Humanized antibodies against CD3

Abstract

The invention provides bispecific antibodies with selective cytotoxicity against malignant B-cells. The bispecific antibodies bind to an effector cell antigen and to a 28/32 kDa heterodimeric protein on the surface of malignant B-cells. The invention also includes the monospecific components of the bispecific antibodies, humanized versions thereof, and humanized bispecific antibodies. The invention further provides therapeutic and diagnostic methods employing these antibodies.

What is claimed is:  
     
         1 . A bispecific antibody that binds to: 
 (a) a first antigen on the surface of effector cells selected from the group consisting of T-cells and natural killer cells, and    (b) a second antigen on a 28/32 kDa heterodimeric protein on the surface of malignant B cells, which second antigen specifically binds to an antibody designated 1D10,    wherein the binding of the bispecific antibody to the first and second antigens results in killing of the malignant B cells.    
     
     
         2 . The bispecific antibody of  claim 1 , wherein the effector cells are T cells.  
     
     
         3 . The bispecific antibody of  claim 2 , wherein the bispecific antibody binds to a CD3 antigen on the T cells.  
     
     
         4 . The bispecific antibody of  claim 3 , wherein the antibody is produced by the cell line ATCC HB 10993.  
     
     
         5 . A cell line producing the bispecific antibody of  claim 1 .  
     
     
         6 . The cell line of  claim 5  that is a hybrid-hybridoma formed from two hybridomas, 
 a first hybridoma producing an antibody that binds to the first antigen, and  
 a second hybridoma producing an antibody that binds to the second antigen.  
 
     
     
         7 . The cell line of  claim 7 , wherein the effector cells are T-cells and the first hybridoma produces an antibody that binds to a CD3 antigen on the T-cells.  
     
     
         8 . A cell line designated ATCC HB 10993.  
     
     
         9 . An antibody designated 1D10.  
     
     
         10 . A humanized version of the antibody of  claim 9 .  
     
     
         11 . A humanized antibody according to  claim 10 , the antibody comprising a humanized heavy chain and a humanized light chain: 
 (1) the humanized light chain comprising three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of the 1D10 immunoglobulin light chain, and a variable region framework from a human kappa light chain variable region framework sequence except in at least one position selected from a first group consisting of L48, L49, L69, and L70 wherein the amino acid position is occupied by the same amino acid present in the equivalent position of the 1D10 immunoglobulin light chain variable region framework; and    (2) the humanized heavy chain comprising three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of 1D10 immunoglobulin heavy chain, and a variable region framework from a human heavy chain variable region framework sequence except in at least one position selected from a second group consisting of H27, H29, H30, H37, H67, H71, H78 and H83, wherein the amino acid position is occupied by the same amino acid present in the equivalent position of the mouse 1D10 immunoglobulin heavy chain variable region framework;    wherein the immunoglobulin specifically binds to a 28/32 kDa heterodimeric protein on the surface of malignant B cells with a binding affinity having a lower limit of about 10 7  M −1  and an upper limit of about five-times the binding affinity of the 1D10 immunoglobulin.    
     
     
         13 . The humanized antibody of claim  12 , wherein 
 the humanized light chain variable region framework is from the light chain variable region framework of the R3.5H5G antibody except in the at least one position from the first group and except at position L43, which is occupied the amino acid present in the equivalent position of a human kappa subgroup I consensus sequence;    the humanized heavy chain is from the heavy chain region variable framework of the IC4 antibody except in at least one position selected from the second group, and except at position H73, wherein the amino acid position is occupied by the same amino acid present in the equivalent position of a human immunoglobulin subgroup II or IV consensus sequence.    
     
     
         14 . The humanized antibody of  claim 13 , wherein 
 the humanized light chain comprises the amino acid sequence of FIG. 4A (upper) and    the humanized heavy chain comprises the amino acid sequence of FIG. 4B (upper).    
     
     
         15 . The humanized antibody of  claim 14 , wherein the humanized light chain further comprises a human kappa constant region, the humanized heavy chain further comprises a human γ1 constant region, and the humanized antibody effects ADCC and complement-mediated lysis of malignant B-cells when bound to a 28/32 kDa heterodimeric protein on the surface of the cells.  
     
     
         16 . A humanized antibody, the antibody comprising a humanized-heavy chain and a humanized light chain: 
 (1) the humanized light chain comprising three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of the mouse M291 immunoglobulin light chain, and a variable region framework from a human kappa light chain variable region framework sequence, and    (2) the humanized heavy chain comprising three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of the mouse M291 immunoglobulin heavy chain, and a variable region framework from a human heavy chain variable region framework sequence except in at least one position selected from a second group consisting of H30, H67, H68, H70, H72 and H74 wherein the amino acid position is occupied by the same amino acid present in the equivalent position of the mouse M291 immunoglobulin heavy chain variable region framework;    wherein the immunoglobulin specifically binds to a CD3 antigen on the surface of T cells with a binding affinity having a lower limit of about 10 7  M −1  and an upper limit of about five-times the binding affinity of the M291 immunoglobulin.    
     
