Fusion Polypeptides Capable of Activating Receptors

Abstract

A fusion polypeptide comprising (A) x -M-(A) y , wherein A and A′ are each polypeptides capable of binding a target receptor. The fusion polypeptides of the invention form multimeric proteins which activate the target receptor. A and A′ may be each be an antibody or fragment derived from an antibody specific for a target receptor, such as the same or different scFv fragments, and/or a ligand or ligand fragment or derivative capable of binding the target protein, M is a multimerizing component, and X and Y are independently a number between 1-10.

1 . A method for activating a Tie-1 receptor (Tie-1), comprising contacting a cell that expresses a Tie-1 with a Tie-1-binding protein, wherein the Tie-1-binding protein comprises a polypeptide that forms a dimer, wherein the polypeptide comprises, from N-terminal to C-terminal,
 a. a first antibody fragment that binds Tie-1, wherein the first antibody fragment comprises a first heavy chain variable region (VH1) and a second light chain variable region (VL1);   b. a multimerizing component comprising an Fc sequence, wherein the Fc sequence comprises a heavy chain IgG constant region; and,   c. either
 i. a second antibody fragment that binds Tie-1, wherein the second antibody fragment comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), or, 
 ii. a fibronectin domain derived from Angiopoeitin 1 (FD1) or Angiopoeitin 2 (FD2); 
   wherein the binding of the first antibody fragment to Tie-1 does not block binding of the second antibody fragment to Tie-1, binding of the second antibody fragment to Tie-1 does not block binding of the first antibody fragment to Tie-1, binding of the first antibody fragment to Tie-1 does not block binding of FD1 or FD2 to Tie-1, and binding of the FD1 or FD2 to Tie-1 does not block binding of the first antibody fragment to Tie-1.   
     
     
         2 . The method of  claim 1 , wherein the IgG is a human IgG1. 
     
     
         3 . The method of  claim 1 , wherein the FD1 or FD2 domain is FD1. 
     
     
         4 . The method of  claim 1 , wherein the cell is a mouse, rat, monkey, ape, or human cell. 
     
     
         5 . The method of  claim 1 , wherein the first and second antibody fragments are scFvs. 
     
     
         6 . The method of  claim 1 , wherein VH1, VL1, VH2, and VL2 comprise human framework regions. 
     
     
         7 . The method of  claim 1 , wherein the VH1, VL1, VH2, and VL2 comprise humanized framework regions. 
     
     
         8 . The method of  claim 1 , wherein the VH1, VL1, VH2, and VL2 comprise humanized CDRs. 
     
     
         9 . The method of  claim 1 , wherein VH1, VL1, VH2, and VL2 comprise humanized variable regions. 
     
     
         10 . A method for activating a Tie-1 receptor (Tie-1), comprising contacting a cell that expresses a Tie-1 to a Tie-1-binding protein, wherein the Tie-1-binding protein comprises a polypeptide that forms a dimer, wherein the polypeptide comprises, from N-terminal to C-terminal,
 a. a first antibody fragment that binds Tie-1, wherein the first antibody fragment comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1);   b. a human IgG1 constant region; and,   c. a second antibody fragment that binds Tie-1, wherein the second antibody fragment comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2),
 wherein the binding of the first antibody fragment to Tie-1 does not block binding of the second antibody fragment to Tie-1, and binding of the second antibody fragment to Tie-1 does not block binding of the first antibody fragment to Tie-1. 
   
     
     
         11 . The method of  claim 10 , wherein the first and second antibody fragments are scFvs. 
     
     
         12 . The method of  claim 10 , wherein VH1, VL1, VH2, and VL2 comprise human framework regions. 
     
     
         13 . The method of  claim 10 , wherein VH1, VL1, VH2, and VL2 comprise humanized framework regions. 
     
     
         14 . The method of  claim 10 , wherein the VH1, VL1, VH2, and VL2 comprise humanized CDRs. 
     
     
         15 . The method of  claim 10 , wherein the VH1, VL1, VH2, and VL2 comprise humanized variable regions. 
     
     
         16 . The method of  claim 10 , wherein the cell is a mouse, rat, monkey, ape, or human cell. 
     
     
         17 . A method for activating a Tie-1 receptor (Tie-1), comprising contacting a cell that expresses a Tie-1 to a Tie-1-binding protein, wherein the Tie-1-binding protein comprises a polypeptide that forms a dimer, wherein the polypeptide comprises, from N-terminal to C-terminal,
 a. an antibody fragment that binds Tie-1, wherein the antibody fragment comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1);   b. a human IgG1 constant region; and,   c. a fibronectin domain selected from Angiopoeitin-1 (FD1) or Angiopoeitin-2 (FD2);
 wherein the binding of the antibody fragment does not block binding of the FD1 or FD2 to Tie-1, and binding of the FD1 or FD2 to Tie-1 does not block binding of the antibody fragment to Tie-1. 
   
     
     
         18 . The method of  claim 17 , wherein the antibody fragment is an scFv. 
     
     
         19 . The method of  claim 17 , wherein VH1 and VL1 comprise human framework regions. 
     
     
         20 . The method of  claim 17 , wherein VH1 and VL1 comprise humanized framework regions. 
     
     
         21 . The method of  claim 17 , wherein the VH1, VL1, VH2, and VL2 comprise humanized CDRs. 
     
     
         22 . The method of  claim 17 , wherein the VH1, VL1, VH2, and VL2 comprise humanized variable regions. 
     
     
         23 . The method of  claim 17 , wherein the FD1 or FD2 domain is an FD1. 
     
     
         24 . The method of  claim 17 , wherein the cell is a mouse, rat, monkey, ape, or human cell.