Therapeutic targeting of non-cellular structures

Abstract

The present invention relates, in part, to chimeric proteins that bind a non-cellular structure and their use as therapeutic agents. The present invention further relates to pharmaceutical compositions comprising the chimeric proteins and their use in the treatment of various diseases.

What is claimed is: 
     
         1 . A chimeric protein comprising:
 (a) one or more modified signaling agents, said one or more modified signaling agent having one or more modifications that confer improved safety as compared to a wild type signaling agent and said signaling agent being an interferon;   (b) one or more targeting moieties, said targeting moieties comprising a recognition domain which specifically binds to tenascin, wherein   
       the targeting moiety and signaling agent are optionally connected with one or more linkers. 
     
     
         2 . The chimeric protein of  claim 1 , wherein the tenascin is selected from a tenascin-C, tenascin-R, tenascin-X, and tenascin-W. 
     
     
         3 . The chimeric protein of  claim 2 , wherein the tenascin is tenascin-CA1. 
     
     
         4 . The chimeric protein of any of the above claims, wherein the recognition domain is a full-length antibody, a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (e.g. cysteine knot protein, knottin), a darpin, an anticalin, an adnectin, an aptamer, a Fv, a Fab, a Fab′, a F(ab′) 2 , a peptide mimetic molecule, a natural ligand for a receptor, or a synthetic molecule. 
     
     
         5 . The chimeric protein of any of the above claims, wherein the recognition domain is a single-domain antibody. 
     
     
         6 . The chimeric protein of any of the above claims, wherein the recognition domain is a V HH , humanized V HH , or camelized V HH . 
     
     
         7 . The chimeric protein of any of the above claims, wherein the recognition domain functionally modulates the antigen or receptor of interest. 
     
     
         8 . The chimeric protein of any of the above claims, wherein the recognition domain binds but does not functionally modulate the antigen or receptor of interest. 
     
     
         9 . The chimeric protein of  claim 1 , wherein the modified signaling agent comprises one or more mutations conferring reduced affinity or activity for a receptor relative to a wild type signaling agent. 
     
     
         10 . The chimeric protein of  claim 1 , wherein the modified signaling agent comprises one or more mutations conferring substantially reduced or ablated affinity or activity for a receptor relative to a wild type signaling agent. 
     
     
         11 . The chimeric protein of  claim 1 , wherein the modified signaling agent comprises both (a) one or more mutations conferring substantially reduced or ablated affinity for a receptor relative to a wild type signaling agent and (b) one or more mutations conferring reduced affinity or activity for a receptor relative to a wild type signaling agent; and wherein the receptors are different. 
     
     
         12 . The chimeric protein of  claim 11 , wherein the one or more mutations allow for attenuation of activity. 
     
     
         13 . The chimeric protein of  claim 12 , wherein agonistic or antagonistic activity is attenuated. 
     
     
         14 . The chimeric protein of  claim 11  or  12 , wherein the signaling agent comprises one or more mutations which convert its activity from agonistic to antagonistic. 
     
     
         15 . The chimeric protein of  claim 9 , wherein the mutation confers reduced affinity or activity that is restorable by attachment to one or more targeting moiety. 
     
     
         16 . The chimeric protein of  claim 10 , wherein the mutation confers substantially reduced or ablated affinity or activity that is not substantially restorable by attachment to one or more targeting moiety. 
     
     
         17 . The chimeric protein of any of the above claims, wherein the modified signaling agent is a modified human interferon. 
     
     
         18 . The chimeric protein of  claim 17 , wherein the modified signaling agent is a modified human IFNα2. 
     
     
         19 . The chimeric protein of  claim 18 , wherein the modified human IFNα2 comprises one or more mutations at positions 144-154, optionally selected from M148A, R149A, and L153A. 
     
     
         20 . The chimeric protein of any one of the above claims, wherein the chimeric protein is suitable for use in a patient having one or more of: cancer, infections, immune disorders, autoimmune diseases, cardiovascular diseases, wound, ischemia-related diseases, neurodegenerative diseases, and/or metabolic diseases. 
     
     
         21 . The chimeric protein of any one of the above claims, wherein the chimeric protein comprises two signaling agents or two targeting moieties or two of both. 
     
     
         22 . The chimeric protein of any one of the above claims, wherein the chimeric protein comprises three signaling agents or three targeting moieties or three of both. 
     
     
         23 . The chimeric protein of any one of the above claims, wherein the chimeric protein comprises a targeting moiety directed against a non-cellular structure and a targeting moiety directed against a cellular structure. 
     
     
         24 . The chimeric protein of any one of the above claims, wherein the chimeric protein comprises two targeting moieties directed against non-cellular structures. 
     
     
         25 . The chimeric protein of  claim 23 , wherein one of the targeting moieties is directed against Clec9A, CD8, or CD3. 
     
     
         26 . The chimeric protein of  claim 23 , wherein one of the targeting moieties is directed against PD-1. 
     
     
         27 . The chimeric protein of  claim 23 , wherein one of the targeting moieties is directed against PD-L1. 
     
     
         28 . A recombinant nucleic acid composition encoding one or chimeric proteins of any one of the above claims. 
     
     
         29 . A host cell comprising a nucleic acid of  claim 28 . 
     
     
         30 . A method for treating cancer, comprising administering an effective amount of the chimeric protein of any of the above claims to a patient in need thereof. 
     
     
         31 . The method of  claim 30 , wherein the method prevents or reduces deregulation and/or disorganization of the non-cellular structure relative to a cancerous state. 
     
