Cysteine engineered antibodies and conjugates

Abstract

Antibodies are engineered by replacing one or more amino acids of a parent antibody with non cross-linked, highly reactive cysteine amino acids. Antibody fragments may also be engineered with one or more cysteine amino acids to form cysteine engineered antibody fragments (ThioFab). Methods of design, preparation, screening, and selection of the cysteine engineered antibodies are provided. Cysteine engineered antibodies (Ab), optionally with an albumin-binding peptide (ABP) sequence, are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered antibody-drug conjugates having Formula I: Ab-(L-D) p   I where p is 1 to 4. Diagnostic and therapeutic uses for cysteine engineered antibody drug compounds and compositions are disclosed.

1 . A cysteine engineered antibody comprising one or more variable region heavy chain sequences with a free cysteine amino acid selected from SEQ ID NOS: 9-16: 
       
         
           
                 
                 
                 
               
                     
                   WVRQCPGKGL 
                   SEQ ID NO: 9 
                 
                     
                     
                 
                     
                   NSLRCEDTAV 
                   SEQ ID NO: 10 
                 
                     
                     
                 
                     
                   LVTVCSASTKGPS 
                   SEQ ID NO: 11 
                 
                     
                     
                 
                     
                   LVTVSCASTKGPS 
                   SEQ ID NO: 12 
                 
                     
                     
                 
                     
                   LVTVSSCSTKGPS 
                   SEQ ID NO: 13 
                 
                     
                     
                 
                     
                   LVTVSSACTKGPS 
                   SEQ ID NO: 14 
                 
                     
                     
                 
                     
                   HTFPCVLQSSGLYS 
                   SEQ ID NO: 15 
                 
                     
                     
                 
                     
                   HTFPAVLQCSGLYS 
                   SEQ ID NO: 16 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         or comprising one or more variable region light chain sequences with a free cysteine amino acid selected from SEQ ID NOS: 17-27: 
       
       
         
           
                 
                 
                 
               
                     
                   SLSASCGDRVT 
                   SEQ ID NO: 17 
                 
                     
                     
                 
                     
                   QKPGKCPKLLI 
                   SEQ ID NO: 18 
                 
                     
                     
                 
                     
                   EIKRTCAAPSV 
                   SEQ ID NO: 19 
                 
                     
                     
                 
                     
                   TCAAPCVFIFPP 
                   SEQ ID NO: 20 
                 
                     
                     
                 
                     
                   FIFPPCDEQLK 
                   SEQ ID NO: 21 
                 
                     
                     
                 
                     
                   DEQLKCGTASV 
                   SEQ ID NO: 22 
                 
                     
                     
                 
                     
                   FYPRECKVQWK 
                   SEQ ID NO: 23 
                 
                     
                     
                 
                     
                   WKVDNCLQSGN 
                   SEQ ID NO: 24 
                 
                     
                     
                 
                     
                   ALQSGCSQESV 
                   SEQ ID NO: 25 
                 
                     
                     
                 
                     
                   VTEQDCKDSTY 
                   SEQ ID NO: 26 
                 
                     
                     
                 
                     
                   GLSSPCTKSFN 
                   SEQ ID NO: 27 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein the cysteine engineered antibody binds to HER2. 
       
     
     
         2 . A cysteine engineered antibody comprising one or more Fc constant region heavy chain sequences with a free cysteine amino acid selected from SEQ ID NOS: 31, 33, 36, 37 and 38: 
       
         
           
                 
                 
                 
               
                     
                   HEDPEVKFNWYVDGCEVHNAKTKPR 
                   SEQ ID NO: 31 
                 
                     
                     
                 
                     
                   HEDPEVKFNWYVDGVEVHNCKTKPR 
                   SEQ ID NO: 33 
                 
                     
                     
                 
                     
                   IEKTISKCKGQPR 
                   SEQ ID NO: 36 
                 
                     
                     
                 
                     
                   KGFYPCDIAVE 
                   SEQ ID NO: 37 
                 
                     
                     
                 
                     
                   PPVLDCDGSFF 
                   SEQ ID NO: 38 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein the cysteine engineered antibody binds to EphB2R. 
       
     
     
         3 . A cysteine engineered antibody comprising one or more variable region heavy chain sequences with a free cysteine amino acid selected from SEQ ID NOS: 40-44: 
       
         
           
                 
                 
                 
               
                     
                   NWIRQCPGNK 
                   SEQ ID NO: 40 
                 
                     
                     
                 
                     
                   LNSCTTEDTAT 
                   SEQ ID NO: 41 
                 
                     
                     
                 
                     
                   GQGTLVTVSACSTKGPSVFPL 
                   SEQ ID NO: 42 
                 
                     
                     
                 
                     
                   HTFPCVLQSSGLYS 
                   SEQ ID NO: 43 
                 
                     
                     
                 
                     
                   HTFPACLQSSGLYS 
                   SEQ ID NO: 44 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
         or comprising one or more variable region light chain sequences with a free cysteine amino acid selected from SEQ ID NOS: 45-49: 
       
       
         
           
                 
                 
                 
               
                     
                   FLSVSCGGRVT 
                   SEQ ID NO: 45 
                 
                     
                     
                 
                     
                   QKPGNCPRLLI 
                   SEQ ID NO: 46 
                 
                     
                     
                 
                     
                   EIKRTCAAPSV 
                   SEQ ID NO: 47 
                 
                     
                     
                 
                     
                   FYPRECKVQWK 
                   SEQ ID NO: 48 
                 
                     
                     
                 
                     
                   VTEQDCKDSTY 
                   SEQ ID NO: 49 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein the cysteine engineered antibody binds to MUC16. 
       
     
     
         4 . The cysteine engineered antibody of any one of  claims 1 - 3  wherein the free cysteine amino acid is conjugated to a drug moiety selected from a maytansinoid, an auristatin, a dolastatin, or a calicheamicin.