Cysteine engineered anti-tenb2 antibodies and antibody drug conjugates

Abstract

Cysteine engineered anti-TENB2 antibodies are engineered by replacing one or more amino acids of a parent anti-TENB2 antibody with non cross-linked, reactive cysteine amino acids. Methods of design, preparation, screening, and selection of the cysteine engineered anti-TENB2 antibodies are provided. Cysteine engineered anti-TENB2 antibodies (Ab) are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered anti-TENB2 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 anti-TENB2 antibody comprising one or more free cysteine amino acids and a sequence selected from SEQ ID NOS:8-23. 
     
     
         2 . The cysteine engineered anti-TENB2 antibody of  claim 1  wherein the cysteine engineered anti-TENB2 antibody binds to a TENB2 polypeptide. 
     
     
         3 . The cysteine engineered anti-TENB2 antibody of  claim 1  prepared by a process comprising replacing one or more amino acid residues of a parent anti-TENB2 antibody by cysteine. 
     
     
         4 - 7 . (canceled) 
     
     
         8 . The cysteine engineered anti-TENB2 antibody of  claim 1  comprising a heavy chain sequence comprising: 
       
         
           
                 
                 
               
                     
                   SEQ ID NO: 3 
                 
                     
                     MAVLGLLLCLVTFPSCVLS DVQLQESGPGLVKPSETLSLTCAVSGY 
                 
                     
                     
                 
                     
                   SITSGYYWSWIRQPPGKGLEWMGFISYDGSNKYNPSLKNRITISRD 
                 
                     
                     
                 
                     
                   TSKNQFSLKLSSVTAADTAVYYCARGLRRGDYSMDYWGQGTLVTVS 
                 
                     
                     
                 
                     
                   SCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 
                 
                     
                     
                 
                     
                   LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN 
                 
                     
                     
                 
                     
                   TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS 
                 
                     
                     
                 
                     
                   RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY 
                 
                     
                     
                 
                     
                   RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP 
                 
                     
                     
                 
                     
                   QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK 
                 
                     
                     
                 
                     
                   TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ 
                 
                     
                     
                 
                     
                   KSLSLSPGK 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         9 . The cysteine engineered anti-TENB2 antibody of  claim 1  comprising a light chain sequence comprising: 
       
         
           
                 
                 
               
                     
                   SEQ ID NO: 2 
                 
                     
                     MDFQVQIFSFLLISASVIMSRG DIQMTQSPSSLSASVGDRVTITCK 
                 
                     
                     
                 
                     
                   ASQNVVTAVAWYQQKPGKAPKLLIYESASNRHTGVPSRFSGSGSGT 
                 
                     
                     
                 
                     
                   DFTLTISSLQPEDGATYYCQQYSSYPFTFGGGTKVEIKRTVAAPSV 
                 
                     
                     
                 
                     
                   FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE 
                 
                     
                     
                 
                     
                   SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK 
                 
                     
                     
                 
                     
                   SFNRGEC 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         10 . The cysteine engineered anti-TENB2 antibody of  claim 1  wherein the parent anti-TENB2 antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody. 
     
     
         11 . The cysteine engineered anti-TENB2 antibody of  claim 1  which is an antibody fragment. 
     
     
         12 - 15 . (canceled) 
     
     
         16 . A method of determining the presence of a TENB2 protein in a sample suspected of containing said protein, said method comprising exposing said sample to a cysteine engineered anti-TENB2 antibody of  claim 1  and determining binding of said antibody to said TENB2 protein in said sample, wherein binding of the antibody to said protein is indicative of the presence of said protein in said sample. 
     
     
         17 . The method of  claim 16  wherein said sample comprises a cell suspected of expressing said TENB2 protein. 
     
     
         18 . The method of  claim 16  wherein said cell is a prostate, ovarian, breast, lung, or pancreatic cancer cell. 
     
     
         19 . The method of  claim 16  wherein the antibody is covalently attached to a label selected from a fluorescent dye, a radioisotope, biotin, or a metal-complexing ligand. 
     
     
         20 . A pharmaceutical formulation comprising the cysteine engineered anti-TENB2 antibody of  claim 1 , and a pharmaceutically acceptable diluent, carrier or excipient. 
     
     
         21 . The cysteine engineered anti-TENB2 antibody of  claim 1  wherein the antibody is covalently attached to an auristatin drug moiety whereby an antibody drug conjugate is formed. 
     
     
         22 . The antibody-drug conjugate of  claim 21  comprising a cysteine engineered anti-TENB2 antibody (Ab), and an auristatin drug moiety (D) wherein the cysteine engineered anti-TENB2 antibody is attached through one or more free cysteine amino acids by a linker moiety (L) to D; the compound having Formula I:
   Ab-(L-D) p   I
 
 where p is 1, 2, 3, or 4. 
 
     
     
         23 - 33 . (canceled) 
     
     
         34 . The antibody-drug conjugate compound of  claim 21  wherein the parent anti-TENB2 antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody. 
     
     
         35 . The antibody-drug conjugate compound of  claim 21  wherein the parent anti-TENB2 antibody is an antibody fragment. 
     
