TARGETED TGFß INHIBITION

Abstract

This invention relates generally to bifunctional molecules including (a) a TGFβRII or fragment thereof capable of binding TGFβ and (b) an antibody, or antigen binding fragment thereof, that binds to an immune checkpoint protein, such as Programmed Death Ligand 1 (PD-L1), uses of such molecules (e.g., for treating cancer), and methods of making such molecules.

1 . A protein comprising:
 a) human Transforming Growth Factor β Receptor II (TGFβRII), or a fragment thereof capable of binding TGFβ; and   b) an antibody, or an antigen-binding fragment thereof, that binds human protein Programmed Death Ligand 1 (PD-L1).   
     
     
         2 . A polypeptide comprising:
 a) at least a variable domain of a heavy chain of an antibody that binds human protein Programmed Death Ligand 1 (PD-L1); and   b) human TGFβRII, or a fragment thereof capable of binding TGFβ.   
     
     
         3 . The polypeptide of  claim 2 , further comprising an amino acid linker connecting the C-terminus of the variable domain to the N-terminus of the human TGFβRII or fragment thereof. 
     
     
         4 . The polypeptide of  claim 3 , comprising the amino acid sequence of SEQ ID NO:3. 
     
     
         5 . A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to  claim 2 . 
     
     
         6 . The nucleic acid of  claim 5 , further comprising a second nucleotide sequence encoding at least a variable domain of a light chain of an antibody which, when combined with the polypeptide, forms an antigen-binding site that binds PD-L1. 
     
     
         7 . The nucleic acid of  claim 6 , wherein the second nucleotide sequence encodes the amino acid sequence of SEQ ID NO:1 (secreted anti-PD-L1 lambda light chain). 
     
     
         8 . A cell comprising the nucleic acid of  claim 5  and a second nucleic acid encoding at least a variable domain of a light chain of an antibody which, when combined with the polypeptide, forms an antigen-binding site that binds PD-L1. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . A method of producing a protein comprising a) TGFβRII, or a fragment thereof capable of binding TGFβ, and b) an antibody, or an antigen-binding fragment thereof, that binds human protein Programmed Death Ligand 1 (PD-L1), the method comprising maintaining a cell according to  claim 8  under conditions permitting expression of the protein. 
     
     
         12 . The method of  claim 11 , further comprising harvesting the protein. 
     
     
         13 . A protein comprising:
 a polypeptide according to  claim 2 ; and   at least a variable domain of a light chain of an antibody which, when combined with the polypeptide, forms an antigen-binding site that binds PD-L1.   
     
     
         14 . The protein of  claim 13 , comprising two polypeptides each having an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:3, and two additional polypeptides each having an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:1. 
     
     
         15 - 25 . (canceled) 
     
     
         26 . A method of promoting localized depletion of TGFβ, the method comprising administering a protein according to  claim 1 , wherein the protein binds TGFβ in solution, binds PD-L1 on a cell surface, and carries the bound TGFβ into the cell. 
     
     
         27 . A method of inhibiting SMAD3 phosphorylation in a cell, the method comprising exposing the cell to a protein according to  claim 1 . 
     
     
         28 . (canceled) 
     
     
         29 . A method of inhibiting tumor growth, the method comprising exposing the tumor to a protein according to  claim 1 . 
     
     
         30 . The method of  claim 29 , further comprising exposing the tumor to radiation or a chemotherapeutic. 
     
     
         31 . (canceled) 
     
     
         32 . A method of treating cancer, the method comprising administering to a cancer patient a protein according to  claim 1 . 
     
     
         33 . The method of  claim 32 , further comprising:
 (a) exposing the cancer patient to radiation;   (b) or administering a chemotherapeutic.   
     
     
         34 . (canceled) 
     
     
         35 . The method according to  claim 29 , wherein the tumor is selected from the group consisting of colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, myeloma, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes. 
     
     
         36 . The protein of  claim 1 , wherein the antibody, or the antigen-binding fragment thereof, that binds human protein Programmed Death Ligand 1 (PD-L1), comprises a heavy chain variable region polypeptide comprising an HVR-H1, HVR-H2, and HVR-H3 sequence and a light chain variable region polypeptide comprising an HVR-L1, HVR-L2, and HVR-L3 sequence, wherein
 the HVR-H1 sequence is X 1 YX 2 MX 3  (SEQ ID NO:21);   the HVR-H2 sequence is SIYPSGGX 4  TFY ADX 5  VKG (SEQ ID NO:22);   the HVR-H3 sequence is IKLGTVTTVX 6 Y (SEQ ID NO:23); and further wherein   X 1  is K, R, T, Q, G, A, W, M, I, or S; X 2  is V, R, K, L, M, or I; X 3  is H, T, N, Q, A, V, Y, W, F, or M; X 4  is F or I; X 5  is S or T; and X 6  is E or D; and wherein the HVR-L1 sequence is TGTX 7 X 8 DVGX 9 YNYVS (SEQ ID NO:28);   the HVR-L2 sequence is X 10 VX 11 X 12 RPS (SEQ ID NO:29);   the HVR-L3 sequence is SSX 13 TX 14 X 15 X 16 X 17 RV (SEQ ID NO:30); and further wherein   X 7  is N or 5; X 8  is T, R, or S; X 9  is A or G; X 10  is E or D; is I, N or S;   X 12  is D, H or N; X 13  is F or Y; X 14  is N or S; X 15  is R, T or S; X 16  is G or S; and   X 17  is I or T.