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 - 60 . (canceled) 
     
     
         61 . A cysteine engineered anti-TENB2 antibody comprising one or more free cysteine amino acids, wherein the cysteine engineered anti-TENB2 antibody comprises a heavy chain sequence comprising: 
       
         
           
                 
               
                   (SEO ID NO: 3) 
                 
                     MAVLGLLLCLVTFPSCVLS DVQLQESGPGLVKPSETLSLTCAVSGYS 
                 
                     
                 
                   ITSGYYWSWIRQPPGKGLEWMGFISYDGSNKYNPSLKNRITISRDTSKN 
                 
                     
                 
                   QFSLKLSSVTAADTAVYYCARGLRRGDYSMDYWGQGTLVTVSSCSTKGP 
                 
                     
                 
                   SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA 
                 
                     
                 
                   VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD 
                 
                     
                 
                   KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED 
                 
                     
                 
                   PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY 
                 
                     
                 
                   KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL 
                 
                     
                 
                   VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW 
                 
                     
                 
                   QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         and a light chain sequence comprising: 
       
       
         
           
                 
               
                   (SEQ ID NO: 2) 
                 
                     MDFQVQIFSFLLISASVIMSRG DIQMTQSPSSLSASVGDRVTITCKAS 
                 
                     
                 
                   QNVVTAVAWYQQKPGKAPKLLIYSASNRHTGVPSRFSGSGSGTDFTLTI 
                 
                     
                 
                   SSLQPEDFATYYCQQYSSYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQ 
                 
                     
                 
                   LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY 
                 
                     
                 
                   SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         62 . The cysteine engineered anti-TENB2 antibody according  claim 61  which is produced in bacteria or CHO cells. 
     
     
         63 . The cysteine engineered anti-TENB2 antibody according to  claim 61  wherein the antibody is covalently attached to an auristatin drug moiety whereby an antibody drug conjugate is formed. 
     
     
         64 . The antibody-drug conjugate of  claim 63  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, 3, 4, or preferably 2. 
 
     
     
         65 . The antibody-drug conjugate compound of  claim 64  wherein L has the formula:
   -A a -W w —Y y —
 
 where: 
 A is a Stretcher unit covalently attached to a cysteine thiol of the cysteine engineered antibody (Ab); 
 a is 0 or 1; 
 each W is independently an Amino Acid unit; 
 w is an integer ranging from 0 to 12; 
 Y is a Spacer unit covalently attached to the drug moiety; and 
 y is 0, 1 or 2. 
 
     
     
         66 . The antibody-drug conjugate compound of  claim 65  having the formula: 
       
         
           
           
               
               
           
         
         where PAB is para-aminobenzylcarbamoyl, and R 17  is a divalent radical selected from (CH 2 ) r , C 3 -C 8  carbocyclyl, O—(CH 2 ) r , arylene, (CH 2 ) r -arylene, -arylene-(CH 2 ) r —, (CH 2 ) r —(C 3 -C 8  carbocyclyl), (C 3 -C 8  carbocyclyl)-(CH 2 ) r , C 3 -C 8  heterocyclyl, (CH 2 ) r —(C 3 -C 8  heterocyclyl), —(C 3 -C 8  heterocyclyl)-(CH 2 ) r —, —(CH 2 ) r C(O)NR b (CH 2 ) r —, —(CH 2 CH 2 O) r —, —(CH 2 CH 2 O) r —CH 2 —, —(CH 2 ) r C(O)NR b (CH 2 CH 2 O) r —, —(CH 2 ) r C(O)NR b (CH 2 CH 2 O) r —CH 2 —, —(CH 2 CH 2 O) r C(O)NR b (CH 2 CH 2 O) r —, —(CH 2 CH 2 O) r C(O)NR b (CH 2 CH 2 O) r —CH 2 —, and —(CH 2 CH 2 O) r C(O)NR b (CH 2 ) r —; where R b  is H, C 1 -C 6  alkyl, phenyl, or benzyl; and r is independently an integer ranging from 1 to 10. 
       
     
     
         67 . The antibody-drug conjugate compound of  claim 65  wherein W w  is valine-citrulline. 
     
