Multimerization technologies

Abstract

The present invention provides, among other things, dimeric multispecific binding agents (e.g., fusion proteins comprising antibody components) that have improved properties over multispecific binding agents without the capability of dimerization.

1 - 36 . (canceled) 
     
     
         37 . A method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific binding agent comprised of two fusion proteins, wherein each of the fusion proteins comprise from N-terminus to C-terminus:
 (i) a first antibody component that binds a tumor antigen;   (ii) a second antibody component that binds an antigen present on an immune effector cell; and   (iii) a dimerization component comprising a human hepatocyte nuclear factor-1 alpha (HNF-1α) element,   wherein the HNF-1α element comprises a sequence that is at least 90% identical to residues 1-32 of SEQ ID NO:1, and   wherein the fusion proteins can dimerize through the dimerization component to form a homodimer.   
     
     
         38 . The method of  claim 37 , wherein the tumor antigen is selected from alpha fetoprotein (AFP), CA15-3, CA27-29, CA19-9, CA-125, calretinin, carcinoembryonic antigen, CD34, CD99, CDI 17, chromogranin, cytokeratin, desmin, epithelial membrane protein (EMA), Factor VIII, CD31 FLI, GD2, GD3, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-I5), HMB-45, human chorionic gonadotropin (hCG), inhibin, keratin, CD45, a lymphocyte marker, MART-I (Melan-A), Myo DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen, S 100 protein, smooth muscle actin (SMA), synaptophysin, thyroglobulin, thyroid transcription factor-I, tumor M2-PK, and vimentin. 
     
     
         39 . The method of  claim 37 , wherein the tumor antigen is GD2. 
     
     
         40 . The method of  claim 37 , wherein the immune effector cell is selected from a T cell, a natural killer (NK) cell, a macrophage, a granulocyte and an antibody-dependent cytotoxic cell. 
     
     
         41 . The method of  claim 37 , wherein the antigen present on an immune effector cell is selected from CD1d, CD3, CD4, CD7, CD8, CD13, CD14, CD16, CD31, CD38, CD56, CD68, MAC-1/MAC-3, IL2Rα, OX40, Ly49 and CD94. 
     
     
         42 . The method of  claim 37 , wherein the antigen present on an immune effector cell is CD3. 
     
     
         43 . The method of  claim 37 , wherein the dimerization component comprises amino acid residues 1-32 of SEQ ID NO:1. 
     
     
         44 . The method of  claim 37 , wherein each of the fusion proteins comprise a sequence that is at least 90% identical to any one of SEQ ID NO: 2-23. 
     
     
         45 . The method of  claim 37 , wherein each of the fusion proteins comprise a sequence that is selected from any one of SEQ ID NO: 2-23. 
     
     
         46 . A method of treating a GD2-positive cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific binding agent comprised of two fusion proteins, wherein each of the fusion proteins comprise from N-terminus to C-terminus:
 a first antibody component that binds to GD2,   a second antibody component that binds to an antigen present on an immune effector cell, and   a dimerization component comprising a sequence that is at least 90% identical to residues 1-32 of SEQ ID NO:1, such that the fusion proteins dimerize to form a homodimeric bispecific binding agent.   
     
     
         47 . The method of  claim 46 , wherein the immune effector cell is selected from a T cell, a natural killer (NK) cell, a macrophage, a granulocyte and an antibody-dependent cytotoxic cell. 
     
     
         48 . The method of  claim 46 , wherein the antigen present on an immune effector cell is selected from CD1d, CD3, CD4, CD7, CD8, CD13, CD14, CD16, CD31, CD38, CD56, CD68, MAC-1/MAC-3, IL2Rα, OX40, Ly49 and CD94. 
     
     
         49 . The method of  claim 46 , wherein the antigen present on an immune effector cell is CD3. 
     
     
         50 . The method of  claim 46 , wherein the dimerization component comprises amino acid residues 1-32 of SEQ ID NO:1. 
     
     
         51 . The method of  claim 46 , wherein each of the fusion proteins comprise a sequence that is at least 90% identical to any one of SEQ ID NO: 2-23. 
     
     
         52 . The method of  claim 46 , wherein each of the fusion proteins comprise a sequence that is selected from any one of SEQ ID NO: 2-23. 
     
     
         53 . A bispecific binding agent comprised of two fusion proteins, wherein each of the fusion proteins comprise from N-terminus to C-terminus:
 (i) a first antibody component that binds a tumor antigen;   (ii) a second antibody component that binds a toxin; and   (iii) a dimerization component comprising a human hepatocyte nuclear factor-1 alpha (HNF-1α) element,   wherein the HNF-1α element comprises a sequence that is at least 90% identical to residues 1-32 of SEQ ID NO:1, and   wherein the fusion proteins can dimerize through the dimerization component to form a homodimer.   
     
