a subject, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to an antigen expressed by cancer, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating, preventing, or minimizing the progression of cancer in the subject. Optionally, the molecule may be conjugated with a radionuclide.

[0051] The present invention also provides various uses of the molecules and compositions described herein. The present invention also provides a method for treating, preventing or minimizing the progression of cancer in a subject, comprising administering to the subject a composition or molecule of the present invention, wherein the composition or molecule comprises an antigen-binding protein that binds to or specifically binds to an antigen expressed by cancer, and the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating, preventing or minimizing the progression of cancer in the subject. Optionally, the molecule may be conjugated with a radionuclide.

[0052] The present invention further provides a method for inhibiting TGFβ activity in a subject having cancer, comprising administering the subject a molecule or composition of the present invention, thereby inhibiting TGFβ activity in cancer. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radionuclide (MTR) treatment, cell therapy, such as CAR T therapy. Optionally, the subject may have received combination therapy for cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Combination therapy for cancer may be selected from the group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, tumor surgery or resection, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy. Optionally, the method may include administering subsequent therapy for cancer, which may be selected from the group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, tumor surgery or resection, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy.

[0053] The present invention provides a method for inhibiting or preventing cancer treatment-associated fibrosis in a subject, the method comprising administering a molecule or composition of the present invention, thereby treating cancer in the subject, and optionally further comprising the method wherein the subject has previously received treatment for cancer, and the prior treatment for cancer increased the expression of TGFβ in the subject. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecular targeted radiation (MTR) treatment, cell therapy, such as CAR T therapy.

[0054] In a particularly preferred embodiment, the present invention provides a method for reducing or inhibiting radioactive TGFβ activity in a subject receiving or requiring radiotherapy for cancer, comprising administering the subject a molecule or composition of the present invention, thereby reducing or inhibiting radioactive TGFβ activity in the subject. Radiotherapy may be provided in the form of external beam radiation or molecularly targeted radiation. In any embodiment, the molecularly targeted radiation source is a molecule or composition of the present invention.

[0055] In further embodiments, the present invention provides a method for enhancing or increasing the likelihood of success of treatment with an immune checkpoint inhibitor in a subject, comprising administering to the subject a molecule or composition of the present invention, thereby enhancing or increasing the likelihood of success of treatment with an immune checkpoint inhibitor in the subject. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for cancer, which may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radiation (MTR) treatment, and cell therapy, such as CAR T therapy.

[0056] The present invention also provides a method for treating, preventing, or minimizing the progression of cancer characterized by the expression of carbonate anhydrase IX (CAIX) in a subject, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to CAIX, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating, preventing, or minimizing the progression of cancer in the subject. Optionally, the molecule may be conjugated with a radionuclide.

[0057] The present invention also provides a method for treating, preventing, or minimizing the progression of cancer characterized by the expression of prostate-specific membrane antigen (PSMA) in a subject, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to PSMA, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating, preventing, or minimizing the progression of cancer in the subject. Optionally, the molecule may be conjugated with a radionuclide.

[0058] The present invention also provides a method for treating, preventing, or minimizing the progression of cancer characterized by the expression of platelet-derived growth factor receptor alpha (PDGFRα) in a subject, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to PDGFRα, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating, preventing, or minimizing the progression of cancer in the subject. Optionally, the molecule may be conjugated with a radionuclide.

[0059] The present invention also provides a method for treating, preventing, or minimizing the progression of cancer characterized by the expression of La / SSB protein in a subject, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to La / SSB, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating, preventing, or minimizing the progression of cancer in the subject. Optionally, the molecule may be conjugated with a radionuclide.

[0060] The present invention provides a method for inhibiting TGFβ activity in a subject having cancer characterized by the expression of carbonate anhydrase IX (CAIX), comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to carbonate anhydrase IX (CAIX), wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby further providing a method for inhibiting TGFβ activity in cancer. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radionuclide (MTR) treatment, cell therapy, e.g., CAR T therapy. At the discretion of the subjects, the subjects may be receiving combination therapy for cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Combination therapy for cancer may be selected from a group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy. At the discretion of the subjects, the subjects may be administered subsequent therapy for cancer, which may be selected from a group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy.

[0061] The present invention provides a method for inhibiting or preventing cancer treatment-associated fibrosis in a subject, comprising administering a molecule comprising an antigen-binding protein that binds to or specifically binds to carbonate anhydrase IX (CAIX), wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating cancer in the subject, and optionally further comprising a method wherein the subject has previously received treatment for cancer, and the prior treatment for cancer has increased the expression of TGFβ in the subject. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agent including CPI, molecular targeted radiation (MTR) treatment, cell therapy, such as CAR T therapy.

[0062] In a particularly preferred embodiment, the present invention provides a method for reducing or inhibiting radioactive TGFβ activity in a subject receiving or requiring radiotherapy for cancer, comprising administering a molecule comprising an antigen-binding protein that binds to or specifically binds to carbonate anhydrase IX (CAIX), wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby reducing or inhibiting radioactive TGFβ activity in the subject. Radiotherapy may be provided in the form of external beam radiation or molecularly targeted radiation. In any embodiment, the molecularly targeted radiation source is the molecule or composition of the present invention.

[0063] In further embodiments, the present invention provides a method for enhancing or increasing the likelihood of success in treatment with an immune checkpoint inhibitor in a subject, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to carbonate anhydrase IX (CAIX), wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby enhancing or increasing the likelihood of success in treatment with an immune checkpoint inhibitor in the subject. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for cancer, which may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radiation (MTR) treatment, cell therapy, such as CAR T therapy.

[0064] The present invention further provides a method for inhibiting TGFβ activity in a subject having cancer characterized by PSMA expression, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to PSMA, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby inhibiting TGFβ activity in the cancer. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for the cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Prior treatment for the cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radionuclides (MTRs), and cell therapy, such as CAR T therapy. At the discretion of the subjects, the subjects may be receiving combination therapy for cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Combination therapy for cancer may be selected from a group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy. At the discretion of the subjects, the subjects may be administered subsequent therapy for cancer, which may be selected from a group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy.

[0065] The present invention provides a method for inhibiting or preventing cancer treatment-associated fibrosis in a subject, comprising administering a molecule comprising an antigen-binding protein that binds to or specifically binds to prostate-specific membrane antigen (PSMA), wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating cancer in the subject, optionally further comprising a method in which the subject has previously received treatment for cancer, and the prior treatment for cancer has increased the expression of TGFβ in the subject. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agent including CPI, molecular targeted radiation (MTR) treatment, cell therapy, such as CAR T therapy.

[0066] In a particularly preferred embodiment, the present invention provides a method for reducing or inhibiting radioactive TGFβ activity in a subject receiving or requiring radiotherapy for cancer, comprising administering a molecule comprising an antigen-binding protein that binds to or specifically binds to prostate-specific membrane antigen (PSMA), wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby reducing or inhibiting radioactive TGFβ activity in the subject. Radiotherapy may be provided in the form of external beam radiation or molecularly targeted radiation. In any embodiment, the molecularly targeted radiation source is the molecule or composition of the present invention.

[0067] In further embodiments, the present invention provides a method for enhancing or increasing the likelihood of success in treatment with an immune checkpoint inhibitor in a subject, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to prostate-specific membrane antigen (PSMA), wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby enhancing or increasing the likelihood of success in treatment with an immune checkpoint inhibitor in the subject. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for cancer, which may be selected from the group consisting of external beam radiation (EBR) therapy, chemotherapy, tumor surgery or resection, immunomodulatory therapy including CPI, molecularly targeted radiation (MTR) therapy, cell therapy, such as CAR T therapy.

[0068] The present invention further provides a method for inhibiting TGFβ activity in a subject having cancer characterized by PDGFRα expression, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to PDGFRα, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby inhibiting TGFβ activity in the cancer. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for the cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Prior treatment for the cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radionuclides (MTRs), and cell therapy, such as CAR T therapy. At the discretion of the subjects, the subjects may be receiving combination therapy for cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Combination therapy for cancer may be selected from a group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy. At the discretion of the subjects, the subjects may be administered subsequent therapy for cancer, which may be selected from a group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy.

[0069] The present invention provides a method for inhibiting or preventing cancer treatment-associated fibrosis in a subject, comprising administering a molecule comprising an antigen-binding protein that binds to or specifically binds to PDGFRα, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating cancer in the subject, and optionally further comprising a subject that has received prior treatment for cancer, the prior treatment for cancer increasing the expression of TGFβ in the subject. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecular targeted radiation (MTR) treatment, cell therapy, such as CAR T therapy.

[0070] In a particularly preferred embodiment, the present invention provides a method for reducing or inhibiting radioactive TGFβ activity in a subject receiving or requiring radiotherapy for cancer, comprising administering a molecule comprising an antigen-binding protein that binds to or specifically binds to PDGFRα, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby reducing or inhibiting radioactive TGFβ activity in the subject. Radiotherapy may be provided in the form of external beam radiation or molecularly targeted radiation. In any embodiment, the molecularly targeted radiation source is the molecule or composition of the present invention.

[0071] In further embodiments, the present invention provides a method for enhancing or increasing the likelihood of success of treatment with an immune checkpoint inhibitor in a subject, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to PDGFRα, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby enhancing or increasing the likelihood of success of treatment with an immune checkpoint inhibitor in the subject. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for cancer, which may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radiation (MTR) treatment, cell therapy, such as CAR T therapy.

[0072] The present invention further provides a method for inhibiting TGFβ activity in a subject having cancer characterized by La / SSB expression, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to La / SSB, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby inhibiting TGFβ activity in the cancer. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for the cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radionuclides (MTRs), and cell therapy, such as CAR T therapy. At the discretion of the subjects, the subjects may be receiving combination therapy for cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Combination therapy for cancer may be selected from a group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy. At the discretion of the subjects, the subjects may be administered subsequent therapy for cancer, which may be selected from a group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, such as CAR T therapy.

[0073] The present invention provides a method for inhibiting or preventing cancer treatment-associated fibrosis in a subject, comprising administering a molecule comprising an antigen-binding protein that binds to or specifically binds to La / SSB, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby treating cancer in the subject, optionally wherein the subject has previously received treatment for cancer, and the prior treatment for cancer has increased the expression of TGFβ in the subject. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radiation (MTR) treatment, cell therapy, such as CAR T therapy.

[0074] In any embodiment, the composition or molecule of the present invention may be used as a neoadjuvant for administration to a patient prior to treatment in the therapies described herein.

[0075] In any embodiment, the composition or molecule of the present invention may be used as an adjuvant for administration to a patient after treatment with the therapy described herein.

[0076] In a particularly preferred embodiment, the present invention provides a method for reducing or inhibiting radioactive TGFβ activity in a subject receiving or requiring radiotherapy for cancer, comprising administering a molecule comprising an antigen-binding protein that binds to or specifically binds to La / SSB, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby reducing or inhibiting radioactive TGFβ activity in the subject. Radiotherapy may be provided in the form of external beam radiation or molecularly targeted radiation. In any embodiment, the molecularly targeted radiation source is the molecule or composition of the present invention.

[0077] In further embodiments, the present invention provides a method for enhancing or increasing the likelihood of success of treatment with an immune checkpoint inhibitor in a subject, comprising administering to the subject a molecule comprising an antigen-binding protein that binds to or specifically binds to La / SSB, wherein the molecule further comprises an ECD or ligand-binding fragment of TGFβR, thereby enhancing or increasing the likelihood of success of treatment with an immune checkpoint inhibitor in the subject. Optionally, the cancer may have high levels of baseline TGFβ expression or activity in the tumor microenvironment. Optionally, the subject may have received prior treatment for cancer, which may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radiation (MTR) treatment, cell therapy, such as CAR T therapy.

[0078] In any embodiment, the antigen-binding protein for binding to CAIX, PSMA, PDGFRα, or LA / SSB may be one of those further described herein. In any aspect or embodiment described herein, the antigen-binding protein that binds to or specifically binds to carbonate anhydrase IX (CAIX) may be an antibody against CAIX. In some embodiments, the anti-CAIX antibody is gylenetuximab or a derivative thereof, and gylenetuximab or a derivative thereof includes a humanized form of gylenetuximab. In some embodiments, the antigen-binding protein that binds to or specifically binds to PSMA may be derived from J591 or huJ591, which are further described herein. In some embodiments, the antigen-binding protein that binds to PDGFRα is olaratumab or a derivative thereof. In some embodiments, the antigen-binding protein that binds to or specifically binds to LA / SSB may be APOMAB or a derivative thereof, and APOMAB or a derivative thereof includes a humanized form of APOMAB.

[0079] In any embodiment, the method further comprises administering a treatment for cancer, wherein the treatment for cancer is suspected to, or is known to, cause an increase in TGFβ activity in the tumor microenvironment when administered to a subject. In a preferred embodiment, the treatment for cancer is a molecularly targeted radionuclide (MTR), for example, in the form of an antibody or antibody fragment conjugated with a radionuclide that binds to or specifically binds to an antigen expressed by cancer. Optionally, the molecules of the present invention do not contain radionuclides.

[0080] Therefore, in further embodiments, the present invention is -Administering the molecule or composition of the present invention to a subject, optionally, the molecule being administered does not contain a radionuclide. - The administration of a cancer treatment to a subject may include administering a cancer treatment that is suspected or known to cause an increase in TGFβ activity in the tumor microenvironment, preferably the cancer treatment being molecularly targeted radiation (MTR), for example, in the form of an antibody or antibody fragment conjugated with a radionuclide that binds to or specifically binds to an antigen expressed by cancer.

[0081] In some embodiments, the antibody or an antibody fragment may be intended to bind to the same cancer antigen as the molecule of the present invention.

[0082] For example, in one embodiment, the method comprises administering a molecule comprising i) a first portion in the form of a tumor antigen-binding protein for binding to CAIX and a second portion in the form of the extracellular domain (ECD) of a transforming growth factor β receptor (TGFβR), and ii) an antibody or its antigen-binding fragment for binding to CAIX. Optionally, the molecule in i) contains a radionuclide, while the antibody in ii) does not. Alternatively, the molecule in i) does not contain a radionuclide, and the antibody in ii) contains a radionuclide.

[0083] In another example, in one embodiment, the method comprises administering a molecule comprising i) a first portion in the form of a tumor antigen-binding protein for binding to PSMA and a second portion in the form of the extracellular domain (ECD) of a transforming growth factor β receptor (TGFβR), and ii) an antibody or its antigen-binding fragment for binding to PSMA. Optionally, the molecule in i) contains a radionuclide, but the antibody in ii) does not. Alternatively, the molecule in i) does not contain a radionuclide, and the antibody in ii) contains a radionuclide. Alternatively, the molecule in i) and the antibody in ii) each contain a radionuclide.

[0084] In another example, in one embodiment, the method comprises administering i) a molecule comprising a first portion in the form of a tumor antigen-binding protein for binding to PDGFRα and a second portion in the form of the extracellular domain (ECD) of a transforming growth factor β receptor (TGFβR), and ii) an antibody or its antigen-binding fragment for binding to PDGFRα. Optionally, the molecule in i) contains a radionuclide, but the antibody in ii) does not. Alternatively, the molecule in i) does not contain a radionuclide, and the antibody in ii) contains a radionuclide. Alternatively, the molecule in i) and the antibody in ii) each contain a radionuclide.

[0085] In another example, in one embodiment, the method comprises administering i) a molecule comprising a first portion in the form of a tumor antigen-binding protein for binding to La / SSB and a second portion in the form of the extracellular domain (ECD) of a transforming growth factor β receptor (TGFβR), and ii) an antibody or its antigen-binding fragment for binding to LA / SSB. Optionally, the molecule in i) contains a radionuclide, but the antibody in ii) does not. Alternatively, the molecule in i) does not contain a radionuclide, and the antibody in ii) contains a radionuclide. Alternatively, the molecule in i) and the antibody in ii) each contain a radionuclide.

[0086] In any embodiment or aspect described herein, the immune checkpoint inhibitor (CPI) may be a PD-1, PD-L1, CTLA-4, TIGIT, VISTA, LAG-3, TIM-3, or CD47 checkpoint inhibitor. In any embodiment, the checkpoint inhibitor may be an antibody or its antigen-binding fragment, a protein, a peptide, or a small molecule. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4 TIGIT, VISTA, LAG-3, TIM-3, or CD47 in the form of an antibody or its antigen-binding fragment. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4 TIGIT, VISTA, LAG-3, TIM-3, or CD47 in the form of a peptide. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, immune checkpoint inhibitor therapy involves administering inhibitors of PD-1 and CTLA-4. In some embodiments, the checkpoint inhibitor is an inhibitor of TIGIT. In some embodiments, the checkpoint inhibitor is an inhibitor of VISTA. In some embodiments, the checkpoint inhibitor is an inhibitor of LAG-3. In some embodiments, the checkpoint inhibitor is an inhibitor of TIM-3. In some embodiments, the checkpoint inhibitor is an inhibitor of CD47.

[0087] In any embodiment of the present invention, the method described herein further includes identifying a subject having cancer. In one embodiment, the cancer may be precancerous or non-metastatic. In another embodiment, the cancer may be malignant or metastatic.

[0088] In any embodiment of the present invention, the immune checkpoint inhibitor (CPI) may be a PD-1, PD-L1, or CTLA-4 checkpoint inhibitor. In any embodiment, the checkpoint inhibitor is an antibody. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4 in the form of an antibody. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, immune checkpoint inhibitor therapy involves administering inhibitors of PD-1 and CTLA-4.

[0089] The present invention also, ●Methods to treat, prevent, or minimize the progression of cancer in the target population. ● A method for inhibiting TGFβ activity in subjects with cancer. ● A method for reducing or inhibiting radioactive TGFβ activity in subjects who accept or require radiotherapy for cancer. ●Methods for inhibiting or preventing cancer treatment-related fibrosis in the subject, and / or ●Compositions or molecules described herein for use in methods to enhance or increase the likelihood of success of immune checkpoint inhibitor therapy in a subject, The present invention provides compositions or molecules comprising an antigen-binding protein that binds to or specifically binds to an antigen expressed by cancer, and further comprising an ECD or ligand-binding fragment of TGFβR. Optionally, the molecule may be conjugated to a radionuclide.

[0090] In any embodiment, the composition or molecule for use described above may be for use after the subject has received prior treatment for cancer, the prior treatment is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radionuclides (MTRs), cell therapy, for example, CAR T therapy.

[0091] In any embodiment, the composition or molecule for use described above may be for use prior to the subject receiving treatment for cancer, the treatment is suspected of causing or causes an increase in TGFβ activity in the tumor microenvironment. The treatment for cancer may be selected from the group consisting of treatment with external beam radiation (EBR), treatment with chemotherapeutic agents, surgery or resection of the tumor, treatment with immunomodulators including CPI, treatment with molecularly targeted radionuclides (MTRs), and cell therapy, such as treatment with CAR T therapy.

[0092] Optionally, use may be for the treatment of a patient who may be receiving combination therapy for cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Combination therapy for cancer may be selected from the group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, e.g., CAR T therapy. Optionally, use may be for the administration of subsequent therapy for cancer, which may be selected from the group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, e.g., CAR T therapy.

[0093] The present invention also, ● To treat, prevent, or minimize the progression of cancer in the target population. ●To inhibit TGFβ activity in subjects with cancer, ● To reduce or inhibit radioactive TGFβ activity in subjects who accept or require radiotherapy for cancer, ● To inhibit or prevent cancer treatment-related fibrosis in the target population, and / or ● Use of molecules or bioconjugates described herein for use in the manufacture of pharmaceuticals to enhance or increase the likelihood of success of treatment with immune checkpoint inhibitors in a target, The present invention provides for the use of the molecule or bioconjugate described herein, wherein the molecule or bioconjugate comprises an antigen-binding protein that binds to or specifically binds to an antigen expressed by cancer, and the molecule further comprises an ECD or ligand-binding fragment of TGFβR. Optionally, the molecule may be conjugated to a radionuclide.

[0094] The present invention also, ● To treat, prevent, or minimize the progression of cancer in the target population. ●To inhibit TGFβ activity in subjects with cancer, ● To reduce or inhibit radioactive TGFβ activity in subjects who accept or require radiotherapy for cancer, ● To inhibit or prevent cancer treatment-related fibrosis in the target population, and / or ●Compositions, bioconjugates, or molecules described herein for use in the manufacture of pharmaceuticals to enhance or increase the likelihood of success of immune checkpoint inhibitor therapy in a target, The present invention provides compositions, bioconjugates, or molecules comprising an antigen-binding protein that binds to or specifically binds to an antigen expressed by cancer, and further comprising an ECD or ligand-binding fragment of TGFβR. Optionally, the molecule may be conjugated to a radionuclide.