     
         32 . The method of  claim 30  or  31 , wherein the method prevents or reduces alterations in the amount, stiffness, composition, or topography of the non-cellular structure which are characteristic of cancer. 
     
     
         33 . The method of any one of  claims 30 - 32 , wherein the method reduces activity of one or more enzymes that promote deregulation and/or disorganization of the non-cellular structure, optionally from matrix metalloproteinases (MMPs), disintegrins, metalloproteinases, optionally having thrombospondin motifs, and the serine proteases, including plasmin. 
     
     
         34 . The method of any one of  claims 30 - 33 , wherein the method reduces oncogenic effects of various growth factor signaling pathways associated with deregulation and/or disorganization of the non-cellular structure. 
     
     
         35 . The method of any one of  claims 30 - 34 , wherein the method reduces or impairs the progression of the cancer and/or tumor cell metastasis. 
     
     
         36 . The method of any one of  claims 30 - 35 , wherein the method prevents or reduces cancer stem cell overexpansion and/or loss of differentiation. 
     
     
         37 . The method of any one of  claims 30 - 36 , wherein the method prevents or reduces the formation of a tumorigenic microenvironment. 
     
     
         38 . The method of any one of  claims 30 - 37 , wherein the method prevents or reduces tumor angiogenesis and/or tumor inflammation. 
     
     
         39 . The method of any one of  claims 30 - 38 , wherein the cancer is selected form one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome. 
     
     
         40 . The method of  claim 39 , wherein the cancer is breast cancer. 
     
     
         41 . A pharmaceutical composition, comprising the chimeric protein of any one of  claims 1 - 27 . 
     
     
         42 . The chimeric protein of any one of  claims 1 - 27 , for use in treatment of cancer. 
     
     
         43 . The chimeric protein of any one of  claims 1 - 27 , for use in treatment of a cancer selected form one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome. 
     
     
         44 . A method for treating cancer, comprising administering an effective amount of a first chimeric protein and a second chimeric protein, wherein the first chimeric protein is a chimeric protein of any one of the above claims, and where the second chimeric protein comprises: one or more modified signaling agents, said one or more modified signaling agent having one or more modifications that confer improved safety as compared to a wild type signaling agent and one or more targeting moieties, said targeting moieties comprising a recognition domain which specifically binds to an antigen or receptor of interest on a cell, to a patient in need thereof. 
     
     
         45 . The method of  claim 44 , wherein the first chimeric protein comprises a modified TNF signaling agent. 
     
     
         46 . The method of  claim 45 , wherein the modified TNF signaling agent is modified human TNF-α and comprises a mutation at position 87, optionally selected from Y87Q, Y87L, Y87A, and Y87F. 
     
     
         47 . The method of any one of  claims 44 - 46 , wherein the second chimeric protein comprises a targeting moiety comprising a recognition domain which specifically binds CD8. 
     
     
         48 . The method of  claim 47 , wherein the first chimeric protein comprises a targeting moiety comprising a recognition domain which specifically binds CD8 and a modified IFN signaling agent. 
     
     
         49 . The method of  claim 48 , wherein the modified IFN signaling agent is a human IFNα2 comprises one or more mutations at positions 144-154, optionally selected from M148A, R149A, and L153A. 
     
     
         50 . The method of any one of  claims 44 - 49 , wherein the cancer is selected form one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome. 
     
     
         51 . A method for treating cancer, comprising administering an effective amount of the chimeric protein of any of the above claims and a chemotherapeutic agent to a patient in need thereof. 
     
     
         52 . The method of  claim 51 , wherein the chimeric protein comprises a modified IFN signaling agent. 
     
     
         53 . The method of  claim 52 , wherein the modified IFN signaling agent is human IFNα2 and comprises one or more mutations at positions 144-154, optionally selected from M148A, R149A, and L153A. 
     
     
         54 . The method of  claim 51 , wherein the chimeric protein comprises a modified TNF signaling agent. 
     
     
         55 . The method of  claim 54 , wherein the modified TNF signaling agent is modified human TNF-α and comprises a mutation at position 87, optionally selected from Y87Q, Y87L, Y87A, and Y87F. 
     
     
         56 . The method of any one of  claims 51 - 55 , wherein the chemotherapeutic agent is a DNA-intercalating agent. 
     
     
         57 . The method of  claim 56 , wherein the DNA-intercalating agent is selected from doxorubicin, cisplatin, daunorubicin, and epirubicin. 
     
     
         58 . The method of  claim 57 , wherein the DNA-intercalating agent is doxorubicin. 
     
     
         59 . The method of any one of  claims 51 - 58 , wherein the cancer is selected form one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome. 
     
     
         60 . A method for treating cancer, comprising administering to a patient in need thereof an effective amount of:
 a) a chimeric protein comprising a targeting moiety comprising a recognition domain which specifically binds TNCA and a modified IFN signaling agent; and   b) TNF.   
     
     
         61 . The method of  claim 60 , wherein the modified IFN signaling agent is human IFNα2 and comprises one or more mutations at positions 144-154, optionally selected from M148A, R149A, and L153A. 
     
     
         62 . The method of  claim 60  or  61 , wherein the cancer is selected form one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome. 
     
     
         63 . The chimeric protein of any one of  claims 1 - 43 , for use in the manufacture of a medicament for treating one or more of cancer, infections, immune disorders, inflammatory diseases or conditions, and autoimmune disease