Multispecific antibodies, multispecific activatable antibodies and methods of using the same

Abstract

The invention relates generally to multispecific antibodies and to multispecific activatable antibodies that specifically bind to two or more different antigens or epitopes, as well as to methods of making and using these multispecific antibodies and/or multispecific activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

What is claimed is: 
     
         1 . A bispecific antibody, wherein said bispecific antibody comprises the following structure:
 a. a full length IgG antibody (AB1) that specifically binds to a first antigen wherein AB1 comprises two antibody heavy chains and two antibody light chains; and wherein the AB1 is linked to:
 i. two first masking moiety peptides (MM1s); and 
 ii. two cleavable moiety peptides (CM1s), each CM1 being a substrate for a first protease;
 wherein each MM1 is linked in an N- to C-terminal direction to a CM1, to form two MM1-CM1 peptides; and 
 wherein the carboxyl terminus of each MM1-CM1 peptide is linked to the amino terminus of a corresponding AB1 light chain; and 
 
   b. two scFvs (each an AB2) that each specifically binds to a second antigen, wherein each AB2 comprises an antibody light chain variable domain and an antibody heavy chain variable domain; and wherein each AB2 is linked to:
 i. a second masking moiety peptide (MM2); and 
 ii. a second cleavable moiety (CM2) peptide that is a substrate for a second protease;
 wherein MM2 is linked in an N- to C-terminal direction to CM2 to form an MM2-CM2 peptide; 
 wherein the carboxyl terminus of the MM2-CM2 peptide is linked to the amino terminus of the AB2 
 
   wherein the carboxyl terminus of each AB2 is linked to the amino terminus of a corresponding heavy chain of AB1,   wherein the bispecific antibody comprises an amino acid sequence as set forth in SEQ ID NOs: 234, 236, 238, 240, 254, 256, 258, 260, 262, 264, 266, 268, or 270; or an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NOs: 233, 235, 237, 239, 253, 255, 257, 259, 261, 263, 265, 267, or 269.   
     
     
         2 . A method of producing a bispecific antibody of  claim 1 , comprising culturing a cell that comprises a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 233, 235, 237, 239, 253, 255, 257, 259, 261, 263, 265, 267, or 269 such that the cell expresses the bispecific antibody, and recovering the bispecific antibody. 
     
     
         3 . A method of alleviating a symptom of a clinical indication associated with a disorder in a subject, the method comprising administering the bispecific antibody of  claim 1  to the subject in need thereof in an amount sufficient to alleviate the symptom of the clinical indication associated with the disorder. 
     
     
         4 . The method of  claim 3 , wherein said subject is a human. 
     
     
         5 . The method of  claim 3 , wherein the disorder is cancer. 
     
     
         6 . The method of  claim 3 , wherein the method comprises administering an additional agent. 
     
     
         7 . The method of  claim 3 , wherein the additional agent is a therapeutic agent. 
     
     
         8 . A bispecific antibody, wherein said bispecific antibody comprises the following structure:
 a. a full length IgG antibody (AB1) that specifically binds to a first antigen wherein AB1 comprises two antibody heavy chains and two antibody light chains; and wherein the AB1 is linked to:
 i. two first masking moiety peptides (MM1s); and 
 ii. two cleavable moiety peptides (CM1s), each CM1 being a substrate for a first protease;
 wherein each MM1 is linked in an N- to C-terminal direction to a CM1, to form two MM1-CM1 peptides; and 
 wherein the carboxyl terminus of each MM1-CM1 peptide is linked to the amino terminus of a corresponding AB1 light chain; and 
 
   b. two scFvs (each an AB2) that each specifically binds to a second antigen, wherein each AB2 comprises an antibody light chain variable domain and an antibody heavy chain variable domain; and wherein each AB2 is linked to:
 i. a second masking moiety peptide (MM2); and 
 ii. a second cleavable moiety (CM2) peptide that is a substrate for a second protease;
 wherein MM2 is linked in an N- to C-terminal direction to CM2 to form an MM2-CM2 peptide; 
 wherein the carboxyl terminus of the MM2-CM2 peptide is linked to the amino terminus of the AB2 
 
   wherein the carboxyl terminus of each AB2 is linked to the amino terminus of a corresponding heavy chain of AB1,   wherein the bispecific antibody comprises an amino acid sequence as set forth in SEQ ID NOs: 254, 256, 292, 294, 300, 304, 306, 308, 310, 314, or 336.   
     
     
         9 . An isolated nucleic acid molecule encoding the bispecific antibody of  claim 8 . 
     
     
         10 . A vector comprising the isolated nucleic acid molecule of  claim 9 . 
     
     
         11 . A method of producing a bispecific antibody of  claim 8 , comprising culturing a cell that comprises an isolated nucleic acid molecule encoding the bispecific antibody, such that the cell expresses the bispecific antibody, and recovering the bispecific antibody. 
     
     
         12 . A method of alleviating a symptom of a clinical indication associated with a disorder in a subject, the method comprising administering the bispecific antibody of  claim 8  to a subject in need thereof in an amount sufficient to alleviate the symptom of the clinical indication associated with the disorder. 
     
     
         13 . The method of  claim 12 , wherein said subject is a human. 
     
     
         14 . The method of  claim 12 , wherein the disorder is cancer. 
     
     
         15 . The method of  claim 12 , wherein the method comprises administering an additional agent. 
     
     
         16 . The method of  claim 12 , wherein the additional agent is a therapeutic agent. 
     
     
         17 . A bispecific antibody wherein the bispecific antibody comprises a sequence as set forth in SEQ ID NOs: 292, 294, or 300. 
     
     
         18 . The bispecific antibody of  claim 17 , comprising the amino acid sequence set forth in SEQ ID NO: 292. 
     
     
         19 . The bispecific antibody of  claim 17 , comprising the amino acid sequence set forth in SEQ ID NO: 294. 
     
     
         20 . The bispecific antibody of  claim 17 , comprising the amino acid sequence set forth in SEQ ID NO: 300. 
     
     
         21 . An isolated nucleic acid molecule encoding the bispecific antibody of  claim 17 . 
     
     
         22 . A vector comprising the isolated nucleic acid molecule of  claim 17 . 
     
     
         23 . A method of producing a bispecific antibody of  claim 17 , comprising culturing a cell that comprises an isolated nucleic acid molecule encoding the bispecific antibody, such that the cell expresses the bispecific antibody, and recovering the bispecific antibody. 
     
     
         24 . A method of alleviating a symptom of a clinical indication associated with a disorder in a subject, the method comprising administering the bispecific antibody of  claim 17  to a subject in need thereof in an amount sufficient to alleviate the symptom of the clinical indication associated with the disorder. 
     
     
         25 . The method of  claim 24 , wherein said subject is a human. 
     
     
         26 . The method of  claim 24 , wherein the disorder is cancer. 
     
     
         27 . The method of  claim 24 , wherein the method comprises administering an additional agent. 
     
     
         28 . The method of  claim 24 , wherein the additional agent is a therapeutic agent.