[0095] In any embodiment, the above-described pharmacopoeia may be for use after the subject has received prior treatment for cancer, the prior treatment is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Prior treatment for cancer may be selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, tumor surgery or resection, immunomodulatory agents including CPI, molecularly targeted radionuclide (MTR) treatment, cell therapy, for example, CAR T therapy.

[0096] In any embodiment, the above-described pharmacopoeia may be for use prior to the subject receiving treatment for cancer, and the treatment is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. The treatment for cancer may be selected from the group consisting of treatment with external beam radiation (EBR), treatment with chemotherapeutic agents, surgery or resection of the tumor, treatment with immunomodulators including CPI, treatment with molecularly targeted radionuclides (MTRs), and cell therapy, such as treatment with CAR T therapy.

[0097] Optionally, the drug may be for the treatment of a patient who may be receiving combination therapy for cancer, which is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment. Combination therapy for cancer may be selected from the group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, e.g., CAR T therapy. Optionally, the drug may be for the administration of subsequent therapy for cancer, which may be selected from the group consisting of external beam radiation (EBR) therapy, chemotherapy therapy, surgery or resection of the tumor, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, cell therapy, e.g., CAR T therapy.

[0098] In any embodiment of the present invention, which includes a tumor antigen-binding protein whose molecule specifically binds to CAIX, the antigen-binding protein is preferably, Includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, FR1, FR2, FR3, and FR4 are each framework domains. CDR1, CDR2, and CDR3 are complementarity determination regions, FR1a, FR2a, FR3a, and FR4a are each framework domains. CDR1a, CDR2a, and CDR3a are complementarity determination regions, One of the sequences in the complementarity-determining region has an amino acid sequence listed in Table 2.

[0099] Preferably, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked via a linker, optionally in the form of a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0100] In any embodiment, the tumor antigen-binding protein comprises a heavy chain HCDR1, HCDR2, HCDR3 containing the amino acid sequence described in any of SEQ ID NOs. 52, 68, 84, 100, or 116, and a light chain LCDR1, LCDR2, and LCDR3 containing the amino acid sequence described in any of SEQ ID NOs. 132, 148, 164, 180, 196, or 212. It is understood that the CDR boundaries and sequences can be determined by any preferred method known to those skilled in the art, including, but not limited to, the Kabat, Chothia, or IMGT methods further described herein.

[0101] Preferably, the tumor antigen-binding protein includes an antigen-binding domain, which essentially consists of or comprises the amino acid sequence of SEQ ID NOs. 52, 68, 84, 100, or 116 (in the order of N to C-terminus or C to N-terminus) and the sequence described in SEQ ID NOs. 132, 148, 164, 180, 196, or 212.

[0102] Preferably, the tumor antigen-binding protein is (i) VH including a complementarity determination region (CDR) 1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NOs. 49, 65, 81, 97, or 113, a CDR2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence set in SEQ ID NOs. 50, 66, 82, 98, or 114, and a CDR3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NOs. 51, 67, 83, 99, or 115. (ii) VH containing a sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the sequence described in sequence numbers 52, 68, 84, 100, or 116, (iii) A VL containing CDR1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NOs. 129, 145, 161, 177, 193, or 209; CDR2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NOs. 130, 146, 162, 178, 194, or 210; and CDR3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NOs. 131, 147, 163, 179, 195, or 211. (iv) VL containing a sequence that is at least approximately 95% identical to the sequence described in sequence numbers 132, 148, 164, 180, 196, or 212, (v) A VH containing a CDR1 containing the sequence described in SEQ ID NOs. 49, 65, 81, 97, or 113, a CDR2 containing the sequence described between SEQ ID NOs. 50, 66, 82, 98, or 114, and a CDR3 containing the sequence described in SEQ ID NOs. 51, 67, 83, 99, or 115. (vi) VH containing the sequence described in sequence numbers 52, 68, 84, 100, or 116, (vii) A VL containing a CDR1 containing the sequence set to sequence number 129, 145, 161, 177, 193, or 209, a CDR2 containing the sequence described in sequence number 130, 146, 162, 178, 194, or 210, and a CDR3 containing the sequence described in sequence number 131, 147, 163, 179, 195, or 211. (viii) VL containing the sequence described in sequence numbers 132, 148, 164, 180, 196, or 212, (ix) VH containing CDR1 containing the sequence described in SEQ ID NOs. 49, 65, 81, 97, or 113, CDR2 containing the sequence described between SEQ ID NOs. 50, 66, 82, 98, or 114, and CDR3 containing the sequence described in SEQ ID NOs. 51, 67, 83, 99, or 115, and VL containing CDR1 containing the sequence set in SEQ ID NOs. 129, 145, 161, 177, 193, or 209, CDR2 containing the sequence described in SEQ ID NOs. 130, 146, 162, 178, 194, or 210, and CDR3 containing the sequence described in SEQ ID NOs. 131, 147, 163, 179, 195, or 211, or (x) comprising at least one of VH containing the sequence described in sequence number 52, 68, 84, 100, or 116, and VL containing the sequence described in sequence number 132, 148, 164, 180, 196, or 212.

[0103] Preferably, the tumor antigen-binding protein includes a variable heavy chain that maintains binding affinity to CAIX and contains the amino acid sequence described in SEQ ID NO: 52, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0104] Preferably, the tumor antigen-binding protein includes a variable heavy chain that maintains binding affinity to CAIX and contains the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 52, and includes the amino acid sequence described in SEQ ID NO: 52, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0105] Preferably, the tumor antigen-binding protein includes a variable light chain that maintains binding affinity to CAIX and contains the amino acid sequence described in SEQ ID NO: 132, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0106] Preferably, the tumor antigen-binding protein includes a variable light chain that maintains binding affinity to CAIX and contains the same CDR sequence as defined for the variable light chain of SEQ ID NO: 132, and includes the amino acid sequence described in SEQ ID NO: 132, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0107] Preferably, the tumor antigen-binding protein includes a variable heavy chain containing the amino acid sequence described in SEQ ID NO: 52, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto, while maintaining binding affinity to CAIX; and a variable light chain containing the amino acid sequence described in SEQ ID NO: 132, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0108] Preferably, the tumor antigen-binding protein maintains binding affinity to CAIX and includes the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 52 and the variable light chain of SEQ ID NO: 132, while also containing the amino acid sequence described in SEQ ID NO: 52, or at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% of it. The variable heavy chain comprises a sequence that is identical by %, 97%, 98%, or 99%, and comprises a variable light chain comprising the amino acid sequence described in Sequence ID No. 132, or a sequence that is identical by at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0109] In any embodiment, the tumor antigen-binding protein further comprises a constant region of a heavy chain containing the amino acid sequence described in SEQ ID NO: 225, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0110] In any embodiment, the tumor antigen-binding protein further comprises a constant region of a light chain containing the amino acid sequence described in SEQ ID NO: 229, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0111] In embodiments of the present invention that include a tumor antigen-binding protein that specifically binds to PSMA, the antigen-binding protein is preferably: Includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, FR1, FR2, FR3, and FR4 are each framework domains. CDR1, CDR2, and CDR3 are complementarity determination regions, FR1a, FR2a, FR3a, and FR4a are each framework domains. CDR1a, CDR2a, and CDR3a are complementarity determination regions, One of the complementarity-determining regions has an amino acid sequence listed in Table 1.

[0112] Preferably, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked via a linker, optionally in the form of a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0113] In any embodiment, the tumor antigen-binding protein comprises a heavy chain HCDR1, HCDR2, HCDR3 containing the amino acid sequence described in any of SEQ ID NOs: 4, 20, or 244, and a light chain LCDR1, LCDR2, and LCDR3 containing the amino acid sequence described in any of SEQ ID NOs: 36 or 245. It is understood that the CDR boundaries and sequences can be determined by any preferred method known to those skilled in the art, including, but not limited to, the Kabat, Chothia, or IMGT methods further described herein.

[0114] Preferably, the tumor antigen-binding protein includes an antigen-binding domain, which is essentially composed of or consists of the amino acid sequences of SEQ ID NOs. 4, 20, or 244 and 36 or 245 (in the order of N to C-terminus or C to N-terminus).

[0115] Preferably, the tumor antigen-binding protein is (i) VH including a complementarity determination region (CDR) 1 containing a sequence that is at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% identical to the sequence described in SEQ ID NO: 1 or 17, a CDR2 containing a sequence that is at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% identical to the sequence set in SEQ ID NO: 2 or 18, and a CDR3 containing a sequence that is at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% identical to the sequence described in SEQ ID NO: 3 or 19. (ii) A VH containing a sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the sequence described in Sequence ID No. 4, 20, or 244. (iii) VL including CDR1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 33, CDR2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 34, and CDR3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 35. (iv) VL containing a sequence that is at least approximately 95% identical to the sequence described in SEQ ID NO: 36 or 245, (v) VH containing CDR1 containing the sequence described in SEQ ID NO: 1 or 17, CDR2 containing the sequence described between SEQ ID NOs: 2 or 18, and CDR3 containing the sequence described in SEQ ID NO: 3 or 19. (vi) VH containing the sequence described in sequence number 4, 20, or 244, (vii) VL containing CDR1 containing the sequence set in SEQ ID NO: 33, CDR2 containing the sequence described in SEQ ID NO: 34, and CDR3 containing the sequence described in SEQ ID NO: 45. (viii) VL containing the sequence described in sequence number 36 or 245, (ix) VH including CDR1 containing the sequence described in SEQ ID NO: 1 or 17, CDR2 containing the sequence described between SEQ ID NOs: 2 or 18, and CDR3 containing the sequence described in SEQ ID NO: 3 or 19, and VL including CDR1 containing the sequence set in SEQ ID NO: 33, CDR2 containing the sequence described in SEQ ID NO: 34, and CDR3 containing the sequence described in SEQ ID NO: 35, or (x) comprising at least one of VH containing the sequence described in SEQ ID NO: 4, 20, or 244, and VL containing the sequence described in SEQ ID NO: 36 or 245.

[0116] Preferably, the tumor antigen-binding protein includes a variable heavy chain that maintains binding affinity to PSMA and contains the amino acid sequence described in SEQ ID NO: 244, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0117] Preferably, the tumor antigen-binding protein includes a variable heavy chain that maintains binding affinity to PSMA and contains the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 244, and includes the amino acid sequence described in SEQ ID NO: 244, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0118] Preferably, the tumor antigen-binding protein includes a variable light chain that maintains binding affinity to PSMA and contains the amino acid sequence described in SEQ ID NO: 245, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0119] Preferably, the tumor antigen-binding protein includes a variable light chain that maintains binding affinity to PSMA and contains the same CDR sequence as defined for the variable light chain of SEQ ID NO: 245, and includes the amino acid sequence described in SEQ ID NO: 245, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0120] Preferably, the tumor antigen-binding protein includes a variable heavy chain containing the amino acid sequence described in SEQ ID NO: 244, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto, while maintaining binding affinity to PSMA; and a variable light chain containing the amino acid sequence described in SEQ ID NO: 245, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0121] Preferably, the tumor antigen-binding protein maintains binding affinity to PSMA and includes the same CDR sequences as defined for the variable heavy chain of SEQ ID NO: 244 and the variable light chain of SEQ ID NO: 245, and contains the amino acid sequence described in SEQ ID NO: 244, or at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 9% of it. The material comprises a variable heavy chain containing a sequence that is identical to 6%, 97%, 98%, or 99%, and a variable light chain containing the amino acid sequence described in Sequence ID No. 245, or a sequence that is identical to it by at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0122] In any embodiment of the present invention, which includes a tumor antigen-binding protein that specifically binds to PDGFRα, the antigen-binding protein is preferably: Includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, FR1, FR2, FR3, and FR4 are each framework domains. CDR1, CDR2, and CDR3 are complementarity determination regions, FR1a, FR2a, FR3a, and FR4a are each framework domains. CDR1a, CDR2a, and CDR3a are complementarity determination regions, One of the sequences in the complementarity-determining region has an amino acid sequence listed in Table 5.

[0123] Preferably, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked via a linker, optionally in the form of a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0124] In any embodiment, the tumor antigen-binding protein comprises a heavy chain HCDR1, HCDR2, HCDR3 having the amino acid sequence described in SEQ ID NO: 265, and a light chain LCDR1, LCDR2, LCDR3 having the amino acid sequence described in SEQ ID NO: 266. It is understood that the CDR boundaries and sequences can be determined by any preferred method known to those skilled in the art, including, but not limited to, the Kabat, Chothia, or IMGT methods further described herein.

[0125] Preferably, the tumor antigen-binding protein includes an antigen-binding domain, which is essentially composed of or consists of the amino acid sequence of SEQ ID NOs. 265 and / or 266 (in the order N to C-terminus or C to N-terminus).

[0126] Preferably, the tumor antigen-binding protein is (i) VH including a complementarity determination region (CDR) 1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 267, a CDR 2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence set in SEQ ID NO: 268, and a CDR 3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 269. (ii) A VH containing a sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the sequence described in Sequence ID No. 265. (iii) VL including CDR1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 270, CDR2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 271, and CDR3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 272. (iv) VL containing a sequence that is at least approximately 95% identical to the sequence described in SEQ ID NO: 266, (v) VH containing CDR1 containing the sequence described in SEQ ID NO: 267, CDR2 containing the sequence described in SEQ ID NO: 268, and CDR3 containing the sequence described in SEQ ID NO: 269. (vi) VH containing the sequence described in Sequence ID No. 265, (vii) VL containing CDR1 containing the sequence set in sequence number 270, CDR2 containing the sequence described in sequence number 271, and CDR3 containing the sequence described in sequence number 272. (viii) VL containing the sequence described in Sequence ID No. 266, (ix) VH containing CDR1 containing the sequence described in SEQ ID NO: 267, CDR2 containing the sequence described in SEQ ID NO: 268, and CDR3 containing the sequence described in SEQ ID NO: 269, and VL containing CDR1 containing the sequence set in SEQ ID NO: 270, CDR2 containing the sequence described in SEQ ID NO: 271, and CDR3 containing the sequence described in SEQ ID NO: 272, or (x) comprising at least one of VH containing the sequence described in SEQ ID NO: 265 and VL containing the sequence described in SEQ ID NO: 266.

[0127] Preferably, the tumor antigen-binding protein includes a variable heavy chain that maintains binding affinity to PDGFRα and contains the amino acid sequence described in SEQ ID NO: 265, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0128] Preferably, the tumor antigen-binding protein includes a variable heavy chain that maintains binding affinity to PDGFRα and contains the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 265, and includes the amino acid sequence described in SEQ ID NO: 265, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0129] Preferably, the tumor antigen-binding protein includes a variable light chain that maintains binding affinity to PDGFRα and contains the amino acid sequence described in SEQ ID NO: 266, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0130] Preferably, the tumor antigen-binding protein includes a variable light chain that maintains binding affinity to PDGFRα and contains the same CDR sequence as defined for the variable light chain of SEQ ID NO: 266, and includes the amino acid sequence described in SEQ ID NO: 266, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0131] Preferably, the tumor antigen-binding protein includes a variable heavy chain containing the amino acid sequence described in SEQ ID NO: 265, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto, while maintaining binding affinity to PDGFRα; and a variable light chain containing the amino acid sequence described in SEQ ID NO: 266, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0132] Preferably, the tumor antigen-binding protein maintains binding affinity to PDGFRα and includes the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 265 and the variable light chain of SEQ ID NO: 266, and contains the amino acid sequence described in SEQ ID NO: 265, or at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or A variable heavy chain comprising a sequence that is 96%, 97%, 98%, or 99% identical thereto, and a variable light chain comprising the amino acid sequence described in Sequence ID No. 266, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0133] In any embodiment of the present invention, which includes a tumor antigen-binding protein that specifically binds to La / SSB, the antigen-binding protein is preferably: Includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, FR1, FR2, FR3, and FR4 are each framework domains. CDR1, CDR2, and CDR3 are complementarity determination regions, FR1a, FR2a, FR3a, and FR4a are each framework domains. CDR1a, CDR2a, and CDR3a are complementarity determination regions, One of the sequences in the complementarity-determining region has an amino acid sequence listed in Table 6.

[0134] Preferably, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked via a linker, optionally in the form of a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0135] In any embodiment, the tumor antigen-binding protein comprises heavy chains HCDR1, HCDR2, and HCDR3 having the amino acid sequence described in SEQ ID NO: 303, and light chains LCDR1, LCDR2, and LCDR3 having the amino acid sequence described in SEQ ID NO: 304. It is understood that the CDR boundaries and sequences can be determined by any preferred method known to those skilled in the art, including, but not limited to, the Kabat, Chothia, or IMGT methods further described herein.

[0136] Preferably, the tumor antigen-binding protein includes an antigen-binding domain, which is essentially composed of or consists of the amino acid sequence of SEQ ID NOs. 303 and / or 304 (in the order N to C-terminus or C to N-terminus).

[0137] Preferably, the tumor antigen-binding protein is (i) VH including a complementarity determination region (CDR) 1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 305, a CDR 2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence set in SEQ ID NO: 306, and a CDR 3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 307, (ii) A VH containing a sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the sequence described in Sequence ID No. 303. (iii) VL including CDR1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 308, CDR2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 309, and CDR3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 310. (iv) VL containing a sequence that is at least approximately 95% identical to the sequence described in SEQ ID NO: 304, (v) VH containing CDR1 containing the sequence described in SEQ ID NO: 305, CDR2 containing the sequence described in SEQ ID NO: 306, and CDR3 containing the sequence described in SEQ ID NO: 307. (vi) VH containing the sequence described in sequence number 303, (vii) VL containing CDR1 containing the sequence set in sequence number 308, CDR2 containing the sequence described in sequence number 309, and CDR3 containing the sequence described in sequence number 310, (viii) VL containing the sequence described in sequence number 304, (ix) VH containing CDR1 containing the sequence described in SEQ ID NO: 305, CDR2 containing the sequence described in SEQ ID NO: 306, and CDR3 containing the sequence described in SEQ ID NO: 307, and VL containing CDR1 containing the sequence set in SEQ ID NO: 308, CDR2 containing the sequence described in SEQ ID NO: 309, and CDR3 containing the sequence described in SEQ ID NO: 310, or (x) comprising at least one of VH containing the sequence described in SEQ ID NO: 303 and VL containing the sequence described in SEQ ID NO: 304.

[0138] Preferably, the tumor antigen-binding protein includes a variable heavy chain containing the amino acid sequence described in SEQ ID NO: 303, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto, while maintaining binding affinity to La / SSB.

[0139] Preferably, the tumor antigen-binding protein includes a variable heavy chain that maintains binding affinity to La / SSB and contains the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 303, and includes the amino acid sequence described in SEQ ID NO: 303, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0140] Preferably, the tumor antigen-binding protein includes a variable light chain that maintains binding affinity to La / SSB and contains the amino acid sequence described in SEQ ID NO: 304, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0141] Preferably, the tumor antigen-binding protein includes a variable light chain that retains binding affinity to La / SSB and contains the same CDR sequence as defined for the variable light chain of SEQ ID NO: 304, and includes the amino acid sequence described in SEQ ID NO: 304, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0142] Preferably, the tumor antigen-binding protein includes a variable heavy chain containing the amino acid sequence described in SEQ ID NO: 303, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto, while maintaining binding affinity to La / SSB; and a variable light chain containing the amino acid sequence described in SEQ ID NO: 304, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

[0143] Preferably, the tumor antigen-binding protein maintains binding affinity to La / SSB and includes the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 303 and the variable light chain of SEQ ID NO: 304, while also containing the amino acid sequence described in SEQ ID NO: 303, or at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or A variable heavy chain comprising a sequence that is 96%, 97%, 98%, or 99% identical thereto, and a variable light chain comprising the amino acid sequence described in SEQ ID NO: 304, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0144] The antigen-binding proteins described herein may include human constant regions, such as IgG constant regions, such as IgG1, IgG2, IgG3, or IgG4 constant regions, or mixtures thereof. In the case of antibodies or proteins containing VH and VL, VH may be linked to the heavy chain constant region, and VL may be linked to the light chain constant region.

