Bi-functional chimeric proteins and uses thereof

Abstract

The present invention relates, in part, to targeted chimeric proteins with beneficial therapeutic effects, including, for example, effects mediated by mutant forms of soluble agents that are part of the chimeric proteins. Pharmaceutical compositions comprising the chimeric proteins are also provided. The present invention finds use in the treatment of various disease and disorders.

What is claimed is: 
     
         1 . A chimeric protein, comprising:
 (a) a modified soluble agent having a reduced affinity for a first receptor and a second receptor and   (b) a targeting moiety, wherein:
 the first receptor is a therapeutic receptor that mediates a therapeutic effect and 
 the modified soluble agent and the targeting moiety are optionally connected with a linker. 
   
     
     
         2 . The chimeric protein of  claim 1 , wherein the modified soluble agent comprises one or more mutations. 
     
     
         3 . The chimeric protein of  claim 2 , wherein the modified soluble agent comprises one or more mutations conferring reduced affinity for the first receptor relative to a wild type soluble agent. 
     
     
         4 . The chimeric protein of  claim 3 , wherein the one or more mutations allow for attenuation of activity. 
     
     
         5 . The chimeric protein of  claim 4 , wherein agonistic activity is attenuated. 
     
     
         6 . The chimeric protein of  claim 4 , wherein antagonistic activity is attenuated. 
     
     
         7 . The chimeric protein of  claim 2 , wherein the modified soluble agent comprises one or more mutations conferring substantially reduced or ablated affinity for the second receptor relative to a wild type soluble agent. 
     
     
         8 . The chimeric protein of  claim 7 , wherein the one or more mutations prevent non-therapeutic signaling or undesirable sequestration of the chimeric protein 
     
     
         9 . The chimeric protein of any one of the above claims, wherein the modified soluble agent is human TNF-α, optionally having mutations at one or more amino acid positions selected from R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, A109, P113, Y115, E127, N137, D143, A145, and E146 with reference to SEQ ID NO:5. 
     
     
         10 . The chimeric protein of  claim 10 , wherein the modified human TNF-α moiety has one or more substitution mutations selected from R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, 197A, 197Q, 197S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145R, A145T, and E146K. 
     
     
         11 . The chimeric protein of  claim 10 , wherein the modified human TNF-α moiety comprises an E146K mutation. 
     
     
         12 . The chimeric protein of  claim 10 , wherein the modified human TNF-α moiety comprises Y87H and E146K mutations. 
     
     
         13 . The chimeric protein of  claim 10 , wherein the modified human TNF-α moiety comprises Y87H and A145R mutations. 
     
     
         14 . The chimeric protein of  claim 10 , wherein the modified human TNF-α moiety comprises R32W and S86T mutations. 
     
     
         15 . The chimeric protein of any one of  claims 9 - 14 , wherein the chimeric protein comprises a single polypeptide chain having three copies of modified human TNF-α. 
     
     
         16 . The chimeric protein of any of the above claims, wherein the targeting moiety comprises an antibody or derivative thereof, 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 (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, or a synthetic molecule. 
     
     
         18 . The chimeric protein of any of the above claims, wherein the targeting moiety is a single-domain antibody (NANOBODY). 
     
     
         19 . The chimeric protein of any of the above claims, wherein the targeting moiety is a V HH , humanized V HH , or camelized V HH . 
     
     
         20 . The chimeric protein of any one of the above claims, wherein the targeting moiety binds to CD20. 
     
     
         21 . The chimeric protein of any of the above claims, wherein the linker is substantially comprised of glycine and serine residues. 
     
     
         22 . The chimeric protein of  claim 1 , wherein the modified soluble agent has a lower side effect profile as compared to a wild type soluble agent. 
     
     
         23 . The chimeric protein of  claim 1 , wherein the modified soluble agent 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. 
     
     
         24 . A method for treating cancer, comprising administering an effective amount of the chimeric protein of any of the above claims. 
     
     
         25 . The method of  claim 24 , wherein the treatment allows for continued administration of the chimeric protein after a cancer patient's chemotherapy is finished. 
     
     
         26 . The method of  claim 24 , wherein the treatment allows for treatment of a cancer patient without dose reduction relative to a non-cancer patient. 
     
     
         27 . The method of  claim 24 , wherein the treatment allows for treatment of a cancer patient receiving chemotherapy and considered curable.