Bispecific signaling agents and uses thereof

Abstract

The present invention relates, in part, to bispecific chimeric proteins that find use in various immunotherapies based on various properties, including, for example, a dual immune cell recruitment and immune signal delivery function.

What is claimed is: 
     
         1 . A chimeric protein comprising:
 (a) two or more targeting moieties, said targeting moieties comprising recognition domains which specifically bind to antigens or receptors of interest; and   (b) a modified signaling agent, said modified signaling agent having one or more mutations that confer improved safety as compared to a wild type signaling agent,   
       wherein the targeting moieties and modified signaling agent are optionally connected with one or more linkers. 
     
     
         2 . The chimeric protein of  claim 1 , wherein one or more of the targeting moieties is directed against a tumor cell. 
     
     
         3 . The chimeric protein of  claim 1 , wherein one or more of the targeting moieties is directed against an immune cell. 
     
     
         4 . The chimeric protein of  claim 3 , wherein the immune cell is selected from a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, and a NK cell. 
     
     
         5 . The chimeric protein of any of the above claims, wherein (i) one or more of the targeting moieties is directed against an immune cell selected from a T cell, a B cell, a dendritic cell, a neutrophil, a macrophage, and a NK cell and (ii) one or more of the targeting moieties is directed against a tumor cell. 
     
     
         6 . 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. 
     
     
         7 . The chimeric protein of any of the above claims, wherein the recognition domain is a single-domain antibody (VHH) or an scFv. 
     
     
         8 . The chimeric protein of any of the above claims, wherein the recognition domain is a V HH , humanized V HH , or camelized V HH . 
     
     
         9 . The chimeric protein of any of the above claims, wherein the recognition domain functionally modulates the antigen or receptor of interest. 
     
     
         10 . 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. 
     
     
         11 . The chimeric protein of any of the above claims, wherein the antigen of interest is selected from one or more of CD8, CD3, CD33, SLAMF7 (CS1), Sirplec, DNAM, and CLEC9A. 
     
     
         12 . The chimeric protein of any of the above claims, wherein the antigen of interest is an immune checkpoint, optionally selected from PD-1, PD-L1, and PD-L2. 
     
     
         13 . 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. 
     
     
         14 . 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. 
     
     
         15 . 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. 
     
     
         16 . The chimeric protein of  claim 13 , wherein the one or more mutations allow for attenuation of activity. 
     
     
         17 . The chimeric protein of  claim 16 , wherein agonistic or antagonistic activity is attenuated. 
     
     
         18 . The chimeric protein of  claim 16  or  17 , wherein the signaling agent comprises one or more mutations which convert its activity from agonistic to antagonistic. 
     
     
         19 . The chimeric protein of  claim 13 , wherein the mutation confers reduced affinity or activity that is restorable by attachment to one or more targeting moiety. 
     
     
         20 . The chimeric protein of  claim 14 , wherein the mutation confers substantially reduced or ablated affinity or activity that is not substantially restorable by attachment to one or more targeting moiety. 
     
     
         21 . The chimeric protein of any of the above claims, wherein the chimeric protein has a molecular mass of about 50 KDa or higher. 
     
     
         22 . The chimeric protein of any of the above claims, wherein the chimeric protein substantially evades filtration by the kidney. 
     
     
         23 . The chimeric protein of any of the above claims, wherein the modified signaling agent is IFNα2a, optionally having one or more mutations at positions L153, R149, and M148. 
     
     
         24 . The chimeric protein of any of the above claims, wherein the modified signaling agent is IL-1β, optionally having one or more mutations at positions R120 and H146. 
     
     
         25 . The chimeric protein of any of the above claims, wherein the modified signaling agent is TNF, optionally having one or more mutations at positions Y87 and Y115. 
     
     
         26 . 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. 
     
     
         27 . A recombinant nucleic acid composition encoding one or chimeric proteins of any one of the above claims. 
     
     
         28 . A host cell comprising a nucleic acid of  claim 27 . 
     
     
         29 . 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. 
     
     
         30 . The method of  claim 29 , 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. 
     
     
         31 . A method for treating an autoimmune disease or disorder, comprising administering an effective amount of the chimeric protein of any of the above claims to a patient in need thereof. 
     
     
         32 . The method of  claim 31 , wherein the autoimmune disease or disorder, is selected from one or more of Crohn's Disease, systemic lupus erythematosis, rheumatoid arthritis or juvenile rheumatoid arthritis, ulcerative colitis immune disorders such as eosinophilic fasciitis, hypoimmunoglobulinemia, or thymoma/thymic carcinoma, graft versus host disease, preleukemia, Nonhematologic syndrome (e.g. Down's, Dubowwitz, Seckel), Felty syndrome, hemolytic uremic syndrome, myelodysplasic syndrome, nocturnal paroxysmal hemoglobinuria, osteomyelofibrosis, pancytopenia, pure red-cell aplasia, Schoenlein-Henoch purpura, malaria, protein starvation, menorrhagia, systemic sclerosis, liver cirrhosis, hypometabolic states, and congestive heart failure. 
     
     
         33 . A chimeric protein of any of the above claims for use in the treatment of one or more of: cancer, infections, immune disorders, autoimmune diseases, cardiovascular diseases, wound, ischemia-related diseases, neurodegenerative diseases, and/or metabolic diseases, as described herein. 
     
     
         34 . Use of a chimeric protein of any of the above claims for the manufacture of a medicament for treating one or more of: cancer, infections, immune disorders, autoimmune diseases, cardiovascular diseases, wound, ischemia-related diseases, neurodegenerative diseases, and/or metabolic diseases, as described herein.