[0145] In one example, the antigen-binding protein described herein comprises a constant region of an IgG4 antibody or a stabilizing constant region of an IgG4 antibody. In one example, the protein or antibody comprises an IgG4 constant region having proline at position 241 (according to Kabat's numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991)).

[0146] For example, an antigen-binding protein described herein, or a composition of an antigen-binding protein described herein, comprises a heavy chain constant region, the heavy chain constant region comprises a stabilized heavy chain constant region, the stabilized heavy chain constant region comprises all or part of a mixture of sequences having or not having a C-terminal lysine residue.

[0147] For example, the antigen-binding protein comprises VH as disclosed herein, where VH is ligated to or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., those considered previously), and VL is ligated to or fused to a kappa light chain constant region.

[0148] The antigen-binding proteins or molecules described herein may be purified, substantially purified, isolated, and / or recombinant.

[0149] In any embodiment, the tumor antigen-binding protein further comprises a constant region of the heavy chain containing the amino acid sequence described in SEQ ID NO: 225, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto. In any embodiment, the constant region of the heavy chain may contain one or more amino acid substitutions to stabilize the linkage of TGFβR to the ECD or ligand-binding domain. Such substitutions are further described herein and known to those skilled in the art with respect to the stabilization of the fusion protein.

[0150] In any embodiment, the tumor antigen-binding protein further comprises a constant region of a light chain containing the amino acid sequence described in SEQ ID NO: 229, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

[0151] In any embodiment, the ECD or ligand-binding domain of TGFβR may comprise or consist of the amino acid sequence described in any one of SEQ ID NOs: 246-254, or any of the amino acid sequences further described herein, including those listed in Table 4. In a preferred embodiment, the molecule of the present invention may comprise the amino acid sequence described in SEQ ID NOs: 249 or 321, or a sequence that is at least about 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99%, which retains the ability to bind to TGFβ.

[0152] In embodiments of the present invention, the molecule is in the form of an antibody, and the antibody comprises a heavy chain linked to the ECD or ligand-binding domain of TGFβR, and a light chain. Accordingly, the present invention provides a fusion protein comprising the sequence of the heavy chain of the antibody described herein and the ECD or ligand-binding domain of TGFβR. In further embodiments, the molecule is in the form of an antibody, and the antibody comprises a heavy chain linked to the ECD or ligand-binding domain of TGFβR, and a light chain. Accordingly, the present invention provides a fusion protein comprising the sequence of the light chain of the antibody described herein and the ECD or ligand-binding domain of TGFβR.

[0153] In a particularly preferred embodiment of the present invention, the molecule is in the form of an antibody, which comprises a heavy chain linked to the ECD or ligand-binding domain of TGFβR, and a light chain. Exemplary heavy-chain and light-chain pairs are shown in Table 5. In a preferred embodiment, the molecule comprises an antibody for binding to CAIX, comprising a heavy chain containing the amino acid sequence described in SEQ ID NO: 257 or 258, and a light chain containing the amino acid sequence described in SEQ ID NO: 259. In a further preferred embodiment, the molecule comprises an antibody for binding to PSMA, comprising a heavy chain containing the amino acid sequence described in SEQ ID NO: 262 or 263, and a light chain containing the amino acid sequence described in SEQ ID NO: 264.

[0154] In addition, the following: (i) The molecule of the present invention or an expression construct encoding it, (iii) A kit comprising one or more of the pharmaceutical compositions of the present invention.

[0155] The kit may additionally include a pharmaceutically acceptable carrier.

[0156] In certain embodiments, the kit may include one or more additional therapeutic agents for pre- or post-treatment administration to a subject with the molecules or compositions of the present invention as described herein. Optionally, the kit may include written instructions on the use of the components of the kit.

[0157] As used herein, unless the context requires otherwise, the term “comprise” and variations such as “comprising,” “comprises,” and “comprised” are not intended to exclude further additives, ingredients, integers, or steps.

[0158] Further aspects of the present invention and further embodiments of the embodiments described in the preceding paragraphs will become apparent from the following description, which is given by example and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0159] [Figure 1] Representative SDS page (A) and SEC-HPLC (B) from the anti-CAIX TGFβ fusion protein (TLX250 trap) of the present invention. [Figure 2] The anti-CAIX TGFβ fusion protein specifically binds to CAIX-expressing cells. (A) Representative histograms from CT26-hCAIX cells incubated with anti-CAIX TGFβ fusion protein (TLX250 trap) or anti-PSMA TGFβ fusion protein (TLX591 trap), followed by incubation with anti-human IgG. The primary antibody concentration was 0.1235 ug / ml. (B) gMFI of signals from CT26-hCAIX cells treated as in (A) over the dose range. The parent antibody, gilentuximab, at a single concentration (white circle) is shown for reference. [Figure 3]The anti-PSMA TGFβ fusion protein specifically binds to PSMA-expressing cells. (A) Representative histograms from LnCap cells incubated with anti-PSMA TGFβ fusion protein (TLX591 trap) or anti-CAIX TGFβ fusion protein (TLX250 trap), followed by incubation with anti-human IgG. The primary antibody concentration was 90 ug / ml. (B) gMFI of signals from LnCap cells treated as in (A) over the dose range. The parent antibody HuJ591 at a single concentration (white circle) is shown for reference. [Figure 4] The anti-CAIX TGFβ fusion protein conjugates to all three isoforms of TGF beta in vitro. For TGF beta 1 and TGF beta 3, TGF beta was coated onto plates and incubated with the anti-CAIX TGFβ fusion protein (TLX250 trap), and binding was determined by ELISA using anti-human IgG. For TGF beta 2, the antibody was coated onto plates and incubated with TGF beta 2, which was detected by ELISA using an anti-TGF beta 2 detection antibody. The parent antibody (gilentuximab) was used as a negative control. [Figure 5] Representative SDS pages (A) and SEC-HPLC (B) from anti-PSMA TGFβ fusion protein (TLX591 trap). [Figure 6] The anti-PSMA TGFβ fusion protein conjugates in vitro to all three isoforms of TGF beta. For TGF beta 1 and TGF beta 3, TGF beta was coated onto plates and incubated with the anti-PSMA TGFβ fusion protein (TLX591 trap), and binding was determined by ELISA using anti-human IgG. For TGF beta 2, the antibody was coated onto plates and incubated with TGF beta 2, which was detected by ELISA using an anti-TGF beta 2 detection antibody. The parent antibody (HuJ591) was used as a negative control. [Figure 7]Radiolabeled anti-PSMA TGFβ fusion protein binds to TGFB1 in vitro with high affinity. Plates were coated with TGFB1 and then incubated with various concentrations of 89Zr radiolabeled antibody. After washing, radioactivity in the wells was determined. Against the anti-PSMA TGFβ fusion protein (TLX591 trap), anti-TGFb refers to the positive control antibody fresolimmab, and HuJ591 refers to the negative control parent antibody. [Figure 8] Anti-PSMA TGFβ fusion proteins capture TGF-beta in the tumor microenvironment. Animals with RM1-hPSMA tumors were treated according to the timeline in (A). Controls or animals previously treated with anti-PSMA TGFβ fusion protein (TLX591 trap) were imaged for TGF-beta using 89Zr-labeled anti-TGFβ antibody (fresolimmab). Animals previously treated with TLX591 trap had substantially reduced PET signal, indicating that TLX591 trap captured TGFβ from 89Zr fresolimmab (B). %ID / ml = % injection dose per 1 ml. n = 5 animals per group. [Figure 9] The anti-PSMA TGFβ fusion protein systemically captures TGF-beta. Animals with RM1-hPSMA tumors were treated according to the timeline in (A). The graph in (B) shows the serum concentration of TGF-beta-1 as measured by ELISA. Animals treated with the anti-PSMA TGFβ fusion protein (TLX591 trap) or a positive control anti-TGFβ mAb had substantially reduced serum levels of TGF-beta-1. EBRT = 10 Gy of external beam radiotherapy. Tx = no treatment. [Modes for carrying out the invention]

[0160] It will be understood that the present invention, as disclosed and defined herein, encompasses all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

[0161] Further aspects of the present invention and further embodiments of the embodiments described in the preceding paragraphs will become apparent from the following description, which is given by example and with reference to the accompanying drawings.

[0162] Hereinafter, we will refer in detail to certain embodiments of the present invention. While the present invention will be described in conjunction with these embodiments, it will be understood that the intent is not to limit the invention to those embodiments. On the contrary, the present invention is intended to encompass all substitutes, modifications, and equivalents that may fall within the scope of the invention as defined by the claims.

[0163] The present invention attempts to overcome at least some of the limitations of existing treatments for cancer and other conditions, in which existing treatments, particularly in the tumor microenvironment, are frequent or result in the induction of TGFβ expression.

[0164] Increased TGFβ expression is a key driving mechanism for wound healing responses to various cancer therapies, including, but not limited to, radiotherapy, chemotherapy, and surgery. TGFβ can promote the activity of inhibitory regulatory cells that suppress antitumor T cells. In addition, increased TGFβ expression can drive fibrosis, which can create a physical barrier against anti-cancer T cells entering the tumor. TGFβ not only limits the T cell response but also promotes DNA damage responses and EMT in tumor cells, making them radioresistant.

[0165] Therefore, the present invention attempts to inhibit or reduce baseline and treatment-inducible TGFβ activity, specifically in the tumor microenvironment. Without wishing to be constrained by theory, the inventors believe that this approach may facilitate reduction in tumor fibrosis and enhancement of anti-cancer immunity. Accordingly, the present invention provides a tumor antigen-binding protein (e.g., an antibody) fused to a “molecular trap” in the form of a TGFβ-binding domain for binding to TGFβ, thereby preventing TGFβ from interacting with its normal counterpart and signaling through it (i.e., inhibiting TGFβ activity). As further described herein, the TGFβ-binding domain is preferably in the form of a ligand-binding domain or extracellular domain of the TGFβ receptor. Such molecules of the present invention may be utilized after any treatment for cancer that results in increased TGFβ expression, such as external beam radiation, molecularly targeted radiation, surgery, chemotherapy, immunotherapy, and immune checkpoint inhibitors. Because the molecules of the present invention include an antigen-binding domain to ensure that the molecular trap specifically targets a site on tumor cells, the molecules are thought to be able to maximize the reduction of TGFβ activity in the tumor microenvironment.

[0166] In certain embodiments of the present invention, the molecules of the present invention may also be conjugated with radioisotopes. Thus, a particular advantage of one approach of the present invention is the ability to physically link a "molecular trap" (for binding to TGFβ) and a radiation source to deliver the molecules to the tumor microenvironment via tumor antigen-binding proteins. Thus, in preferred embodiments of the present invention, the molecules and compositions of the present invention provide simultaneous tumor-targeting radiation and mitigation of important inhibitory compensatory mechanisms that attenuate its activity.

[0167] Furthermore, the approach of the present invention may help prepare the tumor microenvironment for subsequent treatment, such as subsequent treatment with immune checkpoint inhibitors. For example, increased TGFβ activity that may occur after radiation or other cancer treatment may limit the response to treatment with checkpoint inhibitors, thereby contributing at least partially to immune exclusion. Therefore, in further embodiments, the present invention attempts to improve the likelihood of success of downstream treatments for cancer after initial treatment, which may result in increased TGFβ signaling.

[0168] Overview Throughout this Specification, unless otherwise specifically stated or the context requires, references to a single step, composition of a substance, group of steps, or group of compositions of a substance shall be construed as encompassing one and more (i.e., one or more) of those steps, compositions of a substance, group of steps, or group of compositions of a substance. Accordingly, as used herein, the singular forms "a," "an," and "the" include multiple aspects, and vice versa, unless the context clearly indicates otherwise. For example, a reference to "a" includes one and more than one; a reference to "an" includes one and more than one; a reference to "the" includes one and more than one, and so on.

[0169] Those skilled in the art will understand that the present invention is susceptible to modifications and alterations other than those specifically described. It should be understood that the present invention includes all such modifications and alterations. The present invention also includes, individually or collectively, all of the steps, features, compositions and compounds referred to or indicated herein, as well as any and all combinations, or any two or more such steps or features.

[0170] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in carrying out the present invention. The present invention is by no means limited to the methods and materials described.

[0171] All patents and publications referenced herein are incorporated in their entirety by reference.

[0172] The present invention should not be limited to the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention.

[0173] Any example or embodiment of the present invention described herein shall be construed as applying mutatis mutandis to any other example or embodiment of the present invention unless otherwise specifically described.

[0174] Unless otherwise specifically defined, all technical and scientific terms used herein shall be construed to have the same meaning as that generally understood by those skilled in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0175] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are based on the following sources of information: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory (1988); and JEColigan This is described and explained throughout the book *Current Protocols in Immunology*, by John Wiley & Sons (editors), including all updates to date, edited by et al.

[0176] The descriptions and definitions of variable regions and their parts, immunoglobulins, antibodies, and their fragments as used herein can be further clarified by the discussions in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989, and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0177] The term "and / or," for example "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as explicitly supporting both meanings or either meaning.

[0178] As used herein, at least 70% sequence identity should be understood to provide a basis for at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, and at least 80% identity. At least 80% sequence identity should be understood to provide a basis for at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, and at least 90% identity. At least 90% sequence identity should be understood to provide a basis for at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity.

[0179] As used herein, the term "derived from" shall be interpreted as indicating that a given integer may be obtained from a particular source, but not necessarily directly from that source.

[0180] Selected definition As used herein, the term “antigen-binding protein” is used interchangeably with “antigen-binding domain” and is interpreted to mean a region of an antibody capable of specifically binding to an antigen, i.e., VH, or VL, or Fv containing both VH and VL. The antigen-binding domain does not need to be in the context of the entire antibody and can be, for example, isolated (e.g., a domain antibody) or in another form, such as those described herein (e.g., scFv).

[0181] For the purposes of this disclosure, the term “antibody” includes proteins that are capable of specifically binding to one or more closely related antigens by an antigen-binding domain contained within the Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant antibodies, or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-transplant antibodies, primate-transplant antibodies, deimmunized antibodies, synthetic humanized antibodies, hemiantibodies, bispecific antibodies). Antibodies generally include a constant domain that can be located in a constant region or a constant fragment or crystallizable fragment (Fc). Exemplary forms of antibodies include a four-chain structure as their basic unit. Full-length antibodies include two covalently linked heavy chains (about 50–70 kD) and two light chains (about 23 kDa each). The light chains generally include a variable region (if present) and a constant domain, which in mammals are either κ-light chains or λ-light chains. A heavy chain generally consists of a variable region and one or two constant domains linked to additional constant domains by a hinge region. Mammalian heavy chains are of one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains, as well as a heavy chain and a light chain, are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary between different types of antibodies. Each chain has an N-terminal variable region (VH or VL, each about 110 amino acids long) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL, about 110 amino acids long) is aligned and disulfide-bonded to the first constant domain of the heavy chain (CH1, 330-440 amino acids long). The variable region of the light chain is aligned with the variable region of the heavy chain. The antibody heavy chain may contain two or more additional CH domains (such as CH2, CH3), and may include a hinge region between the constant domains of CH1 and CH2. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For example, the antibody may be a mouse (mouse or rat) antibody or a primate (e.g., human) antibody.In one example, the antibody heavy chain lacks a C-terminal lysine residue. In another example, the antibody is humanized, synthetically humanized, chimeric, CDR-transplanted, or deimmunized.

[0182] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably and refer to an antibody in a substantially intact form, as opposed to an antigen-binding fragment. Specifically, whole antibodies include those with heavy and light chains containing the Fc region. The constant domain may be the wild-type sequence constant domain (e.g., the human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0183] As used herein, “variable region” refers to a portion of the light and / or heavy chain of an antibody as defined herein, which is capable of specifically binding to an antigen and includes the amino acid sequences of complementarity-determining regions (CDRs), namely CDR1, CDR2, and CDR3, and the framework region (FR). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) along with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0184] As used herein, the term “complementarity-determining region” (synonym: CDR, i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in the antibody variable region whose presence significantly contributes to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs, identified as CDR1, CDR2, and CDR3. The CDRs of VH are also referred herein as CDR H1, CDR H2, and CDR H3, respectively, with CDR H1 corresponding to CDR1 of VH, CDR H2 to CDR2 of VH, and CDR H3 to CDR3 of VH. Similarly, the CDRs of VL are referred herein as CDR L1, CDR L2, and CDR L3, respectively, with CDR L1 corresponding to CDR1 of VL, CDR L2 to CDR2 of VL, and CDR L3 to CDR3 of VL. In one example, the amino acid positions assigned to CDR and FR are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to CDR and FR are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html).The present invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the classical numbering system, or those of Chothia and Lesk J.Mol.Biol.196:901-917,1987, Chothia et al., Nature 342:877-883,1989, and / or Al-Lazikani et al., J.Mol.Biol.273:927-948,1997; the numbering system of Honnegher and Pluekthun J.Mol.Biol.309:657-670,2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res.25:206-211 1997. For example, CDRs are defined according to the Kabat numbering system. Optionally, heavy chain CDR2s assigned to the Kabat numbering system either do not contain the five C-terminal amino acids listed herein, or one or more of these amino acids are substituted with other naturally occurring amino acids. In this regard, Padlan et al., FASEB J., 9:133-139, 1995 established that the five C-terminal amino acids of heavy chain CDR2s are generally not involved in antigen binding.

[0185] The "framework region" (FR) is a variable region residue other than the CDR residue. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively, with FR H1 corresponding to FR1 of VH, FR H2 to FR2 of VH, FR H3 to FR3 of VH, and FR H4 to FR4 of VH. Similarly, the FRs of VL are referred herein as FR L1, FR L2, FR L3, and FR L4, respectively, with FR L1 corresponding to FR1 of VL, FR L2 to FR2 of VL, FR L3 to FR3 of VL, and FR L4 to FR4 of VL.

[0186] As used herein, the term "Fv" shall be interpreted as meaning any protein in which VL and VH associate to form a complex having an antigen-binding domain (i.e., capable of specifically binding to an antigen), whether composed of multiple polypeptides or a single polypeptide. The VH and VL forming the antigen-binding domain may be on a single polypeptide chain or on different polypeptide chains. Furthermore, the Fv (and any protein in the present invention) may have multiple antigen-binding domains that may or may not bind to the same antigen. The term shall be understood to encompass fragments directly derived from antibodies and proteins corresponding to such fragments produced using recombinant means. In some examples, VH is not linked to the heavy chain constant domain (CH)1 and / or VL is not linked to the light chain constant domain (CL). Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher-order complexes, or any of the aforementioned (e.g., minibodies) linked to their constant regions or domains (e.g., CH2 or CH3 domains). "Fab fragments" consist of monovalent antigen-binding fragments of immunoglobulins and can be produced by digesting the whole antibody with the enzyme papain to obtain a fragment consisting of an intact light chain and a portion of the heavy chain, or they can be produced using recombinant methods. The "Fab' fragment" of an antibody can be obtained by treating the whole antibody with pepsin and then reducing it to obtain a molecule consisting of an intact light chain and a portion of the heavy chain containing VH and a single constant domain. Two Fab' fragments are obtained for each antibody treated in this manner. Fab' fragments can also be produced by recombinant methods. The "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds and can be obtained by treating the whole antibody molecule with the enzyme pepsin without subsequent reduction. The "Fab2" fragment is a recombinant fragment containing two Fab fragments linked using, for example, a leucine zipper or a CH3 domain."Single-chain Fv" or "scFv" is a recombinant molecule containing an antibody variable region fragment (Fv) in which the variable regions of the light chain and the variable regions of the heavy chain are covalently linked by a suitable and plastic polypeptide linker.

[0187] As used herein, the term "Fc region," which may be referred to as "Fc" or "Fc domain," refers to the portion of an IgG molecule that corresponds to a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region consists of the C-terminal half of two heavy chains of an IgG molecule linked by disulfide bonds. It does not have antigen-binding activity but contains a carbohydrate portion and binding sites for complement and Fc receptors (including FcRn receptors). The Fc region contains the entirety of the second constant domain CH2 (residues 231-340 of human IgG1 according to the EU index numbering system, and defined as residues 244-360 in the Kabat system) and the third constant domain CH3 (residues 341-447 EU index / 361-478 Kabat) (see, for example, for the sequence of CH2, see Sequence ID 1 or Figure 1C of WO2015 / 175874, and for the sequence of CH3, see Sequence ID 2, Figure 1D, which is incorporated herein by reference; and for a comparison of the numbering rules used for various residues in the Fc region of immunoglobulins, see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs).