     
         68 . The antibody-drug conjugate compound of  claim 65  having the formula: 
       
         
           
           
               
               
           
         
       
     
     
         69 . The antibody-drug conjugate compound of either  claim 66  or  68  wherein R 17  is (CH 2 ) 5  or (CH 2 ) 2 . 
     
     
         70 . The antibody-drug conjugate compound of  claim 65  having the formula: 
       
         
           
           
               
               
           
         
       
     
     
         71 . The antibody-drug conjugate compound of  claim 64  wherein L is SMCC or BMPEO. 
     
     
         72 . The antibody-drug conjugate compound of  claim 64  wherein D is either MMAE, preferably having the structure: 
       
         
           
           
               
               
           
         
         where the wavy line indicates the attachment site to the linker L; or MMAF, preferably having the structure: 
       
       
         
           
           
               
               
           
         
         where the wavy line indicates the attachment site to the linker L. 
       
     
     
         73 . The cysteine engineered anti-TENB2 antibody of  claim 61  or the antibody-drug conjugate compound of any one of  claims 63  to  72  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. 
     
     
         74 . The cysteine engineered anti-TENB2 antibody of  claim 61  or antibody-drug conjugate compound of any one of  claims 63  to  72  wherein the parent anti-TENB2 antibody is an antibody fragment, preferably a Fab fragment. 
     
     
         75 . The antibody drug conjugate of  claim 63  wherein L is MC-val-cit-PAB or MC, SMCC, SPP, or BMPEO. 
     
     
         76 . 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 61 . 
       
     
     
         77 . A pharmaceutical formulation comprising the cysteine engineered anti-TENB2 antibody of  claim 61  or the antibody drug conjugate of  claim 63 , and a pharmaceutically acceptable diluent, carrier or excipient. 
     
     
         78 . The pharmaceutical formulation comprising an antibody drug conjugate according to  claim 77 , 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. 
     
     
         79 . An article of manufacture comprising the pharmaceutical formulation comprising
 an antibody drug conjugate according to  claim 77 ;   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, wherein the cancer is preferably selected from ovarian cancer, prostate cancer, cancer of the urinary tract, pancreatic cancer, lung cancer, breast cancer, or colon cancer.   
     
     
         80 . 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 61  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. 
     
     
         81 . The method of  claim 80 , wherein the antibody is covalently attached to a label selected from a fluorescent dye, a radioisotope, biotin, or a metal-complexing ligand; or wherein said sample comprises a cell suspected of expressing said TENB2 protein; or wherein said cell is a prostate, ovarian, breast, lung, or pancreatic cancer cell. 
     
     
         82 . An assay for detecting cancer cells comprising:
 (a) exposing cells to an antibody-drug conjugate compound of  claim 63 ; and   (b) determining the extent of binding of the antibody-drug conjugate compound to the cells;   wherein preferably the cells are prostate, pancreatic, lung, breast, colon or ovarian tumor cells.   
     
     
         83 . A method of inhibiting cellular proliferation comprising treating mammalian tumor cells in a cell culture medium with an antibody-drug conjugate compound of  claim 63 , whereby proliferation of the tumor cells is inhibited, wherein the mammalian tumor cells are preferably ovarian tumor cells. 
     
     
         84 . A method of treating cancer comprising administering the pharmaceutical formulation of  claim 77  to the patient, 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. 
     
     
         85 . The method of  claim 84 ,wherein the method comprises administering a chemotherapeutic agent to the patient 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. 
     
     
         86 . A method for making an antibody drug conjugate compound comprising a cysteine engineered anti-TENB2 antibody (Ab) of  claim 61 , 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.   
     
     
         87 . The method of  claim 86 , wherein the method comprises the step of expressing the cysteine engineered antibody in chinese hamster ovary (CHO) cells. 
     
     
         88 . The method of  claim 86  further comprising the step of treating the expressed cysteine engineered antibody with a reducing agent, wherein the reducing agent is selected from TCEP and DTT. 
     
     
         89 . The method of  claim 87  further comprising the step of treating the expressed cysteine engineered antibody with an oxidizing agent, after treating with the reducing agent, wherein the oxidizing agent is selected from copper sulfate, dehydroascorbic acid, and air.