Certain Improved Human Bispecific EGFRvIII Antibody Engaging Molecules

Abstract

We have constructed a polynucleotide encoding a bispecific antibody engaging molecule which has one arm that specifically engages a tumor cell which expresses the human EGFRvIII mutant protein on its surface, and a second arm that specifically engages T cell activation ligand CD3. The polynucleotide is codon optimized for expression in CHO cells. The subunits of the engaging molecules are organized to achieve greater efficiency. These are promising therapeutic agents.

We claim: 
     
         1 . A bispecific polypeptide, comprising:
 a first single chain human variable region which binds to EGFRvIII, and comprises segments encoded by SEQ ID NO: 5 and 6 in amino to carboxy order; in series with   a second single chain human variable region which binds to T cell activation ligand CD3 and comprises segments encoded by SEQ ID NO: 7 and 8 in amino to carboxy order; wherein the first and second single chain human variable regions are in amino to carboxy order.   
     
     
         2 . The bispecific polypeptide of  claim 1  which comprises segments encoded by SEQ ID NOs: 5, 6, 10, 7, and 8, in amino to carboxy order. 
     
     
         3 . The bispecific polypeptide of  claim 1  which comprises segments encoded by SEQ ID NO: 5, 9, 6, 10, 7, 9, and 8, in amino to carboxy order. 
     
     
         4 . The bispecific polypeptide of  claim 1  which comprises segments encoded by SEQ ID NO: 4, 5, 9, 6, 10, 7, 9, and 8, in amino to carboxy order. 
     
     
         5 . A polynucleotide encoding the bispecific polypeptide of  claim 1 . 
     
     
         6 . The polynucleotide of  claim 5  comprising the sequence of SEQ ID NO: 1. 
     
     
         7 . A method of treating a patient with an EGFRvIII-expressing tumor, comprising:
 administering the bispecific polypeptide of  claim 1  to the patient, whereby a cytolytic T cell response to the tumor is induced.   
     
     
         8 . The bispecific polypeptide of  claim 1  wherein a spacer polypeptide segment links the first and second single chain variable regions. 
     
     
         9 . The bispecific polypeptide of  claim 8  wherein the spacer polypeptide segment is (G 4 S) n  wherein n is 1-5. 
     
     
         10 . The bispecific polypeptide of  claim 1  wherein a spacer polypeptide segment links V L  and V H  domains within the first single chain variable region. 
     
     
         11 . The bispecific polypeptide of  claim 1  wherein a spacer polypeptide segment links V H  and V L  domains within the second single chain variable region. 
     
     
         12 . The bispecific polypeptide of  claim 10  wherein the spacer polypeptide segment is (G 4 S) n  wherein n is 1-5. 
     
     
         13 . The bispecific polypeptide of  claim 11  wherein the spacer polypeptide segment is (G 4 S) n  wherein n is 1-5. 
     
     
         14 . The bispecific polypeptide of  claim 1  wherein each single chain variable region comprises a disulfide bond between the V H  and the V L  domain. 
     
     
         15 . A method of making a bispecific polypeptide comprising:
 culturing a cell comprising the polynucleotide of  claim 5  in a culture medium and collecting the bispecific polypeptide from the cells or culture medium.   
     
     
         16 . A method of making a bispecific polypeptide comprising:
 culturing a cell comprising the polynucleotide of  claim 6  in a culture medium and collecting the bispecific polypeptide from the cells or culture medium.