[0188] As used herein, “EU Index” or “EU Numbering Scheme” refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85 (the entire text is incorporated herein by reference)). As used herein, “Kabat System” refers to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991. Those skilled in the art will be able to readily determine whether a given amino acid sequence is numbered according to either the EU or the Kabat System.

[0189] The terms “isolated protein” or “isolated polypeptide” refer to a protein or polypeptide that, by its origin or the source of its derivatives, does not associate with its naturally associated components in its natural state and substantially does not contain other proteins from the same source. Proteins can be made substantially free of naturally associated components by using protein purification techniques known in the art, or substantially purified by isolation. “Substantially purified” means that the protein is substantially free of contaminants, for example, at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% free of contaminants.

[0190] The term "recombinant" shall be understood to mean the product of artificial genetic modification. Therefore, in the context of recombinant proteins containing antibody-antigen-binding domains, this term does not include antibodies that occur naturally within the subject's body, which are products of natural recombination during B cell maturation. However, if such antibodies are isolated, they should be considered isolated proteins containing antibody-antigen-binding domains. Similarly, if a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein containing an antibody-antigen-binding domain. Recombinant proteins also include proteins expressed by artificial recombinant means, for example, if they are present in cells, tissues, or subjects where they are expressed.

[0191] The term "protein" shall be interpreted as including a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other covalently or non-covalently (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0192] The terms "polypeptide" or "polypeptide chain" will be understood from the preceding paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0193] As used herein, the term “binding” in relation to the interaction of an antigen-binding protein or its antigen-binding domain with an antigen means that the interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, antibodies generally recognize and bind to specific protein structures, not proteins. When an antibody binds to epitope “A”, in a reaction involving labeled “A” and a protein, the presence of a molecule containing epitope “A” (or free, unlabeled “A”) reduces the amount of labeled “A” bound to the antibody.

[0194] As used herein, the terms “specifically binds” or “binds specifically” are interpreted to mean that the antigen-binding protein of the present invention reacts or associates with a particular antigen or cell expressing it more frequently, rapidly, for a longer duration, and / or with greater affinity than with alternative antigens or cells. For example, the antigen-binding protein binds to a designated tumor-associated antigen or tumor antigen with substantially greater affinity (e.g., 1.5 times, 2 times, 5 times, 10 times, 20 times, 40 times, 60 times, 80 times to 100 times, 150 times, or 200 times) than with other antigens.

[0195] As used herein, the term “epitope” (synonym: “antigenic determinant”) is understood to mean a region of a cell surface protein (e.g., PSMA, CAIX PDGFRα, and LA / SSB) to which an antigen-binding protein, including the antigen-binding domain of an antibody, binds.

[0196] As used herein, the term “condition” refers to the disruption or interference of normal function and includes, but is not limited to, any specific condition, disease or disorder.

[0197] As used herein, the terms “prevent,” “prevent,” or “prevent” include administering the antigen-binding protein of the present invention to thereby cessate or prevent the onset of at least one symptom of a condition. The term also includes the treatment of a subject in remission to prevent or prevent relapse.

[0198] As used herein, the terms “to treat,” “to cure,” or “to treat” include administering an antigen-binding protein as described herein to thereby reduce or eliminate at least one symptom of a specified disease or condition.

[0199] As used herein, the term “subject” shall be interpreted as meaning any animal, including humans, e.g., mammals. Examples of subjects include, but are not limited to, humans and non-human primates. For example, the subject is humans.

[0200] As used herein, “tumor-associated antigen” (TAA) refers to an antigen expressed by cancer cells. TAA-associated antigens are not unique to tumor cells and are expressed in normal cells under conditions that do not induce a state of immune tolerance to the antigen. Antigen expression in tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed in normal cells during fetal development when the immune system is immature and unable to respond, or TAAs may be antigens that are normally present in normal cells at very low levels but expressed in tumor cells at much higher levels. Cytotoxic T lymphocytes that recognize these antigens may be able to destroy tumor cells before they proliferate or metastasize. Tumor antigens may also be present on the surface of tumors, for example, in the form of mutant receptors, in which case the tumor antigen may be recognized by B cells.

[0201] Antibody production Preferably, the antigen-binding proteins described herein by any example are recombinants.

[0202] In the case of recombinant proteins, the nucleic acid encoding them can be cloned into an expression construct or vector, which is then transfected into host cells such as E. coli cells, yeast cells, insect cells, or mammalian cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or, in other cases, myeloma cells that do not produce the protein. Exemplary cells used to express the protein are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques for achieving these goals are known in the art and are described, for example, in Ausubel et al., (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), or in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art; see, for example, US4816567 or US5530101.

[0203] After isolation, the nucleic acid is operably ligated and inserted into a promoter in an expression construct or expression vector for further cloning (DNA amplification) or expression in a cell-free system or in cells.

[0204] As used herein, the term “promoter” should be understood in its broadest context, including, for example, transcriptional regulatory sequences of genomic genes, including a TATA box or initiation factor element necessary for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that alter nucleic acid expression in response to developmental and / or external stimuli, or in a tissue-specific manner. In this context, the term “promoter” is also used to describe recombinant nucleic acids, synthetic nucleic acids, or fusion nucleic acids, or derivatives that confer, activate, or enhance the expression of a operably linked nucleic acid. An exemplary promoter may contain additional copies of one or more specific regulatory elements for further enhancing the expression of the nucleic acid and / or altering spatial and / or temporal expression.

[0205] As used herein, the term “operably linked to ~” means positioning a promoter relative to a nucleic acid such that the expression of the nucleic acid is controlled by the promoter.

[0206] Many vectors are available for expression in cells. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a protein-coding sequence (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Those skilled in the art will recognize sequences suitable for protein expression. Exemplary signal sequences include prokaryotic secretory signals (e.g., pelB, alkaline phosphatase, penicillinase, IPP, or thermostable enterotoxin II), yeast secretory signals (e.g., invertase reader, α-factor reader, or acid phosphatase reader), or mammalian secretory signals (e.g., herpes simplex gD signal).

[0207] Exemplary promoters active in mammalian cells include the cytomegalovirus early promoter (CMV-IE), human elongation factor 1-α promoter (EF1), small nuclear RNA promoters (U1a and U1b), α-myosin heavy chain promoter, monkey virus 40 promoter (SV40), Roussarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter; and hybrid regulatory elements including the CMV enhancer / β-actin promoter or immunoglobulin promoter or their active fragments. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), or Chinese hamster ovary cells (CHO).

[0208] For example, typical promoters suitable for expression in yeast cells, such as yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and S. pombe, include, but are not limited to, the ADH1 promoter, GAL1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter, or TEF1 promoter.

[0209] Means for introducing isolated nucleic acids or expression constructs containing them into cells are known to those skilled in the art. The techniques used for a given cell depend on known and successful techniques. Means for introducing recombinant DNA into cells include, among others, microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection, e.g., transfection using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and microparticle bombardment, e.g., using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0210] Host cells used to produce proteins can be cultured in a variety of media, depending on the cell type used. Commercial media such as Ham's Fl0 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types considered herein are known in the art.

[0211] Steady-state region The present invention also encompasses antigen-binding proteins and / or antibodies described herein, including a constant region of the antibody. This includes an antigen-binding fragment of the antibody fused to Fc.

[0212] Sequences of the constant region useful for the production of the protein of the present invention can be obtained from several different sources. In some examples, the constant region or portion thereof of the protein is derived from a human antibody. The constant region or portion thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one example, the constant region is the human isotype IgG4 or stabilized IgG4 constant region.

[0213] In one example, the Fc region of the constant region has a reduced ability to induce effector function compared to, for example, the Fc region of natural or wild-type human IgG1 or IgG3. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for evaluating the level of effector function of Fc region-containing proteins are known in the art and / or described herein.

[0214] In one example, the Fc region is an IgG4 Fc region (i.e., from the IgG4 constant region), for example, a human IgG4 Fc region. Suitable sequences of IgG4 Fc regions are obvious to those skilled in the art and / or available in publicly available databases (e.g., available from the National Center for Biotechnology Information).

[0215] In one example, the constant region is the stabilized IgG4 constant region. The term “stabilized IgG4 constant region” would be understood to mean an IgG4 constant region modified to reduce Fab arm exchange or the tendency to undergo Fab arm exchange, or the formation of a semi-antibody or the tendency to form a semi-antibody. “Fab arm exchange” refers to a type of protein modification of human IgG4 in which the IgG4 heavy chain and attached light chain (half-chain) are exchanged with a heavy-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs spontaneously in vivo and can be induced in vivo by purified blood cells or by reducing agents such as reduced glutathione. “Semi-antibodies” are formed when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0216] In one example, the stabilized IgG4 constant region contains proline at position 241 of the hinge region according to Kabat's system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 2001, and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is generally serine. After the serine substitution for proline, the IgG4 hinge region contains the sequence CPPC. In this regard, those skilled in the art will recognize that the “hinge region” is the proline-rich portion of the antibody heavy chain constant region that links the Fc and Fab regions, which confer mobility to the two Fab arms of the antibody. The hinge region contains cysteine ​​residues involved in the inter-heavy-chain disulfide bond. Generally, the hinge region is defined as the Glu226–Pro243 stretch of human IgG1, according to Kabat's numbering system. Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy-chain disulfide (SS) bond at the same position (see, e.g., WO2010 / 080538).

[0217] An example of an additional stabilized IgG4 antibody is one in which the arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU numbering system) is replaced with lysine, threonine, methionine, or leucine (as described, for example, in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively contain a residue selected from the group consisting of alanine, valine, glycine, isoleucine, and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region may contain proline (i.e., a CPPC sequence) at position 241 (as described above).

[0218] In another example, the Fc region is a region modified to have reduced effector function, i.e., a “non-immunostimulated Fc region.” For example, the Fc region is an IgG1 Fc region containing substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In yet another example, the Fc region is an IgG1 Fc region containing one or more of the following changes, deletions of E233P, L234V, L235A, and G236, and / or one or more of the following changes, A327G, A330S, and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J Biol Chem. 276(9):6591-604, 2001). Examples of additional non-immunostimulated Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177:1129-1138 2006, and / or Hezareh J Virol; 75:12161-12168, 2001).

[0219] In another example, the Fc region is, for example, at least one C derived from an IgG4 antibody. H 2 domains and at least one C derived from the IgG1 antibody HThe Fc region is a chimeric region containing three domains, the Fc region containing substitutions at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering) (as described, for example, in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S and 427F.

[0220] Additional modifications The present invention also aims to provide additional modifications to antibody or antigen-binding proteins that include an Fc region or a constant region.

[0221] For example, an antibody contains one or more amino acid substitutions that increase the half-life of a protein. For instance, an antibody contains an Fc region containing one or more amino acid substitutions, which increase the affinity of the Fc region to the neonatal Fc region (FcRn). For example, the Fc region has increased affinity to FcRn at lower pH, e.g., around pH 6.0, facilitating Fc / FcRn binding in endosomes. In one example, the Fc region has increased affinity to FcRn at around pH 6 compared to its affinity at around pH 7.4, which facilitates the re-release of Fc into the bloodstream after cellular recirculation. These amino acid substitutions are useful in extending the half-life of a protein by reducing its clearance from the blood.

[0222] Exemplary amino acid substitutions include, in accordance with the EU numbering system, T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F. Additional or alternative amino acid substitutions are described, for example, in US2007 / 0135620 or US7083784.

[0223] Protein-containing antibody-binding domain Single-domain antibody In some examples, the antigen-binding protein of the invention is a single-domain antibody or comprises one (which is used interchangeably with the term "domain antibody" or "dAb"). A single-domain antibody is a single polypeptide chain that comprises all or a portion of the variable region of an antibody heavy chain. In certain examples, the single-domain antibody is a human single-domain antibody (see, e.g., Domantis, Inc., Waltham, MA; US 6248516).

[0224] Diabody, triabody, tetrabody In some examples, the protein of the invention is a diabody, triabody, tetrabody, or higher-order protein complex, such as those described in WO 98 / 044001 and / or WO 94 / 007921, or comprises one.

[0225] For example, a diabody is a protein that comprises two associated polypeptide chains, each polypeptide chain having the structure V L -X-V H or V H -X-V L where V L is a variable region of an antibody light chain, V H is a variable region of an antibody heavy chain, X is a linker that contains residues insufficient to allow V H and V L in a single polypeptide chain to associate (or form an Fv), or is absent, and V H of one polypeptide chain binds to V L of the other polypeptide chain to form an antigen-binding domain, i.e., an Fv molecule that can specifically bind to one or more antigens. V L and V H can be the same in each polypeptide chain, or V L and V H can be different in each polypeptide chain such that a bispecific diabody (i.e., one that contains two Fvs with different specificities) is formed.

[0226] Single-chain Fv (scFv) One skilled in the art will recognize that an scFv contains a V H and a V L region within a single polypeptide chain, and a polypeptide linker between the V H and the V L such that the scFv can form the desired structure for antigen binding (i.e., the V H and V L of the single polypeptide chain can associate with each other to form an Fv). For example, the linker contains more than 12 amino acid residues, and (Gly4Ser)3 is one of the more preferred linkers for scFv.

[0227] The present invention also contemplates disulfide-stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into the FR of V H and the FR of V L and within cysteine residues linked by disulfide bonds to yield a stable Fv.

[0228] Alternatively or in addition, the present invention encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by non-covalent or covalent linkages, such as by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, the two scFvs are linked by a peptide linker of sufficient length such that both scFvs are formed and capable of binding to an antigen, as described, for example, in US2006 / 0263367.

[0229] Heavy-chain antibody Heavy-chain antibodies are structurally different from many other forms of antibodies in that they contain heavy chains but no light chains. Thus, these antibodies are also referred to as "heavy-chain only antibodies." Heavy-chain antibodies are found, for example, in camelids and cartilaginous fish (also called IgNAR).

[0230] The variable regions present in naturally occurring heavy-chain antibodies are distinct from the heavy-chain variable regions ("VH (referred to as "domain") and the light chain variable region ("V") present in conventional 4-chain antibodies. L In order to distinguish it from what is called a "domain," camelid antibodies are generally called "V HH In the context of "domains" and IgNAR, it is referred to as V-NAR.

[0231] A general description of heavy chain antibodies and their variable regions derived from camelid animals, as well as methods for their production and / or isolation and / or use, can be found, in particular, in the following references: WO94 / 04678, WO97 / 49805, and WO97 / 49805.

[0232] A general description of heavy chain antibodies derived from cartilaginous fish and their variable regions, as well as methods for their production and / or isolation and / or use, can be found, in particular, in WO2005 / 118629.

[0233] Other antibodies and proteins containing antigen-binding domains The present invention also relates to other antibodies and proteins containing antigen-binding domains, for example, (i) “Key and hole” bispecific proteins as described in US5731168, (ii) For example, heteroconjugate proteins described in US4676980, (iii) For example, heteroconjugate proteins produced using chemical crosslinking agents, as described in US4676980, and (iv) Conceive Fab3 (for example, as described in EP1993 / 0302894).

[0234] Transformed growth factor β receptor (TGFβR), its extracellular domain (ECD), or ligand-binding fragment The present invention provides a molecule, optionally in the form of a fusion protein, comprising an antigen-binding protein containing a tumor antigen-binding domain, and a transforming growth factor β receptor (TGFβR), its extracellular domain (ECD), or a ligand-binding fragment.

[0235] In any embodiment of the present invention, the transformed growth factor β receptor (TGFβR), its extracellular domain (ECD), or ligand-binding fragment may be derived from TGFβRI, TGFβRII, or TGFβRIII. Typically, the ECD or its ligand-binding fragment contains a polypeptide sequence sufficient to bind to a TGFβ polypeptide (e.g., TGFβ1, TGFβ2, or TGFβ3).

[0236] a. TGFβ receptor I (TGFβRI) In one embodiment, the ECD or its ligand-binding fragment may be derived from TGFβRI (e.g., isoform 1) and may contain all or part of the TGFβRI extracellular domain (amino acid residues 34-126). In some cases, the ECD or its ligand-binding fragment contains an amino acid sequence having at least 70, at least 80, at least 90, at least 100, or 103 amino acid residues of the following TGFβRI extracellular domain (ECD) amino acid sequence and at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity: LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEL (SEQ ID NO: 246).

[0237] b. TGFβ receptor II (TGFβRII) In any embodiment, the ECD or its ligand-binding fragment may be derived from TGFβRII (e.g., isoform A) and may comprise all or part of the TGFβRII ECD sequence (amino acid residues 24-177). In some cases, a preferred TGFβRII isoform A polypeptide may comprise an amino acid sequence having at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 154 amino acid residues of the following TGFβRII isoform A ECD sequence and at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity. IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE(Sequence ID 247).

[0238] In any embodiment, the ECD or its ligand-binding fragment may be derived from TGFβRII (e.g., isoform B) and may comprise all or part of the TGFβRII ECD sequence (amino acid residues 24-166). In some cases, a preferred TGFβRII isoform B polypeptide comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 100, or 103 amino acid residues of the TGFβRII isoform B ECD sequence and at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity. JPEG2026510493000001.jpg19152 (SEQ ID NO: 248). One or more of F30, D32, S52, E55, or D118 (underlined in the above sequence) may be substituted with an amino acid other than the naturally occurring aa at these positions (e.g., alanine).

[0239] In any embodiment, the TGFβRII isoform B polypeptide may comprise the polypeptide of SEQ ID NO: 248 having the D118A and / or D118R substitution. Preferred TGFβRII isoform B polypeptides may also comprise the peptide of SEQ ID NO: 248 having the D118A and / or D118R substitution, as well as one or more of the following substitutions: F30A, D32N, S52L, or E55A.

[0240] In any embodiment, the ECD or its ligand-binding fragment may be derived from TGFβRII (e.g., isoform B) and may comprise all or part of the TGFβRII ECD sequence (amino acid residues 24-166). In some cases, a preferred TGFβRII isoform B polypeptide comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 100, or 103 amino acid residues of the TGFβRII isoform B ECD sequence and at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity. JPEG2026510493000002.jpg19152 (SEQ ID NO: 249) or JPEG2026510493000003.jpg19152 (SEQ ID NO: 321). One or more of the following positions may be substituted with an amino acid other than the naturally occurring aa at these positions (e.g., alanine): F30, D32, S52, E55, or D118 (underlined in the above sequence) (numbered according to SEQ ID NO: 249), or equivalent positions (for example, in SEQ ID NO: 321, these residues are located at F31, D33, S53, E56, and D119, respectively).

[0241] In any embodiment, the TGFβRII isoform B polypeptide may comprise the polypeptide of SEQ ID NO: 249 (or SEQ ID NO: 321 with the substitution at an equivalent position) having the D118A and / or D118R substitution. A preferred TGFβRII isoform B polypeptide may also comprise the peptide of SEQ ID NO: 249 having the D118A and / or D118R substitution, and one or more of the following substitutions: F30A, D32N, S52L, or E55A. A preferred TGFβRII isoform B polypeptide may also comprise the peptide of SEQ ID NO: 321 having the D119A and / or D119R substitution, and one or more of the following substitutions: F31A, D33N, S53L, or E56A.

[0242] In any embodiment, the ECD derived from TGFβRII may include modifications, such as deletions of one to 25 aa lengths within the first 25 amino acids at the N-terminus (e.g., Δ14, Δ25), and / or substitutions in one or more of L27, F30, D32, S49, 150, T51, S52, I53, E55, V77, D118, and / or E119, the substitutions including any one of the substitutions in L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E55A, V77A, D118A, D118R, E119A, and / or E119Q in SEQ ID NO: 248 or s249. For example, see J. Groppe et al. Mol Cell 29, 157-168, (2008) and De Crescenzo et al. JMB 355, 47-62 (2006).

[0243] In one aspect, the aspartic acid at position 118 (D118) of the mature TGFβRII B isoform (SEQ ID NO: 248 or 249) can be replaced by an amino acid other than Asp or Glu, such as Ala, to result in a D118A substitution, or can be replaced by Arg to result in a D118R substitution. The Asp residue corresponding to D118 is shown in SEQ ID NO: 248 and 249. N-terminal deletions of lengths of 1 to 25 aa (e.g., Δ25 deletion) and / or substitutions at F24 (e.g., F24A substitution) can also be combined with the D118 substitution (e.g., D118A or D118R). N-terminal deletions of lengths of 1 to 25 amino acids (e.g., Δ25 deletion) and / or substitutions at F24 (e.g., F24A substitution) can also be combined with substitutions at any of L27, F30, D32, S49, I50, T51, S52, I53, E55, V77, D118, and / or E119, particularly with any of the specific substitutions listed for these positions in SEQ ID NO: 248 or 249 above.