     
         17 . The humanized antibody of  claim 16 , wherein 
 the humanized light chain variable region framework is from the light chain variable region framework of the HF2-1/17 antibody in subgroup I;    the humanized heavy chain region framework is from the heavy chain region variable framework of the 21/28 antibody except in at least one position selected from the second group, and except at position 44, wherein the amino acid position is occupied by the same amino acid present in the equivalent position of a human immunoglobulin subgroup I consensus sequence.    
     
     
         18 . The humanized antibody of  claim 17 , wherein 
 the humanized light chain comprises the amino acid sequence of FIG. 5A (upper) and    the humanized heavy chain comprises the amino acid sequence of FIG. 5B (upper).    
     
     
         19 . The bispecific antibody of  claim 1  that is humanized.  
     
     
         20 . The bispecific antibody of  claim 18 , wherein the first antigen is the CD3 antigen.  
     
     
         21 . The bispecific antibody of  claim 20 , comprising: 
 a first binding fragment comprising: 
 a humanized form of the heavy chain variable region of the M291 antibody;  
 a humanized form of the light chain variable region of the M291 antibody; and  
   a second binding fragment, which is linked to the first binding fragment, comprising: 
 a humanized form of the heavy chain variable region from the 1D10 antibody;  
 a humanized form of the light chain variable region from the 1D10 antibody;  
 wherein the first binding fragment specifically binds to the CD3 antigen and the second binding fragment specifically binds to the 28/32 kDa heterodimeric antigen on the surface of the malignant B cells.  
   
     
     
         22 . The bispecific antibody of  claim 21 , wherein: 
 the humanized form of the heavy chain variable region of the M291 antibody comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of M291 immunoglobulin heavy chain, and a variable region framework from a human heavy chain variable region framework sequence except in at least one position selected from a second group consisting of H30, H67, H68, H70, H72 and H74 wherein the amino acid position is occupied by the same amino acid present in the equivalent position of the mouse M291 immunoglobulin heavy chain variable region framework;    the humanized form of the light chain variable region of the M291 antibody comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of the M291 immunoglobulin light chain, and a variable region framework from a human kappa light chain variable region framework sequence;    the humanized form of the heavy chain variable region from the 1D10 antibody comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of 1D10 immunoglobulin heavy chain, and a variable region framework from a human heavy chain variable region framework sequence except in at least one position selected from a second group consisting of H27, H29, H30, H37, H67, H71, H78 and H83, wherein the amino acid position is occupied by the same amino acid present in the equivalent position of the mouse 1D10 immunoglobulin heavy chain variable region framework; and    the humanized form of the light chain variable region from the 1D10 antibody comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of the 1D10 immunoglobulin light chain, and a variable region framework from a human kappa light chain variable region framework sequence except in at least one position selected from a first group consisting of L48, L49, L69, and L70 wherein the amino acid position is occupied by the same amino acid present in the equivalent position of the 1D10 immunoglobulin light chain variable region framework.    
     
     
         23 . The bispecific antibody of  claim 22 , wherein the first binding fragment comprises the heavy chain variable region shown in FIG. 5B (upper) and the light chain variable region shown in FIG. 5A (upper), and the second binding fragment comprises the heavy chain variable region shown in FIG. 4B (upper) and the light chain variable region shown in FIG. 4A (upper).  
     
     
         24 . The bispecific antibody of  claim 23 , wherein the first and second binding fragments each further comprises a segment of a constant region fused to the respective heavy chain variable regions, and the binding fragments are linked by association of the constant regions.  
     
     
         25 . The bispecific antibody of  claim 24 , wherein the binding fragments are Fab or Fab′.  
     
     
         26 . The bispecific antibody of  claim 25 , wherein the first and second binding fragments are Fab's and the bispecific antibody is a F(ab′) 2 .  
     
     
         27 . The bispecific antibody of  claim 26 , wherein the first and second binding fragments further comprise first and second leucine zippers fused to the respective constant regions.  
     
     
         28 . The bispecific antibody of  claim 27 , wherein the first binding fragment comprises a heavy chain having the amino acid sequence shown in FIG. 5D and the second binding fragment comprises a heavy chain having the amino acid sequence shown in FIG. 4D.  
     
     
         29 . A method of treating a patient suffering from malignant B-cells, the method comprising treating the patient with a therapeutically effective amount of the bispecific antibody of  claim 1 .  
     
     
         30 . The method of  claim 29 , wherein the bispecific antibody comprises: 
 a first binding fragment comprising: 
 a humanized form of the heavy chain variable region of the M291 antibody;  
 a humanized form of the light chain variable region of the M291 antibody; and  
   a second binding fragment, which is linked to the first binding fragment, comprising: 
 a humanized form of the heavy chain variable region from the 1D10 antibody;  
 a humanized form of the light chain variable region from the 1D10 antibody;  
 wherein the first binding fragment specifically binds to the CD3 antigen on the surface of T-cells and the second binding fragment specifically binds to the 28/32 kDa heterodimeric antigen on the surface of the malignant B cells.  
   
     
     
         31 . The method of  claim 30  further comprising the step of administering an agent to activate T-cells in the patient.  
     
     
         33 . The method of  claim 31 , wherein the agent is IL-2.  
     
     
         33 . A pharmaceutical composition comprising the bispecific antibody of  claim 1.