Dual Specific Immunotoxin for Brain Tumor Therapy

Abstract

We tested the in vitro and in vivo efficacy of a recombinant bispecific immunotoxin that recognizes both EGFRwt and tumor-specific EGFRvIII receptors. A single chain antibody was cloned from a hybridoma and fused to toxin, carrying a C-terminal peptide which increases retention within cells. The binding affinity and specificity of the recombinant bispecific immunotoxin for the EGFRwt and the EGFRvIII proteins was measured. In vitro cytotoxicity was measured. In vivo activity of the recombinant bispecific immunotoxin was evaluated in subcutaneous models and compared to that of an established monospecific immunotoxin. In our preclinical studies, the bispecific recombinant immunotoxin, exhibited significant potential for treating brain tumors.

1 . A single chain variable region antibody which binds with a binding affinity that is at least 5×10 8  M −1  as measured by surface plasmon resonance to both (a) EGFR found on normal human cells and (b) EGFR variant III mutant. 
     
     
         2 . The single chain variable region antibody of  claim 1  which is cloned from a hybridoma producing a monoclonal antibody selected from the group consisting of D2C7, F2A2, B10B11, and H11. 
     
     
         3 . The single chain variable region antibody of  claim 1  which is covalently linked to a cytotoxic agent selected from the group consisting of a toxin, a chemotherapeutic agent, and a radionuclide. 
     
     
         4 . The single chain variable region antibody of  claim 3  wherein the agent is a toxin which is produced as a fusion protein with the single chain variable region antibody. 
     
     
         5 . The single chain variable region antibody of  claim 4  wherein the monoclonal antibody is D2C7. 
     
     
         6 . The single chain variable region antibody of  claim 3  wherein the agent is a form of  Pseudomonas  exotoxin A. 
     
     
         7 . The single chain variable region antibody of  claim 6  further comprising a KDEL peptide. 
     
     
         8 . The single chain variable region antibody of  claim 1  which has a VH sequence as shown in  FIG. 1A   
     
     
         9 . The single chain variable region antibody of  claim 1  which as a VL sequence as shown in  FIG. 1B . 
     
     
         10 . The single chain variable region antibody of  claim 1  which has CDR1, CDR2, and CDR3 regions as shown in  FIGS. 1A and 1B . 
     
     
         11 . The single chain variable region antibody of  claim 1  wherein said binding affinity is at least 6×10 8  M −1 . 
     
     
         12 . The single chain variable region antibody of  claim 1  wherein said binding affinity is between 5×10 8  M −1  and 5×10 9  M −1 . 
     
     
         13 . The single chain variable region antibody of  claim 1  which has an IC50 of less than 2 ng/ml for human cells expressing EGFR as found on normal cells or EGFRvIII. 
     
     
         14 . The single chain variable region antibody of  claim 1  which has an IC 50  of less than 1.5 ng/ml for human cells expressing EGFR as found on normal cells or EGFRvIII. 
     
     
         15 . The single chain variable region antibody of  claim 1  which has an IC 50  of less than 1 ng/ml for human cells expressing EGFR as found on normal cells or EGFRvIII. 
     
     
         16 . The single chain variable region antibody of  claim 1  which has an IC 50  of less than 0.5 ng/ml for human cells expressing EGFR as found on normal cells or EGFRvIII. 
     
     
         17 . A method of treating a tumor in a human, comprising:
 administering a single chain variable region antibody according to  claim 1  to the human, whereby tumor cells are killed.   
     
     
         18 . The method of  claim 17  wherein the tumor is a squamous cell head and neck tumor. 
     
     
         19 . The method of  claim 17  wherein the tumor is a brain tumor. 
     
     
         20 . The method of  claim 17  wherein the tumor is a breast tumor. 
     
     
         21 . The method of  claim 17  wherein the tumor is a glioblastoma multiforme. 
     
     
         22 . The method of  claim 17  wherein the tumor is an astrocytoma. 
     
     
         23 . The method of  claim 17  wherein the tumor contains an EGFRvIII allele. 
     
     
         24 . The method of  claim 17  wherein the administering is directly to the central nervous system. 
     
     
         25 . The method of  claim 17  wherein the administering is directly to the brain. 
     
     
         26 . The method of  claim 17  wherein the administering is directly to a surgically-created tumor resection cavity. 
     
     
         27 . The method of  claim 17  wherein the administering is directly to a natural tumor cyst. 
     
     
         28 . The method of  claim 17  wherein the administering is directly to tumor parenchyma. 
     
     
         29 . A monoclonal antibody which binds with a binding affinity that is at least 5×10 8  M −1  to both (a) EGFR found on normal human cells and (b) EGFR variant III mutant, wherein said antibody has a VH sequence as shown in  FIG. 1A , a VL sequence as shown in  FIG. 1B , or CDR1, CDR2, and CDR3 regions as shown in  FIGS. 1A and 1B . 
     
     
         30 . The monoclonal antibody of  claim 29  which is designated D2C7. 
     
     
         31 . The monoclonal antibody of  claim 29  which comprises a human IgG. 
     
     
         32 . A monoclonal antibody which binds with a binding affinity that is at least 1×10 8  M −1  to both (a) EGFR found on normal human cells and (b) EGFR variant III mutant, wherein said antibody is selected from the group consisting of F2A2 and B10B11. 
     
     
         33 . A method of treating a tumor in a human, comprising:
 administering an antibody according to  claim 29 ,  30 ,  31 , or  32 , to the human whereby tumor cells are killed.   
     
     
         34 . A method of determining therapeutic plan to treat a tumor in a human, comprising:
 contacting tissue of the tumor with an antibody according to  claim 1 ,  29 ,  30 ,  31 , or  32 ;   determining amount of cells in the tissue which bind to the antibody, wherein greater amounts of cells which bind are a positive factor to recommend using the antibody therapeutically for the patient.