[0244] Deletions at the N-terminus of the TGFβRII polypeptide can also result in a loss of TGFβRI interaction, preventing the ECD containing the TGFβRII polypeptide or its ligand-binding fragment from acting as a constitutively active complex involved in and activating TGFβRI signaling. A 14-amino acid N-terminal deletion (Δ14) of the TGFβRII polypeptide substantially reduces interaction with TGFβRI, while a Δ25 aa N-terminal deletion appears to completely eliminate interaction with TGFβRI. N-terminal deletions also substantially alter the protein's pI, with the Δ14 TGFβRII ECD mutant exhibiting a pI of approximately 4.5–5.0 (approximately 4.74). Therefore, TGFβ constructs or complexes may contain TGFβRII ECD polypeptides with N-terminal deletions of 14–25 amino acids in length (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids). Modified ECD sequences that may be used for ECD or its ligand-binding fragments, including those that limit interaction with TGFβRI, are described in the following paragraphs.

[0245] In any embodiment, the ECD or its ligand-binding fragment comprises a sequence having at least 60%, at least 70%, at least 90, at least 100, or 103 amino acid residues of the following TGFβRII isoform B ECD sequence: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE (SEQ ID NO: 250). One or more of F30, D32, S52, E55, or D118 may be substituted with a naturally occurring non-aa amino acid (e.g., alanine) at these positions.

[0246] In one embodiment, the ECD or its ligand-binding fragment comprises the peptide of SEQ ID NO: 250 having the D118A substitution. In another embodiment, the ECD or its ligand-binding fragment comprises the polypeptide of SEQ ID NO: 250 having the D118A substitution and one or more of the F30A, D32N, S52L, and / or E55A substitutions.

[0247] N-terminal deletions of TGFβRII, e.g., combinations of 14–25 amino acid residues (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues), which block incidental cellular signaling resulting from the interaction of the bound TGFβ / TGFβRII complex with endogenous TGFβRI, can also be combined with other TGFβRII ECD substitutions, including one or more of F30, D32, S52, E55, and / or D118. The combinations of deletions and substitutions restrict cellular signaling to that which occurs via the cell's endogenous TGFβRI and TGFβRII receptor.

[0248] In one embodiment, the ECD or its ligand-binding fragment comprises a sequence having at least 60%, at least 70%, at least 90, at least 100, or 103 amino acid residues of the following TGFβRII isoform B ECD sequence: TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE (SEQ ID NO: 251), which has an N-terminal deletion (Δ14) of amino acids 1-14. One or more of F30, D32, S52, E55, or D118 may be substituted with an amino acid other than the naturally occurring aa at these positions (e.g., alanine).

[0249] In one embodiment, the ECD or its ligand-binding fragment comprises the peptide of SEQ ID NO: 250 having the D118A substitution. In another embodiment, the ECD or its ligand-binding fragment comprises the polypeptide of SEQ ID NO: 251 having the D118A substitution, and is further substituted with one or more of F30A, D32N, S52L, or E55A. In one embodiment, the ECD or its ligand-binding fragment comprises a sequence having at least 60%, at least 70%, at least 90, at least 100, or 103 amino acid residues of the following sequences and at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity. QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE (SEQ ID NO: 252) is missing residues 1-25 (Δ25). One or more of F30, D32, S52, E55, or D118 may be substituted with an amino acid other than the naturally occurring aa at these positions (e.g., alanine).

[0250] In one embodiment, the ECD or its ligand-binding fragment comprises the polypeptide of SEQ ID NO: 252 having the D118A substitution. In one embodiment, the ECD or its ligand-binding fragment comprises the peptide of SEQ ID NO: 252 having the D118A substitution and one or more of the following substitutions: F30A, D32N, S52L, or E55A. In one embodiment, the ECD or its ligand-binding fragment comprises the peptide of SEQ ID NO: 252 having the D118A and F30A substitutions. In another embodiment, the ECD or its ligand-binding fragment comprises the peptide of SEQ ID NO: 252 having the D118A and D32N substitutions. In another embodiment, the ECD or its ligand-binding fragment comprises the peptide of SEQ ID NO: 252 having the D118A and S52L substitutions. In one embodiment, the ECD or its ligand-binding fragment comprises the peptide of SEQ ID NO: 252 having the D118A and E55A substitutions.

[0251] c. TGFβ receptor III (TGFβRIII) In one embodiment, the ECD or its ligand-binding fragment may be derived from TGFβRIII (e.g., isoforms A and B) and may comprise all or part of the TGFβRIII ECD (amino acids 27-787 of isoform A or 27-786 of isoform B). In some cases, the ECD or its ligand-binding fragment comprises at least 70, at least 80, at least 90, at least 100, or 120 amino acids of the TGFβRIII A isoform or B isoform ECD sequence (i.e., SEQ ID NO: 253 or 254) and an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity.

[0252] Linkers and other means for linking antigen-binding proteins and the extracellular domain (ECD) or ligand-binding fragment of TGFβR. In any embodiment of the present invention, the extracellular domain (ECD) or ligand-binding fragment of the transformed growth factor β receptor (TGFβR) is directly ligated to the tumor antigen-binding protein or ligated via a linker.

[0253] Typically, tumor antigen-binding proteins and the extracellular domain (ECD) or ligand-binding fragment of the transforming growth factor β receptor (TGFβR) are linked via the C-terminus of the heavy chain of the antigen-binding protein. However, it is understood that the ECD or ligand-binding fragment of TGFβR may also be linked via the C-terminus of the light chain of the tumor antigen-binding protein or via any non-antigen-binding region. For example, in cases where the tumor antigen-binding protein is an antibody or Fab, the ECD or ligand-binding fragment of TGFβR may be linked to any amino acid in either the constant region of the protein or in any region of the variable domain that is not directly involved in antigen binding.

[0254] It is understood that any number of different linkers (or "spacers") may be used, including those via peptide linkers, carbohydrate linkers (e.g., PEG-based linkers), or chemical linkers.

[0255] In this specification, “linker” is also referred to as “linker sequence,” “spacer,” “tethering sequence,” or their grammatical equivalents. Known homo- or hetero-bifunctional linkers (see the 1994 Pierce Chemical Company catalog, technical section on crosslinking agents, pp. 155–200, which is incorporated entirely by reference). Several strategies may be used to covalently link molecules together. These include, but are not limited to, polypeptide linking between the N and C terminals of proteins or protein domains, linking via disulfide bonds, and linking via chemical crosslinking agents. In one aspect of this embodiment, the linker is a peptide bond produced by recombinant techniques or peptide synthesis. The linker peptide may mainly consist of the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length suitable for linking the two molecules in such a manner that they exhibit the correct conformations relative to each other and thereby maintain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids long, preferably about 1 to 30 amino acids long. In one embodiment, a linker of 1 to 20 amino acids long may be used. In certain embodiments of the present invention, the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids, or more.

[0256] Useful linkers include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other mobile linkers, where the glycine-serine polymer comprises, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, with n being at least one integer in the sequence. Alternatively, a variety of non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers.

[0257] The linkers may be mobile linkers (e.g., those containing repeats of glycine and serine residues), rigid linkers (e.g., those containing glutamic acid and lysine residues, adjacent alanine repeats), and / or cleavable linkers (e.g., sequences sensitive to protease cleavage). Examples of such linkers are known to those skilled in the art and are described, for example, in Chen et al., (2013) Advanced Drug Delivery Reviews, 65:1357-1369, and Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, and Poljak, RJ, et al. (1994) Structure 2:1121-1123, both of which are incorporated herein by reference.

[0258] In some embodiments, the linker may contain the amino acids glycine and serine in various lengths and combinations. In some embodiments, the peptide linker may include the sequences Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Gly-Ser (GGGGS), and variations or repeats thereof. In some embodiments, the peptide linker may include the amino acid sequence GGGGS (a 6-amino acid linker) or even longer. The linker may consist of a series of repeating glycine and serine residues (GS) of different lengths, i.e., (GS) nIn the array, n is any number from 1 to 15 or more. For example, the linker is (GS)3 (i.e., GSGSGS) or longer, (GS) 11 Or it may be longer than that. n It is understood that this can be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more. Fusion proteins having linkers of such length are within the scope of the present invention. Similarly, the linker can be a series of repeating glycine residues separated by serine residues. For example, (GGGGS)3 (i.e., the linker may include the amino acid sequence GGGGSGGGGSGGGGS(G4S)3) and its variants.

[0259] A peptide linker may consist of a series of repeats of Thr-Pro(TP), each repeat containing one or more additional amino acids at the N and C-terminuses relative to the repeat sequence. For example, a linker may contain or consist of the sequence GTPTPTPTPTGE.

[0260] In a further embodiment, the linker may be a short and / or alpha-helix rigid linker (e.g., A(EAAAK)3A, PAPAP, or dipeptide, e.g., LE).

[0261] In certain embodiments, the linker may be mobile and cleavable. Such a linker preferably includes one or more recognition sites for which a protease can cleave.

[0262] In certain embodiments, the linker may be derived from an antibody hinge region. Hinge region sequences from any antibody isotype may be used, for example, including hinge sequences from IgG1, IgG2, IgG3, and / or IgG4. The linker sequence may also include any sequence of any length of the CL / CH1 domain, rather than all residues of the CL / CH1 domain, for example, the first 5 to 12 amino acid residues of the CL / CH1 domain. The linker may be derived from an immunoglobulin heavy chain of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. The linker may be derived from an immunoglobulin light chain, for example, Cκ or Cλ. The linker sequence may also be derived from other proteins, for example, Ig-like proteins (e.g., TCR, FcR, KIR), hinge region-derived sequences, and other native sequences from other proteins.

[0263] In further embodiments, the antigen-binding protein and the extracellular domain (ECD) or ligand-binding fragment of TGFβR may be provided as separate proteins that can be linked in vivo. For example, providing a complementary binding sequence in one part and a binding sequence in the other part to facilitate protein-protein interaction between the two parts is intended within the scope of the invention. In one example, the invention intends to use a leucine zipper (LZ) protein-protein interaction domain in both parts so that the parts can be linked in vivo to form a fusion comprising a first part containing a tumor antigen-binding domain and a second part containing the extracellular domain (ECD) or ligand-binding fragment of TGFβR. The design and use of LZ zippers are well known to those skilled in the art.

[0264] Another approach to facilitating the linkage between antigen-binding proteins and the extracellular domain (ECD) or ligand-binding fragment of TGFβR may involve using cysteine ​​residues to facilitate the formation of a disulfide bond between the two parts.

[0265] Protein isolation Methods for isolating proteins are known in the art and / or described herein.

[0266] If antigen-binding proteins are secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of accidental contaminants. Alternatively or additionally, the supernatant can be filtered and / or separated from the protein-expressing cells, for example, using serial centrifugation.

[0267] The antigen-binding proteins or fusion proteins of the present invention, prepared from cells, can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination thereof. These methods are known in the art and are described, for example, in WO99 / 57134 or in Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0268] Those skilled in the art will also recognize that proteins can be modified to include tags for facilitating purification or detection, such as polyhistidine tags, such as hexahistidine tags, or influenza virus hemagglutinin (HA) tags, or Simian virus 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags. The resulting proteins are then purified using methods known in the art, such as affinity purification. For example, proteins containing hexahistidine tags are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to hexahistidine tags immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and then eluting the bound proteins. Alternatively or additionally, ligands or antibodies that bind to the tags are used in affinity purification methods.

[0269] Linking of radioactive isotopes to antibodies In any embodiment of the present invention, the molecules or fusion proteins described herein may be directly or indirectly linked to a therapeutic agent. In any embodiment, the bioconjugates described herein may be indirectly linked to a therapeutic agent via a chelator moiety or a linker group. Preferably, the therapeutic agent is a radioisotope. In a preferred embodiment, the therapeutic agent is an alpha-emitting radioisotope or a beta-emitting radioisotope.

[0270] Suitable isotopes include actinium-225 (225Ac), astatine-211 (211At), bismuth-212, and bismuth-213. 212 Bi, 213 Bi), Copper 67( 67 Cu), iodine-123, 124, 125, or 131 ( 123 I, 124 I, 125 I, 131 I) 123 I) Lead 212 ( 212 Pb), Lutetium-177 ( 177 Lu), radium-223 and radium-224 ( 223Ra, 224 Ra), Samarium-153 153 Sm), Scandium-47 ( 47 Sc), Strontium-90 ( 90 Sr), and Yttrium 90 ( 90 Y is one example. In some embodiments, the radionuclide conjugated with the molecule is lutetium. 177 That is the case.

[0271] It is understood that radioactive isotopes can be conjugated to the molecules or fusion proteins of the present invention either directly (via chelating agents, prosthetic groups, or linkers) or indirectly via binding to one or more amino acid residues in the antibody (e.g., halogenation of tyrosine residues).

[0272] In alternative embodiments, chelating agents or linkers may be used to conjugate radioisotopes to antibodies. For example, an antibody can be conjugated to a chelate selected from the following group: TMT (6,6''-bis[N,N'',N'''-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2''-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N'' (N'''-tetraacetic acid, also known as tetraxetan), TCMC (tetraprimary amide of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide), CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), NOTA (1,4,7-triazaciconanane) - Triacetic acid) Diamsar (3,6,10,13,16,19-Hexaazabicyclo[6.6.6]icosane-1,8-diamine), DTPA (Pentetic acid or diethylenetriaminepentaacetic acid), CHX-A''-DTPA ([(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diaminepentaacetic acid), TETA (1,4, 8,11-Tetraazacyclotetradecane-1,4,8), 11-tetraacetic acid, Te2A(4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), HBED, DFO(desferrioxamine), DFOsq(DFO-squalamide), and HOPO(3,4,3-(LI-1,2-HOPO)), or other chelating agents as described herein.

[0273] Cherators having radioactive metals and other halide radioisotopes may be conjugated to the molecules of the present invention described herein, or antibodies, or antibody fragments thereof, via one or more amino acid residues or reactive moieties in the antibody, the amino acid residues or reactive moieties including, but not limited to, one or more lysine residues, tyrosine residues, or thiol moieties.

[0274] In another example, an antibody, an antibody fragment, or a molecule is conjugated to a bifunctional linker, such as bromoacetyl, thiol, succinimide ester, TFP ester, or maleimide, or conjugated using any amine or thiol modification chemistry known in the art.

[0275] Those skilled in the art will be familiar with standard methods for conjugating chelating agents to antibodies and their derivatives or fragments. In addition, those skilled in the art will be familiar with approaches for selecting relevant chelating agents for pairing with radiometals, such as those described, for example, in Chem.Soc.Rev.,2014,43,260 (incorporated herein by reference).

[0276] Analysis of antigen-binding protein activity It is within the understanding of those skilled in the art to utilize the molecules of the present invention by identifying relevant antigen-binding proteins for use in binding to cancer antigens. Examples of various cancer antigens are further described herein and are more known in the art. Those skilled in the art can determine which antigens are preferentially expressed by the cancer being treated.

[0277] After identifying (one or more) antigens that characterize the cancer to be treated, a person skilled in the art can then identify various antigen-binding proteins for binding to those antigens and subsequently generate the molecules of the present invention containing those antigen-binding proteins. Examples of known antigen-binding proteins for binding to known antigens associated with cancer are further described herein. It is well within the skill of a person skilled in the art that, by utilizing the general skills in the art in accordance with the information provided herein, any ECD of TGFβR, including those described herein, can be linked to any desired antigen-binding protein. Furthermore, a person skilled in the art can identify the ability of molecules to bind to both cancer antigens and TGFβ.

[0278] Methods for evaluating protein binding are known in the art, for example, as described in Scopes (Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve immobilizing an antigen-binding protein and contacting it with a labeled antigen. After washing to remove nonspecific binding proteins, the amount of label and, consequently, the amount of bound antigen are detected. Of course, antigen-binding proteins can be labeled and antigens immobilized. Panning assays can also be used. Alternatively, or additionally, surface plasmon resonance assays can be used.

[0279] Treatment method The molecules of the present invention are useful for treating several conditions in which TGFβ inhibition is required in specific tissue or cell types. Typically, such conditions include cancer.

[0280] In any embodiment, the tumor antigen-binding protein may be for binding to an antigen expressed by any cancer, and optionally, the tumor or cancer is selected from cystic and solid tumors, bone and soft tissue tumors, which include tumors of the anal tissue, bile duct, bladder, blood cells, intestine, brain, breast, carcinoid, neck, eye, esophagus, head and neck, kidney, larynx, leukemia, liver, lung, lymph node, lymphoma, melanoma, mesothelioma, myeloma, ovary, pancreas, penis, prostate, skin (e.g., squamous cell carcinoma), sarcoma, stomach, testis, thyroid, vagina, and vulva. Soft tissue tumors include benign Schwannoma monosomy, desmoid tumor, lipoblastoma, lipoma, uterine leiomyoma, clear cell sarcoma, dermatofibrosarcoma, Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, myxoid liposarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma. Specific bone tumors include non-ossifying fibromas, solitary bone cysts, enchondromas, aneurysmal bone cysts, osteoblastomas, chondroblastomas, chondromyxofibromas, ossifying fibromas and adamantinomas, giant cell tumors, fibrous dysplasia, Ewing's sarcoma, eosinophilic granuloma, osteosarcoma, chondroma, chondrosarcoma, malignant fibrous histiocytoma, and metastatic cancers. Leukemia includes acute lymphoblastic, acute myeloblastic, chronic lymphocytic, and chronic myeloid leukemia.

[0281] Other examples include breast tumors, colorectal tumors, adenocarcinomas, mesotheliomas, bladder tumors, prostate tumors, germ cell tumors, hepatocellular carcinomas / bile duct tumors, carcinomas, neuroendocrine tumors, pituitary neoplasms, small round cell tumors, squamous cell carcinomas, melanomas, atypical fibroxanthomas, seminomas, non-seminomas, stromal Leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, gastric tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilms' tumor.

[0282] In some embodiments, the cancer is metastatic cancer. The primary source of the metastatic cancer may be any type of cancer known in the art, including those described herein.

[0283] Preferably, the cancer is a solid tumor.

[0284] Non-specific examples of tumor antigens include TSA or TAA antigens, which are: 17-IA antigen, alpha-fetoprotein (AFP), alpha-actinin 4, antigens specific to A3 and A33 antibodies, ART-4, B7, Ba 733, BAGE, bcl-2, bcl-6, BCMA, BrE3 antigen, CA125, CAMEL, CAP-1, carbonate anhydrase IX (CAIX), CASP-8 / m, CCL19, CCL21, CD1, CDla, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, and CD23. , CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD 64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD 138, CD 147, CD 154, CD171, CDC27, CDK-4 / m, CDKN2A, CEA, CEACAM5, CEACAM6, complement factors (e.g., C3, C3a, C3b, C5a, and C5), colon-specific antigen p (CSAp), c-Met, CTLA-4, CXCR4, CXCR7, CXCL12, DAM, Dickkopf-related protein (DKK), ED-B fibronectin, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, EphA2, EphA3, fibroblast-activating protein (FAP), fibroblast growth factor (FGF), Flt-1, Flt-3, folate-binding protein, folate receptor, G250 antigen, gangliosides (e.g., GC2, GD3, and GM2), GAGE, GD2, gplOO, GPC3, GRO-13, HLA-DR, HM1.24. Human chorionic gonadotropin (HCG) and its subunits, HER2, HER3, HMGB-1, hypoxia-inducible factor (HIF-1), HIF-la, HSP70-2M, HST-2, la, IFN gamma, IFN alpha, IFN beta, IFN-X, IL-4R, IL-6R, IL-13R, IL13R alpha 2, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-2 3, IL-25, ILGF, ILGF-1R, insulin-like growth factor-1 (IGF-1), IGF-1R, integrin ανβ3, integrin α5β1, KC4 antigen, killer cell immunoglobulin-like receptor (KIR), Kras, KS-1 antigen, KS1-4, La / SSB, LDR / FUT, Le1, macrophage migration inhibitor (MIF), MAGE, MAGE-3, MART-1, MART-2, mCRP, MCP-1, melanoma glycoprotein, mesothelin, MIP-1 A, MIP-1B, MIF, mucin (e.g., MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, and MUM-3), NCA66, NCA95, NCA90, NY-ESO-1, PAM4 antigen, pancreatic cancer mucin, PD-1, PD-L1, PD-1 receptor, placental growth factor, platelet-derived growth factor receptor alpha (PDGFRa), p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, RS5, RANTES, SAGE, 5100, Survivin, Survivin-2B, T101, TAC, TAG-72, tenascin, Thomson-Friedenreich antigen, Tn antigen, TNF-alpha, tumor necrosis antigen, TRAG-3, TRAIL receptor, VEGF, VEGFR, and WT-1. Therefore, it is understood that in any embodiment, the tumor antigen-binding protein may be one that is capable of specifically binding to any such antigen.