     
         54 . The binding agent of  claim 53 , wherein the tumor antigen is selected from alpha fetoprotein (AFP), CA15-3, CA27-29, CA19-9, CA-125, calretinin, carcinoembryonic antigen, CD34, CD99, CDI 17, chromogranin, cytokeratin, desmin, epithelial membrane protein (EMA), Factor VIII, CD31 FLI, GD2, GD3, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-I5), HMB-45, human chorionic gonadotropin (hCG), inhibin, keratin, CD45, a lymphocyte marker, MART-I (Melan-A), Myo DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen, S 100 protein, smooth muscle actin (SMA), synaptophysin, thyroglobulin, thyroid transcription factor-I, tumor M2-PK, and vimentin. 
     
     
         55 . The binding agent of  claim 53 , wherein the tumor antigen is GD2. 
     
     
         56 . The binding agent of  claim 53 , wherein the second antibody component binds a toxin selected from saporin and vinca alkaloid. 
     
     
         57 . The binding agent of  claim 53 , wherein the dimerization component comprises amino acid residues 1-32 of SEQ ID NO:1. 
     
     
         58 . A pharmaceutical composition comprising the dimeric bispecific binding agent of  claim 53  and a pharmaceutically acceptable carrier. 
     
     
         59 . The pharmaceutical composition of  claim 58 , further comprising the toxin. 
     
     
         60 . A method of preparing a pharmaceutical composition, the method comprising steps of:
 providing a bispecific binding agent as recited in  claim 53 ; and   combining the provided bispecific binding agent with at least one pharmaceutically acceptable carrier or excipient to prepare a pharmaceutical composition.   
     
     
         61 . A method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific binding agent comprised of two fusion proteins, wherein each of the fusion proteins comprise from N-terminus to C-terminus:
 (i) a first antibody component that binds a tumor antigen;   (ii) a second antibody component that binds to a toxin; and   (iii) a dimerization component comprising a human hepatocyte nuclear factor-1 alpha (HNF-1α) element,   wherein the HNF-1α element comprises a sequence that is at least 90% identical to residues 1-32 of SEQ ID NO:1, and   wherein the fusion proteins can dimerize through the dimerization component to form a homodimer.   
     
     
         62 . The method of  claim 61 , wherein the tumor antigen is selected from alpha fetoprotein (AFP), CA15-3, CA27-29, CA19-9, CA-125, calretinin, carcinoembryonic antigen, CD34, CD99, CDI 17, chromogranin, cytokeratin, desmin, epithelial membrane protein (EMA), Factor VIII, CD31 FLI, GD2, GD3, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-I5), HMB-45, human chorionic gonadotropin (hCG), inhibin, keratin, CD45, a lymphocyte marker, MART-I (Melan-A), Myo DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen, S 100 protein, smooth muscle actin (SMA), synaptophysin, thyroglobulin, thyroid transcription factor-I, tumor M2-PK, and vimentin. 
     
     
         63 . The method of  claim 61 , wherein the tumor antigen is GD2. 
     
     
         64 . The method of  claim 61 , wherein the second antibody component binds a toxin selected from saporin and vinca alkaloid. 
     
     
         65 . The binding agent of  claim 61 , wherein the dimerization component comprises amino acid residues 1-32 of SEQ ID NO:1. 
     
     
         66 . A method of treating a GD2-positive cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific binding agent comprised of two fusion proteins, wherein each of the fusion proteins comprise from N-terminus to C-terminus:
 a first antibody component that binds to GD2,   a second antibody component that binds to a toxin, and   a dimerization component comprising a sequence that is at least 90% identical to residues 1-32 of SEQ ID NO:1, such that the fusion proteins dimerize to form a homodimeric bispecific binding agent.   
     
     
         67 . The method of  claim 66 , wherein the second antibody component binds a toxin selected from saporin and vinca alkaloid. 
     
     
         68 . The method of  claim 66 , wherein the dimerization component comprises amino acid residues 1-32 of SEQ ID NO:1. 
     
     
         69 . A bispecific binding agent comprised of two fusion proteins, wherein each of the fusion proteins comprise from N-terminus to C-terminus:
 (i) a first antibody component that binds a tumor antigen;   (ii) a second antibody component that binds an antigen present on an immune effector cell; and   (iii) a dimerization component comprising a human hepatocyte nuclear factor-1 alpha (HNF-1α) element,   wherein the HNF-1α element comprises a sequence that is at least 90% identical to residues 1-32 of SEQ ID NO:1, and   wherein the fusion proteins can dimerize through the dimerization component to form a homodimer.   
     
     
         70 . A method of preparing a pharmaceutical composition, the method comprising steps of:
 providing a bispecific binding agent as recited in  claim 69 ; and   combining the provided bispecific binding agent with at least one pharmaceutically acceptable carrier or excipient to prepare a pharmaceutical composition.