     
         36 . (canceled) 
     
     
         37 . The antibody drug conjugate of  claim 21  wherein the auristatin is MMAE or MMAF. 
     
     
         38 . The antibody drug conjugate of  claim 21  wherein L is MC-val-cit-PAB or MC. 
     
     
         39 . The antibody drug conjugate of  claim 21  wherein L is SMCC, SPP, or BMPEO. 
     
     
         40 . An antibody-drug conjugate compound selected from the structures: 
       
         
           
           
               
               
           
         
         wherein Val is valine; Cit is citrulline; p is 1, 2, 3, or 4; and Ab is a cysteine engineered anti-TENB2 antibody of  claim 1 . 
       
     
     
         41 . The antibody drug conjugate of  claim 40  wherein Ab comprises SEQ ID NO:1. 
     
     
         42 . The antibody drug conjugate of  claim 40  wherein Ab comprises SEQ ID NO:2. 
     
     
         43 . The antibody drug conjugate of  claim 40  wherein Ab comprises SEQ ID NO: 1 and SEQ ID NO:2. 
     
     
         44 . An assay for detecting cancer cells comprising:
 (a) exposing cells to an antibody-drug conjugate compound of  claim 21 ; and   (b) determining the extent of binding of the antibody-drug conjugate compound to the cells.   
     
     
         45 . The assay of  claim 44  wherein the cells are prostate, pancreatic, lung, breast, colon or ovarian tumor cells. 
     
     
         46 . A method of inhibiting cellular proliferation comprising treating mammalian tumor cells in a cell culture medium with an antibody-drug conjugate compound of  claim 21 , whereby proliferation of the tumor cells is inhibited. 
     
     
         47 . The method of  claim 46  wherein the mammalian tumor cells are ovarian tumor cells. 
     
     
         48 . A pharmaceutical formulation comprising the antibody drug conjugate of  claim 21 , and a pharmaceutically acceptable diluent, carrier or excipient. 
     
     
         49 . The pharmaceutical formulation of  claim 48  further comprising a therapeutically effective amount of a chemotherapeutic agent selected from letrozole, oxaliplatin, doxetaxel, 5-FU, lapatinib, capecitabine, leucovorin, erlotinib, pertuzumab, bevacizumab, and gemcitabine. 
     
     
         50 . A method of treating cancer comprising administering to a patient the pharmaceutical formulation of  claim 48 . 
     
     
         51 . The method of  claim 50  wherein the cancer is selected from the group consisting of prostate cancer, cancer of the urinary tract, pancreatic cancer, lung cancer, breast cancer, colon cancer and ovarian cancer. 
     
     
         52 . The method of  claim 50  wherein the patient is administered a chemotherapeutic agent in combination with the antibody-drug conjugate compound, where the chemotherapeutic agent is selected from letrozole, cisplatin, carboplatin, taxol, paclitaxel, oxaliplatin, doxetaxel, 5-FU, leucovorin, erlotinib, pertuzumab, bevacizumab, lapatinib, and gemcitabine. 
     
     
         53 . An article of manufacture comprising
 the pharmaceutical formulation of  claim 48 ;   a container; and   a package insert or label indicating that the compound can be used to treat cancer characterized by the overexpression of a TENB2 polypeptide.   
     
     
         54 . The article of manufacture of  claim 53  wherein the cancer is ovarian cancer, prostate cancer, cancer of the urinary tract, pancreatic cancer, lung cancer, breast cancer, or colon cancer. 
     
     
         55 . A method for making an antibody drug conjugate compound comprising a cysteine engineered anti-TENB2 antibody (Ab) of  claim 1 , and an auristatin drug moiety (D) wherein the cysteine engineered antibody is attached through the one or more engineered cysteine amino acids by a linker moiety (L) to D; the compound having Formula I:
   Ab-(L-D) p   I
   where p is 1, 2, 3, or 4; the method comprising the steps of:   (a) reacting an engineered cysteine group of the cysteine engineered antibody with a linker reagent to form antibody-linker intermediate Ab-L; and   (b) reacting Ab-L with an activated drug moiety D; whereby the antibody-drug conjugate is formed;   or comprising the steps of:   (c) reacting a nucleophilic group of a drug moiety with a linker reagent to form drug-linker intermediate D-L; and   (d) reacting D-L with an engineered cysteine group of the cysteine engineered antibody; whereby the antibody-drug conjugate is formed.   
     
     
         56 . The method of  claim 55  further comprising the step of expressing the cysteine engineered antibody in chinese hamster ovary (CHO) cells. 
     
     
         57 . The method of  claim 56  further comprising the step of treating the expressed cysteine engineered antibody with a reducing agent. 
     
     
         58 . The method of  claim 57  wherein the reducing agent is selected from TCEP and DTT. 
     
     
         59 . The method of  claim 57  further comprising the step of treating the expressed cysteine engineered antibody with an oxidizing agent, after treating with the reducing agent. 
     
     
         60 . The method of  claim 59  wherein the oxidizing agent is selected from copper sulfate, dehydroascorbic acid, and air.