[0285] In preferred embodiments of the present invention, the TAA is selected from carbonate anhydrase IX (CAIX) prostate-specific membrane antigen (PSMA) and PDGFR alpha-La / SSB.

[0286] In certain embodiments of the present invention, the tumor antigen is not EGFR or EGFRvIII.

[0287] In certain embodiments of the present invention, the tumor antigen is not PD-1, PD-L1, or a PD-L1 receptor.

[0288] Optionally, the tumor antigen-binding protein may be selected from or may contain antigen-binding domains, including: LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), bertuzumab (hA20, anti-CD20), ritucumab (anti-CD20), obinutuzumab (GA101, anti-CD20), daratumumab (anti-CD38), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-TROP-2), PAM4 or KC4 (both, anti-mucin), MN-14 (anti-CEA), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen p), Immu 31 (anti-alpha-fetoprotein), Rl (anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), L243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab (anti-VEG F) Derived from one of the following: cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), tositumomab (anti-CD20), PAM4 (also known as cribatuzumab, anti-mucin), trastuzumab (anti-HER2), pertuzumab (anti-HER2), polatuzumab (anti-CD79b), and anetuzumab (anti-mesotelin).

[0289] In further embodiments, the tumor antigen-binding protein may be selected from or include an antigen-binding domain, the antigen-binding domain being derived from any one of the following: APOMAB(DAB4), atezolizumab, avelumab, bevacizumab, semiprimab, cetuximab, dataumumab, dinutuximab, durvalumab, elotuzumab, girentuximab, ipilimumab, isatuximab, J591 or huJ591, mogamulizumab, nectimumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, pertizimab, ramucirumab, rituximab, or trastuzumab.

[0290] Preferably, the molecules of the present invention are useful for treating cancers characterized by the presence of PSMA, CAIX, PDGFRα, or La / SSB. For example, a molecule that binds to PSMA is useful for treating cancers characterized by increased expression of PSMA, which include prostate cancer, bladder cancer, testicular embryonic carcinoma, neuroendocrine cancer, renal cell carcinoma, and breast cancer. A molecule that binds to CAIX may be useful for treating cancers characterized by increased expression of CAIX, which include renal cell carcinoma (including clear cell renal cell carcinoma), colon cancer, breast cancer, lung cancer, cervical cancer, and melanoma. A molecule that binds to PDGFRα may be useful for treating cancers characterized by increased expression of PDGFRα, which include gastrointestinal stromal tumors (GISTs) and other soft tissue sarcomas. Molecules that bind to La / SSB may be useful in treating cancers characterized by increased La / SSB expression, which include cancer cells that have been treated with chemotherapy and / or radiation.

[0291] In a preferred embodiment, the method of the present invention is - Identifying subjects with cancer who have received, are receiving, or will receive cancer treatment, where the treatment is suspected to cause, or is known to cause, an increase in TGFβ activity in the tumor microenvironment, or where the tumor microenvironment of the cancer has high baseline levels of TGFβ activity. - The administration of a molecule to a target comprising an antigen-binding protein that binds to or specifically binds to a cancer antigen (optionally selected, the antigen being CAIX, PSMA, PDGFRα, or La / SSB), wherein the molecule further comprises the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer.

[0292] In further embodiments, the method of the present invention is - Identifying subjects with cancer characterized by the expression of carbonate anhydrase IX (CAIX), -Administering a primary treatment for cancer to a subject, where the treatment is suspected to cause an increase in TGFβ activity in the tumor microenvironment, or is known to cause such an increase. - The administration of a molecule to a subject comprising an antigen-binding protein that binds to or specifically binds to carbonate anhydrase IX (CAIX), wherein the molecule further comprises the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer.

[0293] In further embodiments, the method of the present invention is - Identifying subjects with cancer preferably characterized by the expression of prostate-specific membrane antigen (PSMA), -Administering a primary treatment for cancer to a subject, where the treatment is suspected to cause an increase in TGFβ activity in the tumor microenvironment, or is known to cause such an increase. - The administration of a molecule to a subject comprising an antigen-binding protein that binds to or specifically binds to PSMA, wherein the molecule further comprises the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer.

[0294] In further embodiments, the method of the present invention is - Identifying subjects with cancer characterized by PDGFRα expression, -Administering a primary treatment for cancer to a subject, where the treatment is suspected to cause an increase in TGFβ activity in the tumor microenvironment, or is known to cause such an increase. - The administration of a molecule to a subject comprising an antigen-binding protein that binds to or specifically binds to PDGFRα, wherein the molecule further comprises the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer.

[0295] In further embodiments, the method of the present invention is - Identifying subjects with cancer characterized by La / SSB expression, -Administering a primary treatment for cancer to a subject, where the treatment is suspected to cause an increase in TGFβ activity in the tumor microenvironment, or is known to cause such an increase. - The administration of a molecule to a subject comprising an antigen-binding protein that binds to or specifically binds to La / SSB, wherein the molecule further comprises the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer.

[0296] Optionally, treatments for cancer that are suspected of or cause an increase in TGFβ activity in the tumor microenvironment may be selected from the group consisting of external beam radiation (EBR) therapy, chemotherapy, tumor surgery or resection, immunomodulatory therapy including CPI, molecularly targeted radionuclide (MTR) therapy, and cell therapy, such as CAR T therapy.

[0297] The administered molecule or composition may optionally contain a radionuclide.

[0298] In any embodiment, the method of the present invention may inhibit or prevent cancer treatment-associated fibrosis in a subject, reduce or inhibit radioactive TGFβ activity in a subject receiving cancer treatment, and / or enhance or increase the likelihood of success of subsequent cancer treatment, such as treatment with immune checkpoint inhibitors.

[0299] It is understood that the molecules or compositions of the present invention may be administered as a first-line treatment for cancer. In such embodiments, the molecules or compositions of the present invention may include an antigen-binding protein for binding to a cancer antigen (e.g., CAIX, PSMA, PDGFRα, or La / SSB), which is linked to the ECD or ligand-binding fragment of TGFβR (preferably the ECD of TGFβR II). In further embodiments, the molecules or compositions of the present invention may include an antigen-binding protein for binding to a cancer antigen (e.g., CAIX, PSMA, PDGFRα, or La / SSB), which is linked to the ECD or ligand-binding fragment of TGFβR (preferably the ECD of TGFβR II), and the molecules may be further conjugated to a radionuclide, thereby enabling molecularly targeted radionuclide therapy at the tumor site. Alternatively or in addition, the molecules or compositions may be administered in combination with external beam radiation directed at the tumor site.

[0300] In alternative embodiments, as should be apparent from the above, the molecules or compositions of the present invention may be administered after initial cancer treatment. In preferred embodiments, the initial cancer treatment is one that induces increased levels of TGFβ in the tumor microenvironment (e.g., EBR or alternative molecular targeted therapy). In such examples, the molecules or compositions administered to the target may not contain radionuclides. In alternative examples, the administered molecule is conjugated to a radionuclide, thereby enabling molecularly targeted radionuclide therapy at the tumor site. In cases where the prior treatment is with an MTR, the method of the present invention may include a subsequent MTR with the molecules of the present invention, and both the prior and subsequent MTRs contain antigen-binding proteins for binding to the same tumor antigen. Alternatively, both the prior and subsequent MTRs contain antigen-binding proteins for binding to different tumor antigens in the same tumor type. Furthermore, while both the earlier and subsequent MTRs contain antigen-binding proteins for binding to the same tumor antigen, the antigen-binding proteins either bind to different epitopes in the antigen, or the antigen-binding proteins bind to the same epitopes in the antigen but contain different amino acid sequences.

[0301] The administration of two or more therapies by any method of the present invention may include the simultaneous, separate, or sequential administration of two or more different therapies. Accordingly, in any embodiment of the method of the present invention, the administration of two or more molecules, bioconjugates, compositions, antibodies, their antibody fragments, and other cancer therapies may be provided in the same or different dosage forms and may be administered simultaneously, separately, or sequentially in any order.

[0302] In further embodiments, the present invention relates to a method for reducing or inhibiting radiation-induced TGFβ activity in a subject, - Identifying subjects with cancer characterized by the expression of carbonate anhydrase IX (CAIX), -Administering a molecule to a subject containing an antigen-binding protein that binds to or specifically binds to CAIX, wherein the molecule further contains the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer, and optionally, the molecule may be administered without radionuclides. -Administering to a subject an antibody or antibody fragment that binds to or specifically binds to CAIX, and optionally administering an antibody or antibody fragment that is conjugated to a radionuclide. This provides a method for reducing or inhibiting radiation-induced TGFβ activity in a target.

[0303] In further embodiments, the present invention relates to a method for reducing or inhibiting radiation-induced TGFβ activity in a subject, - Identifying subjects with cancer characterized by the expression of prostate-specific membrane antigen (PSMA), -Administering a molecule to a subject containing an antigen-binding protein that binds to or specifically binds to PSMA, wherein the molecule further comprises the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer, and optionally, the molecule may be administered without radionuclides. - The administration of an antibody or antibody fragment that binds to or specifically binds to PSMA to the subject, and optionally, the administration of an antibody or antibody fragment that is conjugated to a radionuclide, This provides a method for reducing or inhibiting radiation-induced TGFβ activity in a target.

[0304] In further embodiments, the present invention relates to a method for reducing or inhibiting radiation-induced TGFβ activity in a subject, - Identifying subjects with cancer characterized by PDGFRα expression, -Administering a molecule to a subject containing an antigen-binding protein that binds to or specifically binds to PDGFRα, wherein the molecule further contains the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer, and optionally, the molecule may be administered without radionuclides. - The procedure involves administering to a subject an antibody or antibody fragment that binds to or specifically binds to PDGFRα, and optionally, administering an antibody or antibody fragment that is conjugated to a radionuclide. This provides a method for reducing or inhibiting radiation-induced TGFβ activity in a target.

[0305] In further embodiments, the present invention relates to a method for reducing or inhibiting radiation-induced TGFβ activity in a subject, - Identifying subjects with cancer characterized by La / SSB expression, -Administering a molecule to a subject containing an antigen-binding protein that binds to or specifically binds to La / SSB, wherein the molecule further contains the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer, and optionally, the molecule may be administered without radionuclides. - The procedure involves administering to a subject an antibody or antibody fragment that binds to or specifically binds to La / SSB, and optionally, the antibody or antibody fragment being conjugated to a radionuclide. This provides a method for reducing or inhibiting radiation-induced TGFβ activity in a target.

[0306] In further embodiments, the method of the present invention is - Identifying subjects with cancer characterized by the expression of carbonate anhydrase IX (CAIX), -Administering to a subject a molecule containing an antigen-binding protein that binds to or specifically binds to CAIX, wherein the molecule further contains the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer, and optionally, the molecule is conjugated to a radionuclide. - This includes administering immune checkpoint inhibitor therapy (CPI therapy) to the target group.

[0307] In further embodiments, the method of the present invention is - Identifying subjects with cancer characterized by the expression of prostate-specific membrane antigen (PSMA), -Administering to a subject a molecule containing an antigen-binding protein that binds to or specifically binds to PSMA, wherein the molecule further comprises the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer, and optionally, the molecule is conjugated to a radionuclide. - This includes administering immune checkpoint inhibitor therapy (CPI therapy) to the target group.

[0308] In further embodiments, the method of the present invention is - Identifying subjects with cancer characterized by PDGFRα expression, -Administering to a subject a molecule containing an antigen-binding protein that binds to or specifically binds to PDGFRα, wherein the molecule further contains the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer, and optionally, the molecule is conjugated to a radionuclide. - This includes administering immune checkpoint inhibitor therapy (CPI therapy) to the target group.

[0309] In further embodiments, the method of the present invention is - Identifying subjects with cancer characterized by La / SSB expression, -Administering to a subject a molecule containing an antigen-binding protein that binds to or specifically binds to La / SSB, wherein the molecule further contains the ECD or ligand-binding fragment of TGFβR described herein, thereby inhibiting TGFβ activity in cancer, and optionally, the molecule is conjugated to a radionuclide. - This includes administering immune checkpoint inhibitor therapy (CPI therapy) to the target group.

[0310] Optionally, the amount of CPI administered may be lower than the amount administered for successful monotherapy with CPI, as further described herein.

[0311] In any embodiment, the administration of the molecule of the present invention and the immune checkpoint inhibitor may be separate, sequential, or sequential.

[0312] Examples of immune checkpoint and antibody inhibitors that target these checkpoints include anti-CTLA-4 (e.g., ipilimumab, tremelimumab, KAHR-102), anti-TIM3 (e.g., F38-2E2, ENUM005), anti-LAG3 (e.g., BMS-986016, IMP701, IMP321, C9B7W), anti-KIR (e.g., lirirumab, IPH2101, IPH4102), and anti-PD-1 (e.g., nivolumab, pizilizumab, pembrolizumab, BMS-936559, atezolizumab, lambrolizumab, MK-3475). AMP-224, AMP-514, STI-A1110, TSR-042), anti-PD-L1 (e.g., KY-1003 (EP2012 / 0194977), MCLA-145, atezolizumab, BMS-936559, MEDI-4736, MSB0010718C, AUR-012, STI-A1010, PCT / US2001 / 020964, MPDL3280A, AMP-224, dapirolizumab pegol (CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42 (OSU-HDAC42, HDAC-42, AR42, AR Examples include 42, OSU-HDAC 42, OSU-HDAC-42, NSC D736012, HDAC-42, HDAC 42, HDAC42, NSCD736012, NSC-D736012), MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD47 (e.g., CC-90002, TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1, anti-B7-H4, anti-CD52 (e.g., alemtuzumab), anti-IL-10, anti-IL-35, anti-CSF1R (e.g., FPA008), anti-NKG2A (e.g., monalizumab), anti-MICA (e.g., IPH43), and anti-CD39.

[0313] Examples of suitable PD-1 inhibitors that may be used in the present invention include Keytruda (pembrolizumab), Opdivo (nivolumab), AGEN 2034, BGB-A317, BI-754091, CBT-501 (genolimusumab), MEDI0680, MGA012, PDR001, PF-06801591, REGN2810 (SAR439684), and TSR-042, or those disclosed in U.S. Patent No. 8,008,449. Other anti-PD-1 mAbs are described, for example, in U.S. Patents No. 6,808,710, No. 7,488,802, No. 8,168,757, and No. 8,354,509, and PCT International Publication No. 2012 / 145493.

[0314] Nivolumab (also known as "Opdivo®," and previously referred to as 5C4, BMS-936558, MDX-1106, or ONO4538) is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor (Ab) that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of antitumor T cell function (U.S. Patent No. 8,008,449).

[0315] Pembrolizumab (also known as "Keytruda®," lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587. Pembrolizumab is approved by the FDA for the treatment of relapsed or refractory melanoma.

[0316] Other suitable PD-1 inhibitors include Libtayo (semiprimab), Blincyto (blinatumomab), dostallimab, spartalizumab, cetrelimab, pizilizumab, and BI-754091.

[0317] The anti-PD-1 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and / or PD-L2, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, the anti-PD-1 antibody comprises an antigen-binding moiety or fragment that binds to the PD-1 receptor and exhibits functional properties similar to the whole antibody in inhibiting ligand binding and upregulating the immune system.

[0318] In certain embodiments, the anti-PD-1 antibody used in the method can be replaced with another PD-1 or anti-PD-L1 antagonist. For example, an anti-PD-L1 antibody can replace the use of an anti-PD-1 antibody in the methods disclosed herein, since it prevents the interaction between PD-1 and PD-L1 and thereby exerts a similar effect to the PD-1 signaling pathway. In any embodiment, suitable PD-L1 inhibitors include Imfinzi (durvalumab or MEDI4736), Tecentriq (atezolizumab or MPDL3280A), Bavencio (avelumab, MSB0010718C), MS-936559 (12A4 or MDX-1105), and CX-072.

[0319] The anti-CTLA-4 antibody of the present invention binds to human CTLA-4 and disrupts the interaction between CTLA-4 and the human B7 receptor. Since the interaction between CTLA-4 and B7 transduces a signal that leads to the inactivation of T cells possessing the CTLA-4 receptor, it will be understood that disrupting this interaction effectively induces, enhances, or prolongs the activation of such T cells, thereby inducing, enhancing, or prolonging the immune response.

[0320] Suitable CTLA-4 inhibitors that can be used in the present invention include Yervoy (ipilimumab), tremelimumab, and AGEN 1884, or those disclosed in U.S. Patent Nos. 6,984,720 and 7,605,238. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation. Tremelimumab is a human IgG2 monoclonal anti-CTLA-4 antibody.

[0321] As is to be understood, CPI may be any other CPI known in the art, and other CPIs include inhibitors of PD-1, PD-L1, CTLA-4, TIGIT, VISTA, LAG-3, or CD47.

[0322] Immune checkpoint inhibitors may be administered in the form of pharmaceutical compositions, including combinations with any pharmaceutically acceptable excipients, carriers, and / or diluents described herein. Typically, immune checkpoint inhibitors are administered in formulations known in the art.

[0323] The disclosures herein are not limited to the use of specific immune checkpoint inhibitors described herein, but are understood to include the use of the fusion proteins or compositions of the present invention before, in combination with, or after administration of any immune checkpoint inhibitor, including non-antibody inhibitors.

[0324] Evaluating the therapeutic effectiveness of the above methods is well within the understanding of those skilled in the art. For example, with respect to tumor treatment, successful treatment may result in inhibition of tumor growth of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, compared to untreated subjects. Alternatively, the treatments described herein may cause complete regression of tumor volume. In other embodiments of the present invention, tumor regression may be observed and continued for at least about 10 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, or longer.

[0325] The terms “subject” and “patient” will be understood to be interchangeable. While the present invention is applicable to humans, it is also useful for veterinary therapeutic purposes. The present invention is useful for livestock such as cattle, sheep, horses, and poultry, companion animals such as cats and dogs, and zoo animals.

[0326] composition In some cases, the molecules described herein may be administered orally, parenterally, by inhalation spray, by adsorption, absorption, topically, rectally, nasally, buccally, vaginally, intraventricularly, via an implantable reservoir in a dosage formulation containing a conventional non-toxic, pharmaceutically acceptable carrier, or by any other convenient dosage form. As used herein, the term “parenteral” includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0327] Methods for preparing the molecules of the present invention into forms suitable for administration to a target (e.g., pharmaceutical compositions) are known in the art and include, for example, the methods described in Remington's Pharmaceutical Sciences (18th edition, Mack Publishing Co., Easton, Pa., 1990) and USPharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0328] The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, such as intravenous administration, or administration into body cavities or tubules of organs or joints. The compositions for administration generally comprise a pharmaceutically acceptable carrier, such as a solution of antigen-binding protein dissolved in an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, and toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of the antigen-binding site of the present invention in these formulations can vary widely and is selected mainly based on fluid volume, viscosity, and body weight, according to the specific mode of administration and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as mixed oils and ethyl oleate, may also be used. Liposomes can also be used as carriers. The vehicle may contain small amounts of additives, such as buffers and preservatives, to enhance isotonicity and chemical stability.

[0329] Dosage and timing of administration The preferred dosage of the molecule of the present invention varies depending on the specific molecule, the condition being treated, and / or the target being treated. For example, determining a preferred dosage by starting with a dose below the optimal dose and progressively adjusting the dose to determine the optimal or useful dose is within the capabilities of those skilled in the art. Alternatively, to determine an appropriate dosage for treatment / prevention, data from cell culture assays or animal studies may be used, and the preferred dose may be determined by the ED of the active compound having little or no toxicity. 50 The circulating concentration is within the range of [specific concentration]. The dose may vary within this range depending on the dosage form used and the route of administration utilized. The therapeutic / prophylactic effective dose can be initially estimated from the cell culture assay. IC determined in cell culture 50 Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes (i.e., the concentration or amount of the compound that achieves the half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0330] In some examples, the methods of the present invention include administering a prophylactic or therapeutically effective amount of the protein described herein.

[0331] The term "therapeutic dose" refers to the amount administered to a subject in need of treatment that improves the subject's prognosis and / or condition, and / or reduces or inhibits one or more symptoms of the clinical condition described herein to a level below the level observed and recognized as a clinical diagnosis or characteristic of that condition. The amount administered to the subject depends on the specific characteristics of the condition being treated, the type and stage of the condition being treated, the mode of administration, and the characteristics of the subject, such as overall health, other diseases, age, sex, genotype, and body weight. Those skilled in the art can determine an appropriate dose depending on these and other factors. Therefore, this term should not be interpreted as limiting the invention to a specific amount, such as the weight or quantity of protein, but rather the invention encompasses any amount of antigen-binding protein sufficient to achieve the described results in the subject.

[0332] As used herein, the term “preventive effective dose” is to mean an amount of protein sufficient to prevent, inhibit, or delay the onset of one or more detectable symptoms of a clinical condition. Those skilled in the art will recognize that such an amount will vary depending, for example, the specific antigen-binding protein administered, and / or the particular subject, and / or the type, severity, or level of the condition, and / or the predisposition (genetic or other) to the condition. Therefore, this term is not to be interpreted as limiting the invention to a specific amount, for example, the weight or quantity of antigen-binding protein; rather, the invention encompasses any amount of antigen-binding protein sufficient to achieve the described results in the subject.

[0333] In some embodiments, when the present invention relates to combination therapy with the molecule of the present invention, followed by or in combination with the administration of an immune checkpoint inhibitor, the molecule of the present invention and the immune checkpoint inhibitor are administered together in a “therapeutic effective dose” to a subject in need. This therapeutic effective dose may include amounts in which either or both of the molecule of the present invention or the immune checkpoint inhibitor are less than a therapeutic effective dose on their own. In some embodiments, the molecule of the present invention is administered in an amount less than a therapeutic effective dose in the absence of the immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is administered in an amount less than a therapeutic effective dose in the absence of the molecule of the present invention. In some embodiments, the “therapeutic effective dose” for the combination of the molecule of the present invention and the immune checkpoint inhibitor may be a synergistic dose. The synergistic dose may be synergistic to monotherapy with either the molecule of the present invention or the immune checkpoint inhibitor.

[0334] kit In addition, the following: (i) The molecule of the present invention or an expression construct encoding it, (ii) To provide a kit comprising one or more of the pharmaceutical compositions of the present invention.

[0335] The kit may additionally include one or more therapeutic agents for administration to a subject before or after administration of the molecule or composition of the present invention.

[0336] Optionally, the kit of the present invention is packaged with instructions for use in the methods described herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 3-1 [Table 3-2] [Table 3-3] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2] [Examples]

[0337] Example 1: Anti-CAIX-TGFβ Trap and external beam radiation (EBRT) to enhance the efficacy of checkpoint inhibitors A molecule containing an antigen-binding domain for binding to CAIX and an ECD derived from TGFβR II is obtained in mammalian cell lines by expressing a first expression construct (encoding a fusion protein containing the sequence of SEQ ID NO: 255) and a second expression construct (encoding the antibody light chain described in SEQ ID NO: 256). During protein expression, the protein encoded by the first construct and the protein encoded by the second construct associate to form a single molecule via an intramolecular disulfide bond. This molecule is called the anti-CAIX-TGFβ TRAP.

[0338] Standard in vitro assays of the molecule will be performed to confirm binding to CAIX (including CAIX-expressing tumor cells) and TGFβ.

[0339] To establish a mouse model in which tumors expressing human CAIX engraft within mice, the mice will be divided into the following five groups. 1) Control: No treatment, 2) anti-PD-1, 3) Treatment with EBRT, 4) Treatment with EBRT + anti-PD-1, 5) Treatment with EBRT + anti-PD-1 + anti-CAIX TGFβ-trap.

[0340] Mice will be monitored for tumor growth. Some mice will be sacrificed at fixation time to monitor for T cell infiltration and fibrosis.

[0341] The degree of fibrosis and T-cell infiltration at the tumor site was evaluated among the treatment groups. The degree of radiation-induced fibrosis in treatment group 5 was lower than in groups 3 and 4. The degree of T-cell infiltration in group 5 was higher than in all other groups.

[0342] Example 2: Anti-CAIX-TGFβ TRAP and molecular targeted radiation to enhance checkpoint inhibitor therapy A molecule comprising an antigen-binding domain for binding to CAIX and an ECD derived from TGFβR II is obtained according to the method of Example 1. In this example, the molecule is further conjugated with radionuclide 177 lutetium.

[0343] Standard in vitro assays of the molecule will be performed to confirm its binding to CAIX (including CAIX-expressing tumor cells) and TGFβ.

[0344] To establish a mouse model in which tumors expressing human CAIX engraft within mice, the mice will be divided into the following eight groups. 1) Control: No treatment, 2) anti-PD-1, 3) 177 Treatment with Lu-labeled anti-CAIX binding protein, 4) 177 Treatment with Lu-labeled anti-CAIX-TGFβ-trap 5) 177 Treatment with Lu-labeled anti-CAIX binding protein + anti-PD-1. 6) 177 Treatment with Lu-labeled anti-CAIX-TGFβ-trap + anti-PD-1, 7) 177 Lu-labeled anti-CAIX binding protein, followed by treatment with anti-PD-1 + anti-CAIX-TGFβ-trap. 8) 177 Lu-labeled anti-CAIX TGFβ-trap, followed by treatment with anti-PD-1 + anti-CAIX-TGFβ-trap.

[0345] Mice will be monitored for tumor growth. Some mice will be sacrificed at fixation time to monitor for T cell infiltration and fibrosis.

[0346] The degree of fibrosis and T-cell infiltration at the tumor site was evaluated across treatment groups. The degree of radiation-induced fibrosis was lower in treatment groups 4, 6, 7, and 8 compared to groups 3 and 5. The degree of T-cell infiltration was higher in the irradiated and TGFβ trap-treated groups than in the irradiated but not TGFβ trap-treated groups.

[0347] Example 3: Characterization of anti-CAIX-TGFβ TRAP (TLX250 Trap) A molecule containing an antigen-binding domain for binding to CAIX and an ECD derived from TGFβR II was obtained according to the method of Example 1 (referred to as TLX250 Trap).

[0348] Figure 1 shows the dimerization and purity analysis of the obtained TLX250 trap-containing sample.

[0349] In vitro, the TLX250 trap was shown to bind specifically to CT26-hCAIX cells in a dose-dependent manner (see Figures 2 and 3). The TLX250 trap was also shown to bind specifically to TGFβ1, TGFβ2, and TGFβ3 (Figure 4).

[0350] Example 4: Anti-PSMA-TGFβ Trap and external beam radiation (EBRT) to enhance the efficacy of checkpoint inhibitors A molecule containing an antigen-binding domain for binding to PSMA and an ECD derived from TGFβR II is obtained in mammalian cell lines by expressing a first expression construct (encoding a fusion protein containing the sequence of SEQ ID NO: 260) and a second expression construct (encoding the antibody light chain described in SEQ ID NO: 261). During protein expression, the protein encoded by the first construct and the protein encoded by the second construct associate to form a single molecule via an intramolecular disulfide bond. This molecule is called the anti-PSMA-TGFβ TRAP.

[0351] Standard in vitro assays of the molecule will be performed to confirm binding to PSMA (including PSMA-expressing tumor cells) and TGFβ.

[0352] To establish a mouse model in which tumors expressing human PSMA engraft within mice, the mice will be divided into the following five groups. 1) Control: No treatment, 2) anti-PD-1, 3) Treatment with EBRT, 4) Treatment with EBRT + anti-PD-1, 5) Treatment with EBRT + anti-PD-1 + anti-PSMA TGFβ-trap.

[0353] Mice will be monitored for tumor growth. Some mice will be sacrificed at fixation time to monitor for T cell infiltration and fibrosis.

[0354] The degree of fibrosis and T-cell infiltration at the tumor site was evaluated among the treatment groups. The degree of radiation-induced fibrosis in treatment group 5 was lower than in groups 3 and 4. The degree of T-cell infiltration in group 5 was higher than in all other groups.

[0355] Example 5: Anti-PSMA-TGFβ TRAP and molecular targeted radiation to enhance checkpoint inhibitor therapy A molecule comprising an antigen-binding domain for binding to PSMA and an ECD derived from TGFβR II is obtained according to the method of Example 1. In this example, the molecule is further conjugated with radionuclide 177 lutetium.

[0356] Standard in vitro assays of the molecules will be performed to confirm their binding to PSMA (including PSMA-expressing tumor cells) and TGFβ.

[0357] To establish a mouse model in which tumors expressing human PSMA engraft within mice, the mice will be divided into the following eight groups. 1) Control: No treatment, 2) anti-PD-1, 3) 177 Treatment with Lu-labeled anti-PSMA binding protein, 4) 177 Treatment with Lu-labeled anti-PSMA-TGFβ-trap 5) 177 Treatment with Lu-labeled anti-PSMA binding protein + anti-PD-1. 6) 177 Treatment with Lu-labeled anti-PSMA-TGFβ-trap + anti-PD-1 7) 177 Lu-labeled anti-PSMA binding protein, followed by treatment with anti-PD-1 + anti-PSMA-TGFβ-trap. 8) 177 Lu-labeled anti-PSMA TGFβ-trap, followed by treatment with anti-PD-1 + anti-PSMA-TGFβ-trap.

[0358] Mice will be monitored for tumor growth. Some mice will be sacrificed at fixation time to monitor for T cell infiltration and fibrosis.

[0359] The degree of fibrosis and T-cell infiltration at the tumor site was evaluated across treatment groups. The degree of radiation-induced fibrosis was lower in treatment groups 4, 6, 7, and 8 compared to groups 3 and 5. The degree of T-cell infiltration was higher in the irradiated and TGFβ trap-treated groups than in the irradiated but not TGFβ trap-treated groups.

[0360] Example 6: Characterization of anti-PSMA-TGFβ TRAP (TLX591 Trap) A molecule containing an antigen-binding domain for binding to CAIX and an ECD derived from TGFβR II was obtained according to the method of Example 4 (referred to as TLX591 Trap).

[0361] Figure 5 shows the dimerization and purity analysis of the obtained TLX591 trap-containing sample.

[0362] In vitro, the TLX591 trap was shown to specifically bind to PSMA-expressing LNCap cells in a dose-dependent manner (see Figures 2 and 3). The TLX591 trap was also shown to specifically bind to TGFβ1, TGFβ2, and TGFβ3 (Figure 6).

[0363] Furthermore, 89 The Zr-labeled TLX591 trap exhibits high binding affinity to TGFβ1 in vitro (Figure 7).

[0364] Example 7: TLX591 Trap captures TGFβ in the tumor microenvironment. In in vivo experiments, mice carrying RM1-hPSMA tumors were divided into the following groups. 1) Control group 89 Zr Tracer (standalone) 89 Zr-labeled anti-TGFβ antibody (fresolimmab) 2) TLX591 trap, followed by 6 days later 89 Zr Tracer. PET imaging, 89 The procedure was performed in both groups three days after administration of the Zr tracer. The protocol timeline is shown in Figure 8A.

[0365] As shown in Figure 8B, administration of the TLX591 trap significantly reduced the detection of TGFβ in the tumor microenvironment compared to the control.

[0366] Example 8: TLX591 Trap captures circulating TGFβ after external beam radiotherapy (EBRT). In another experiment, mice carrying RM1-hPSMA tumors were divided into the following five groups. 1) No treatment on day 0. 2) EBRT treatment alone on day 1, 3) On day 0, TLX591 trap alone, 4) TLX591 trap on day 0, followed by EBRT on day 1, and 5) Anti-TGFβ mAb on day 0, followed by EBRT on day 1. Serum samples were collected on day 9 (see Figure 9A for the protocol timeline).

[0367] As shown in Figure 9B, administration of TLX591 trap prior to external beam radiotherapy (EBRT) significantly reduced serum TGFβ1 concentrations compared to no prior treatment (Figure 9B). The reduction in serum TGFβ1 concentrations was also comparable to treatment with a control anti-TGFβ monoclonal antibody prior to EBRT (Figure 9B).

[0368] The incidence of radiation-induced fibrosis in treatment group 4 is expected to be lower compared to group 2.

[0369] Example 9: Administration of anti-CAIX-TGFβ trap before external beam radiation (EBRT) to reduce EBRT side effects. A molecule containing an antigen-binding domain for binding to CAIX and an ECD derived from TGFβR II is obtained in mammalian cell lines by expressing a first expression construct (encoding a fusion protein containing the sequence of SEQ ID NO: 255) and a second expression construct (encoding the antibody light chain described in SEQ ID NO: 256). During protein expression, the protein encoded by the first construct and the protein encoded by the second construct associate to form a single molecule via an intramolecular disulfide bond. This molecule is called the anti-CAIX-TGFβ TRAP.

[0370] Standard in vitro assays of the molecule will be performed to confirm binding to CAIX (including CAIX-expressing tumor cells) and TGFβ.

[0371] To establish a mouse model in which tumors expressing human CAIX engraft within mice, the mice will be divided into the following five groups. 1) No treatment, 2) EBRT treatment alone, 3) Anti-CAIX TGFβ-trap trap alone, 4) Anti-CAIX TGFβ-trap trap, followed by EBRT, and 5) Anti-TGFβ mAb, followed by EBRT.

[0372] Mice will be monitored for tumor growth. Some mice will be sacrificed at fixation time to monitor for T cell infiltration and fibrosis.

[0373] The degree of fibrosis and T-cell infiltration at the tumor site was evaluated among the treatment groups. The degree of radiation-induced fibrosis in treatment group 4 was lower compared to group 2.

[0374] It will be understood that the present invention, as disclosed and defined herein, encompasses all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

Claims

1. A molecule comprising a tumor antigen-binding protein and the extracellular domain (ECD) or ligand-binding fragment of a transforming growth factor β receptor (TGFβR).

2. The molecule according to claim 1, wherein the tumor antigen-binding protein is selected from the antigen-binding domain of an immunoglobulin, antibody, bispecific or multispecific antibody, antibody fragment, single-strand variable fragment (scFv), dimeric scFv, bivalent or polyvalent scFv, Fab, F(ab')2, Fv, or Fc-containing polypeptide.

3. The molecule according to claim 1 or 2, wherein the tumor antigen-binding protein is an antibody or an antigen-binding fragment thereof, and the ECD or ligand-binding fragment of TGFβR is linked to the C-terminus of the heavy chain of the antibody or fragment thereof.

4. The molecule according to claim 1 or 2, wherein the tumor antigen-binding protein is an antibody or an antigen-binding fragment thereof, and the ECD or ligand-binding fragment of TGFβR is linked to the C-terminus of the light chain of the antibody or fragment thereof.

5. The molecule according to claim 1 or 2, wherein the tumor antigen-binding protein is an antibody or an antigen-binding fragment thereof, and the ECD or ligand-binding fragment of TGFβR is linked to the non-antigen-binding portion of the antibody or the antigen-binding fragment.

6. The molecule according to any one of claims 1 to 5, wherein the tumor antigen-binding protein binds to prostate-specific membrane antigen (PSMA), carbonate anhydrase IX (CAIX), PDGFRα, or La / SSB.

7. The molecule according to any one of claims 1 to 6, wherein the TGFβR is type I (TGFβRI), type II (TGFβRII), or type III (TGFβRIIII) TGFβR.

8. The molecule according to any one of claims 1 to 7, wherein the tumor antigen-binding domain and the ECD or ligand-binding fragment of TGFβR are linked via a linker.

9. The molecule according to any one of claims 1 to 8, wherein the molecule is optionally conjugated to a therapeutic agent via a linker or chelator portion.

10. The molecule according to claim 9, wherein the therapeutic agent is a radioactive isotope.

11. The radioactive isotope is selected from the group consisting of actinium 225 (225Ac), astatine 211 (211At), bismuth 212 and bismuth 213 ( ２１２ Bi, ２１３ Bi), copper 67 ( ６７ Cu), iodine 123, 124, 125, or 131 ( １２３ I, １２４ I, １２５ I, １３１ I)( １２３ I), lead 212 ( ２１２ Pb), lutetium 177 ( １７７ Lu), radium 223 and radium 224 ( ２２３ Ra, ２２４ Ra), samarium 153 ( １５３ Sm), scandium 47 ( ４７ Sc), strontium 90 ( ９０ Sr), and yttrium 90 ( ９０ Y), and optionally, the radionuclide conjugated to the molecule is lutetium １７７ as described in claim 10, the molecule.

12. The molecule according to any one of claims 9 to 11, wherein the therapeutic agent is conjugated to the portion of the molecule containing the tumor antigen-binding protein.

13. The molecule according to any one of claims 9 to 11, wherein the therapeutic agent is conjugated to the portion of the molecule containing the ECD or ligand-binding fragment of TGFβR.

14. The tumor antigen-binding protein specifically binds to CAIX, and the antigen-binding protein, It includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, FR1, FR2, FR3, and FR4 are each framework domains. CDR1, CDR2, and CDR3 are complementarity-determining regions, FR1a, FR2a, FR3a, and FR4a are each framework domains. CDR1a, CDR2a, and CDR3a are complementarity-determining regions, The molecule according to any one of claims 1 to 13, wherein any sequence of the complementarity-determining regions has an amino acid sequence listed in Table 2.

15. The molecule according to claim 14, wherein FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked via a linker in the form of a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues, which can be optionally used.

16. The molecule according to claim 14 or 15, wherein the tumor antigen-binding protein comprises an antigen-binding domain, the antigen-binding domain essentially consists of or comprises the amino acid sequence of SEQ ID NO: 52, 68, 84, 100, or 116 (VH) and / or the sequence described in SEQ ID NO: 132, 148, 164, 180, 196, or 212 (VL).

17. The tumor antigen-binding protein comprises an antigen-binding domain, and the antigen-binding domain is (i) VH including a complementarity determination region (CDR) 1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in Sequence ID No. 49, a CDR 2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence set in Sequence ID No. 50, and a CDR 3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in Sequence ID No.

51. (ii) A VH containing a sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the sequence described in sequence numbers 52, 68, 84, 100, or 116. (iii) VL including CDR1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 129, CDR2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 130, and CDR3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO:

131. (iv) A VL containing a sequence that is at least approximately 95% identical to the sequence described in sequence numbers 132, 148, 164, 180, 196, or 212. (v) VH containing CDR1 containing the sequence described in SEQ ID NO: 49, CDR2 containing the sequence described in SEQ ID NO: 50, and CDR3 containing the sequence described in SEQ ID NO:

51. (vi) VH containing the sequence described in sequence numbers 52, 68, 84, 100, or 116, (vii) VL containing CDR1 containing the sequence set in SEQ ID NO: 129, CDR2 containing the sequence described in SEQ ID NO: 130, and CDR3 containing the sequence described in SEQ ID NO:

131. (viiii) VL containing the sequence described in sequence numbers 132, 148, 164, 180, 196, or 212, (ix) VH including CDR1 containing the sequence described in SEQ ID NO: 49, CDR2 containing the sequence described in SEQ ID NO: 50, and CDR3 containing the sequence described in SEQ ID NO: 51, and VL including CDR1 containing the sequence set in SEQ ID NO: 129, CDR2 containing the sequence described in SEQ ID NO: 130, and CDR3 containing the sequence described in SEQ ID NO: 131, or (x) The molecule according to claim 14 or 15, comprising at least one of VH containing the sequence described in SEQ ID NO: 52, 68, 84, 100, or 116, and VL containing the sequence described in SEQ ID NO: 132, 148, 164, 180, 196, or 212.

18. The tumor antigen-binding protein contains a variable heavy chain that maintains binding affinity to CAIX and includes the amino acid sequence described in SEQ ID NO: 52, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or CAIX A molecule according to any one of claims 14, 15, or 17, comprising a variable light chain that maintains binding affinity to X and includes the amino acid sequence described in SEQ ID NO: 132, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

19. The molecule according to claim 18, wherein the tumor antigen-binding protein comprises the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 52 and / or the same CDR sequence as defined for the variable light chain of SEQ ID NO:

132.

20. The tumor antigen-binding protein specifically binds to PSMA, and the antigen-binding protein, It includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, FR1, FR2, FR3, and FR4 are each framework domains. CDR1, CDR2, and CDR3 are complementarity-determining regions, FR1a, FR2a, FR3a, and FR4a are each framework domains. CDR1a, CDR2a, and CDR3a are complementarity-determining regions, The molecule according to any one of claims 1 to 13, wherein one of the sequences in the complementarity-determining regions has an amino acid sequence listed in Table 1.

21. The molecule according to claim 20, wherein FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked via a linker in the form of a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues, which can be optionally used.

22. The molecule according to claim 20 or 21, wherein the tumor antigen-binding protein comprises an antigen-binding domain, and the antigen-binding domain essentially consists of or comprises the amino acid sequence of SEQ ID NOs: 4, 20, or 244 (VH) and 36 or 245 (VL) (in the order of N to C-terminus or C to N-terminus).

23. The tumor antigen-binding protein, (i) VH including a complementarity determination region (CDR) 1 containing at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 1, a CDR 2 containing at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence set in SEQ ID NO: 2, and a CDR 3 containing at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO:

3. (ii) A VH containing a sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the sequence described in Sequence ID No. 4, 20, or 244. (iii) VL including CDR1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in Sequence ID No. 33, CDR2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in Sequence ID No. 34, and CDR3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in Sequence ID No.

35. (iv) A VL containing a sequence that is at least about 95% identical to the sequence described in Sequence ID No. 36 or 245, (v) VH containing CDR1 containing the sequence described in SEQ ID NO: 1, CDR2 containing the sequence described in SEQ ID NO: 2, and CDR3 containing the sequence described in SEQ ID NO:

3. (vi) VH containing the sequence described in sequence number 4, 20, or 244, (vii) VL containing CDR1 containing the sequence set in sequence number 33, CDR2 containing the sequence described in sequence number 34, and CDR3 containing the sequence described in sequence number 45. (viiii) VL containing the sequence described in sequence number 36 or 245, (ix) VH including CDR1 containing the sequence described in SEQ ID NO: 1, CDR2 containing the sequence described in SEQ ID NO: 2, and CDR3 containing the sequence described in SEQ ID NO: 3, and VL including CDR1 containing the sequence set in SEQ ID NO: 33, CDR2 containing the sequence described in SEQ ID NO: 34, and CDR3 containing the sequence described in SEQ ID NO: 35, or (x) The molecule according to claim 20 or 21, comprising at least one of VH containing the sequence described in SEQ ID NO: 4, 20, or 244, and VL containing the sequence described in SEQ ID NO: 36 or 245.

24. The tumor antigen-binding protein contains a variable heavy chain that maintains binding affinity to PSMA and includes the amino acid sequence described in SEQ ID NO: 244, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or The molecule according to claim 20, 21, or 23, comprising a variable light chain that maintains binding affinity to PSMA and includes the amino acid sequence described in SEQ ID NO: 245, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

25. The molecule according to claim 24, wherein the tumor antigen-binding protein comprises the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 244 and / or the same CDR sequence as defined for the variable light chain of SEQ ID NO:

245.

26. The tumor antigen-binding protein specifically binds to PDGFRα, and the antigen-binding protein, It includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, FR1, FR2, FR3, and FR4 are each framework domains. CDR1, CDR2, and CDR3 are complementarity-determining regions, FR1a, FR2a, FR3a, and FR4a are each framework domains. CDR1a, CDR2a, and CDR3a are complementarity-determining regions, The molecule according to any one of claims 1 to 13, wherein one of the sequences in the complementarity-determining regions has an amino acid sequence listed in Table 5.

27. The molecule according to claim 26, wherein FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked via a linker in the form of a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues, which can be optionally used.

28. The molecule according to claim 26 or 27, wherein the tumor antigen-binding protein comprises an antigen-binding domain, and the antigen-binding domain essentially consists of or comprises the amino acid sequence of SEQ ID NOs. 265 and / or 266 (in the order N to C-terminus or C to N-terminus).

29. The tumor antigen-binding protein, (i) VH including a complementarity determination region (CDR) 1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 267, a CDR 2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence set in SEQ ID NO: 268, and a CDR 3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO:

269. (ii) A VH containing a sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the sequence described in Sequence ID No.

265. (iii) VL including CDR1 containing at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 270, CDR2 containing at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 271, and CDR3 containing at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO:

272. (iv) VL containing a sequence that is at least about 95% identical to the sequence described in Sequence ID No. 266, (v) VH containing CDR1 containing the sequence described in SEQ ID NO: 267, CDR2 containing the sequence described in SEQ ID NO: 268, and CDR3 containing the sequence described in SEQ ID NO:

269. (vi) VH containing the sequence described in Sequence ID No. 265, (vii) VL containing CDR1 containing the sequence set in SEQ ID NO: 270, CDR2 containing the sequence described in SEQ ID NO: 271, and CDR3 containing the sequence described in SEQ ID NO:

272. (viiii) VL containing the sequence described in sequence number 266, (ix) VH including CDR1 containing the sequence described in SEQ ID NO: 267, CDR2 containing the sequence described in SEQ ID NO: 268, and CDR3 containing the sequence described in SEQ ID NO: 269, and VL including CDR1 containing the sequence set in SEQ ID NO: 270, CDR2 containing the sequence described in SEQ ID NO: 271, and CDR3 containing the sequence described in SEQ ID NO: 272, or (x) The molecule according to claim 26 or 27, comprising at least one of VH containing the sequence described in SEQ ID NO: 265 and VL containing the sequence described in SEQ ID NO:

266.

30. The tumor antigen-binding protein contains a variable heavy chain that maintains binding affinity to PDGFRα and includes the amino acid sequence described in SEQ ID NO: 265, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or The molecule according to claim 26, 27, or 29, comprising a variable light chain that maintains binding affinity to PDGFRα and includes the amino acid sequence described in SEQ ID NO: 266, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical thereto.

31. The molecule according to claim 30, wherein the tumor antigen-binding protein comprises the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 265 and / or the same CDR sequence as defined for the variable light chain of SEQ ID NO:

266.

32. The tumor antigen-binding protein specifically binds to La / SSB, and the antigen-binding protein, It includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, FR1, FR2, FR3, and FR4 are each framework domains. CDR1, CDR2, and CDR3 are complementarity-determining regions, FR1a, FR2a, FR3a, and FR4a are each framework domains. CDR1a, CDR2a, and CDR3a are complementarity-determining regions, The molecule according to any one of claims to 13, wherein any sequence of the complementarity-determining regions has an amino acid sequence listed in Table 6.

33. The molecule according to claim 26, wherein FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked via a linker in the form of a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues, which can be optionally used.

34. The molecule according to claim 32 or 33, wherein the tumor antigen-binding protein comprises an antigen-binding domain, and the antigen-binding domain essentially consists of or comprises the amino acid sequence of SEQ ID NO: 303 and / or 304 (in the order of N to C-terminus or C to N-terminus).

35. The tumor antigen-binding protein, (i) VH including a complementarity determination region (CDR) 1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 305, a CDR 2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence set in SEQ ID NO: 306, and a CDR 3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 307, (ii) A VH containing a sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the sequence described in Sequence ID No.

303. (iii) VL including CDR1 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 308, CDR2 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO: 309, and CDR3 containing a sequence identical to at least approximately 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, and at least 99% of the sequence described in SEQ ID NO:

310. (iv) VL containing a sequence that is at least about 95% identical to the sequence described in Sequence ID No. 304, (v) VH containing CDR1 containing the sequence described in SEQ ID NO: 305, CDR2 containing the sequence described in SEQ ID NO: 306, and CDR3 containing the sequence described in SEQ ID NO:

307. (vi) VH containing the sequence described in Sequence ID No. 303, (vii) VL containing CDR1 containing the sequence set in SEQ ID NO: 308, CDR2 containing the sequence described in SEQ ID NO: 309, and CDR3 containing the sequence described in SEQ ID NO:

310. (viiii) VL containing the sequence described in sequence number 304, (ix) VH including CDR1 containing the sequence described in SEQ ID NO: 305, CDR2 containing the sequence described in SEQ ID NO: 306, and CDR3 containing the sequence described in SEQ ID NO: 307, and VL including CDR1 containing the sequence set in SEQ ID NO: 308, CDR2 containing the sequence described in SEQ ID NO: 309, and CDR3 containing the sequence described in SEQ ID NO: 310, or (x) The molecule according to claim 33 or 33, comprising at least one of VH containing the sequence described in SEQ ID NO: 303 and VL containing the sequence described in SEQ ID NO:

304.

36. The tumor antigen-binding protein contains a variable heavy chain that maintains binding affinity to La / SSB and includes the amino acid sequence described in SEQ ID NO: 303, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or The molecule according to claim 33, 34, or 35, comprising a variable light chain that maintains binding affinity to La / SSB and includes the amino acid sequence described in SEQ ID NO: 304, or a sequence that is at least 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or 97%, or 98%, or 99% identical thereto.

37. The molecule according to claim 36, wherein the tumor antigen-binding protein comprises the same CDR sequence as defined for the variable heavy chain of SEQ ID NO: 303 and / or the same CDR sequence as defined for the variable light chain of SEQ ID NO:

304.

38. The molecule according to any one of claims 1 to 37, wherein the antigen-binding protein includes a human constant region.

39. The molecule according to claim 38, wherein the heavy chain constant region comprises one or more amino acid substitutions for stabilizing the linkage of the TGFβR to the ECD or ligand-binding domain, and / or one or more amino acid substitutions for reducing the serum half-life of the molecule, as described herein.

40. The molecule according to any one of claims 1 to 39, wherein the ECD or ligand-binding domain of the TGFβR includes or consists of the amino acid sequence described in any one of SEQ ID NOs. 246 to 254 or the amino acid sequence described in Table 4.

41. The molecule according to any one of claims 1 to 40, wherein the ECD or ligand-binding domain of the TGFβR contains or comprises at least about 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in SEQ ID NO: 249 or 321, or a sequence identical thereto by at least about 80%, 81%, 82%, 84%, 84%, 85%, 86%, 87%, 88%, or 99%, and which retains the ability to bind to TGFβ.

42. The molecule according to any one of claims 1 to 19, wherein the molecule comprises an antibody for binding to CAIX, a heavy chain comprising the amino acid sequence described in SEQ ID NO: 257 or 258, and a light chain comprising the amino acid sequence described in SEQ ID NO:

259.

43. The molecule according to any one of claims 1 to 13 and 20 to 25, wherein the molecule comprises an antibody for binding to PSMA, a heavy chain comprising the amino acid sequence described in SEQ ID NO: 262 or 263, and a light chain comprising the amino acid sequence described in SEQ ID NO:

264.

44. - A heavy chain and a light chain, each chain comprising a variable region and a constant region, wherein at least one variable region binds to a tumor antigen, - A radioactive isotope conjugated to one or more amino acid residues of the heavy chain or light chain, A therapeutic IgG molecule comprising the extracellular domain (ECD) or ligand-binding fragment of a transformed growth factor β receptor (TGFβR).

45. The therapeutic IgG molecule according to claim 44, wherein the variable region of the heavy chain comprises the amino acid sequence described in any one of SEQ ID NOs: 52, 68, 84, 100, and 116.

46. The therapeutic IgG molecule according to claim 44 or 45, wherein the variable region of the light chain comprises the amino acid sequence described in any one of SEQ ID NOs: 132, 148, 164, 180, 196, and 212.

47. The therapeutic IgG molecule according to claim 44, wherein the variable region of the heavy chain comprises the amino acid sequence described in SEQ ID NO: 4, 20, or 244.

48. The therapeutic IgG molecule according to claim 44 or 47, wherein the variable region of the light chain comprises the amino acid sequence described in SEQ ID NO: 36 or 245.

49. The therapeutic IgG molecule according to claim 44, wherein the variable region of the heavy chain comprises the amino acid sequence described in SEQ ID NO:

265.

50. The therapeutic IgG molecule according to claim 44 or 49, wherein the variable region of the light chain comprises the amino acid sequence described in SEQ ID NO:

266.

51. The therapeutic IgG molecule according to claim 44, wherein the variable region of the heavy chain comprises the amino acid sequence described in SEQ ID NO:

303.

52. The therapeutic IgG molecule according to claim 44 or 51, wherein the variable region of the light chain comprises the amino acid sequence described in SEQ ID NO:

304.

53. The therapeutic IgG molecule according to any one of claims 44 to 52, wherein the heavy chain constant region of the molecule comprises one or more amino acid substitutions for altering the serum half-life of the molecule, optionally, the substitutions are located at position His310 or His435, preferably, both His310 and His435, or at equivalent positions thereto.

54. A therapeutic IgG molecule according to any one of claims 44 to 53, wherein the molecule further comprises (a) one or more amino acid substitutions that reduce the affinity of the molecule to one or more Fc gamma receptors compared to a wild-type antibody of class IgG, and / or (b) one or more amino acid substitutions that increase the stability of the CH1-CH2 hinge region in the molecule compared to a wild-type antibody of class IgG.

55. The therapeutic IgG molecule according to claim 54, wherein the one or more amino acid substitutions that reduce the affinity of the molecule to the Fc gamma receptor include substitutions at position Leu235 or an equivalent position.

56. The therapeutic IgG molecule according to claim 54 or 55, wherein the one or more amino acid substitutions that increase the stability of the CH1-CH2 hinge region in the molecule include substitutions in an IgG molecule.

57. The therapeutic antibody according to claim 55 or 56, wherein the heavy chain comprises the amino acid sequence described in SEQ ID NO:

231.

58. The therapeutic IgG molecule according to any one of claims 44 to 57, wherein the light chain comprises the amino acid sequence described in SEQ ID NO:

234.

59. The therapeutic antibody according to any one of claims 44 to 58, wherein the heavy chain comprises the amino acid sequence described in SEQ ID NO:

53.

60. The therapeutic IgG molecule according to any one of claims 44 to 59, wherein the light chain comprises the amino acid sequence described in Sequence ID No.

57.

61. The therapeutic IgG molecule according to any one of claims 44 to 60, wherein the antibody comprises the amino acid sequence described in SEQ ID NO: 53, and the antibody comprises the amino acid sequence described in SEQ ID NO:

57.

62. The aforementioned radioactive isotopes are actinium-225 (225Ac), astatine-211 (211At), bismuth-212 and bismuth-213 ( ２１２ Bi, ２１３ Bi), copper 67 ( ６７ Cu), iodine 123, 124, 125, or 131 ( １２３ I, １２４ I, １２５ I, １３１ I) ( １２３ I) Lead 212 ( ２１２ Pb), Lutetium-177 ( １７７ Lu), radium-223 and radium-224 ( ２２３ Ra, ２２４ Ra), Samarium-153 ( １５３ Sm), Scandium-47 ( ４７ Sc), Strontium-90 ( ９０ Sr), and yttrium 90 ( ９０ A therapeutic IgG molecule according to any one of claims 44 to 61, selected from the group consisting of Y).

63. The radioactive element is actinium-225 ( ２２５ Ac), Astatine 211 ( ２１１ At), and Lutetium-177 ( １７７ A therapeutic IgG molecule according to any one of claims 44 to 62, selected from Lu.

64. The therapeutic IgG molecule according to any one of claims 44 to 63, wherein the ECD or ligand-binding domain of TGFβR contains or consists of an amino acid sequence derived from TGFβRII.

65. The therapeutic IgG molecule according to any one of claims 44 to 64, wherein the ECD or ligand-binding domain of the TGFβR includes or consists of the amino acid sequence described in any one of SEQ ID NOs. 246 to 254 or the amino acid sequence described in Table 4.

66. A composition comprising a molecule according to any one of claims 1 to 43 or a therapeutic IgG molecule according to any one of claims 44 to 65, and a pharmaceutically acceptable excipient or carrier.

67. A bioconjugate comprising the molecule according to claims 1 to 8, conjugated to a chelator portion or a linker group.

68. The bioconjugate according to claim 67, wherein the chelator portion is selected from TMT (6,6''-bis[N,N'',N'''-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2''-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N''(N'''-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, Diamsar, DTPA, CHX-A''-DTPA, TETE, Te2A, HBED, DFO, DFOsq, and HOPO.

69. A method for treating cancer in an individual, comprising administering to an individual in need of such treatment a molecule according to any one of claims 1 to 43, or a therapeutic IgG molecule according to any one of claims 44 to 65, or a composition according to claim 66.

70. Use of a molecule according to any one of claims 1 to 43, or a therapeutic IgG molecule according to any one of claims 44 to 65, or a bioconjugate according to claim 67 or 68, in the manufacture of a pharmaceutical product for the treatment of cancer in a subject.

71. A molecule according to any one of claims 1 to 43, or a therapeutic IgG according to any one of claims 44 to 65, or a composition according to claim 66, for use in the treatment of cancer in a subject.

72. A method for treating cancer in an individual, the method comprising administering to a subject in need of the method (a) a molecule according to any one of claims 1 to 43, or a therapeutic IgG molecule according to any one of claims 44 to 54, or a composition according to claim 66, and (b) a molecule comprising an immunoglobulin moiety and a non-protein agent conjugated thereto, wherein the immunoglobulin moiety specifically binds to a tumor antigen, and the non-protein agent comprises the therapeutic moiety, preferably comprising a cytotoxin or a radioactive element.

73. The method according to claim 72, wherein the molecule or therapeutic molecule in (a) does not contain a radionuclide.

74. The method according to any one of claims 69, 72, or 73, or the use according to claim 70, or the use according to claim 71, wherein the subject has previously received treatment for cancer, and the previous treatment for cancer is suspected to cause or causes an increase in TGFβ activity in the tumor microenvironment.

75. The method or use according to claim 74, wherein the prior treatment for the cancer is selected from the group consisting of treatment with external beam radiation (EBR), treatment with chemotherapeutic agents, surgery or resection of the tumor, treatment with immunomodulatory agents including CPIs, treatment with molecularly targeted radionuclides (MTRs), and cell therapy, such as treatment with CART therapy.

76. A method for reducing or inhibiting radioactive TGFβ activity in a subject receiving or requiring radiotherapy for cancer, wherein the method comprises administering a molecule according to any one of claims 1 to 43, a therapeutic IgG molecule according to any one of claims 44 to 65, or a composition according to claim 66.

77. The method or use according to claim 76, wherein the method is for reducing or inhibiting radiation-induced fibrosis in the subject.

78. A method for enhancing or increasing the likelihood of success in treatment with an immune checkpoint inhibitor in a subject, the method comprising administering to a subject requiring treatment with an immune checkpoint inhibitor a molecule according to any one of claims 1 to 43, or a therapeutic IgG molecule according to any one of claims 44 to 65, or a composition according to claim 66.

79. The method or use of claim 78, wherein the subject has previously received or is receiving treatment with external beam radiation or molecular targeted radiation, or any other treatment that increases the activity of TGFβ in the tumor microenvironment.

80. The method according to any one of claims 69, 72, or 73, or the use according to claim 70, or the use according to claim 71, wherein the method comprises administering a treatment for cancer in combination with or afterward, wherein the treatment for cancer is selected from the group consisting of external beam radiation (EBR) treatment, chemotherapy, surgery or resection of a tumor, immunomodulatory agent including an immune checkpoint inhibitor, molecularly targeted radionuclide (MTR) treatment, cell therapy, for example, CART therapy, or the molecular or therapeutic IgG for the use according to claim 71.

81. A method for reducing or inhibiting radiation-induced TGFβ activity in an individual, - Administering the individual a molecule according to any one of claims 1 to 43, or a therapeutic IgG molecule according to any one of claims 44 to 65, or a composition according to claim 66, - The administration of a cancer treatment to the subject, wherein the cancer treatment is suspected to cause an increase in TGFβ activity in the tumor microenvironment, or is known to cause such an increase, A method for reducing or inhibiting radiation-induced TGFβ activity in the subject.

82. The method according to claim 81, wherein the treatment for the cancer is a molecularly targeted radionuclide (MTR).

83. The method according to claim 82, wherein the MTR comprises a radiolabeled antigen-binding protein for binding to CAIX.

84. The method according to claim 82, wherein the MTR comprises a radiolabeled antigen-binding protein for binding to PSMA.

85. The method according to claim 82, wherein the MTR comprises a radiolabeled antigen-binding protein for binding to PDGFRα.

86. The method according to claim 82, wherein the MTR comprises a radiolabeled antigen-binding protein for binding to La / SSB.

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