Cysteine engineered antibodies and conjugates

Abstract

Cysteine engineered antibodies comprising a free cysteine amino acid in the heavy chain or light chain are prepared by mutagenizing a nucleic acid sequence of a parent antibody and replacing one or more amino acid residues by cysteine to encode the cysteine engineered antibody; expressing the cysteine engineered antibody; and isolating the cysteine engineered antibody.

1 . A cysteine engineered antibody comprising a cysteine amino acid in the heavy chain selected from the group consisting of T114C, A140C, L174C, L179C, T187C, T209C, V262C, G371C, Y373C, E382C, S424C, N434C and Q438C according to EU numbering, or a cysteine amino acid in the light chain selected from the group consisting of 1106C, R108C, R142C, and K149C according to Kabat numbering. 
     
     
         2 . The cysteine engineered antibody of  claim 1 , wherein the cysteine engineered antibody comprises a sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   SEQ ID NO.: 5 
                 
                     
                   HC-T114C QGTLV   C   VSSAS, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 6 
                 
                     
                   HC-A140C TSGGT   C   ALGCL, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 7 
                 
                     
                   HC-L174C TFPAV   C   QSSGL, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 8 
                 
                     
                   HC-L179C LQSSG   C   YSLSS, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 9 
                 
                     
                   HC-T187C LSSVV   C   VPSSS, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 10 
                 
                     
                   HC-T209C HKPSN   C   KVDKK, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 11 
                 
                     
                   HC-V262C PEVTC   C   VVDVS, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 12 
                 
                     
                   HC-G371C TCLVK   C   FYPSD, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 13 
                 
                     
                   HC-Y373C LVKGF   C   PSDIA, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 14 
                 
                     
                   HC-E382C IAVEW   C   SNGQP, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 15 
                 
                     
                   HC-S424C QGNVE   C   CSVMH, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 16 
                 
                     
                   HC-N434C HEALH   C   HYTQK, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 17 
                 
                     
                   HC-Q438C HNHYT   C   KSLSL, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 1 
                 
                     
                   LC-I106C GTKVE   C   KRTVA, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 2 
                 
                     
                   LC-R108C KVEIK   C   TVAAP, 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 3 
                 
                     
                   LC-R142C NNFYP   C   EAKVQ,  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID NO.: 4 
                 
                     
                   LC-K149C AKVQW   C   VDNAL . 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         3 . The cysteine engineered antibody of  claim 1 , wherein the in the cysteine mutation in the heavy chain is selected from the group consisting of L174C, A140C, and Y373C according to EU numbering. 
     
     
         4 .- 7 . (canceled) 
     
     
         8 . The cysteine engineered antibody of  claim 1  prepared by a process comprising:
 (i) mutagenizing a nucleic acid sequence of a parent antibody by replacing one or more amino acid residues by cysteine to encode the cysteine engineered antibody; 
 (ii) expressing the cysteine engineered antibody; and 
 (iii) isolating the cysteine engineered antibody. 
 
     
     
         9 . The cysteine engineered antibody of  claim 8  wherein the mutagenizing comprises site directed mutagenesis. 
     
     
         10 . The cysteine engineered antibody of  claim 8  wherein the cysteine is a free cysteine and wherein the process further comprises:
 (i) reacting the free cysteine of the cysteine engineered antibody with a thiol-reactive affinity reagent to generate an affinity labelled, cysteine engineered antibody comprising the affinity reagent covalently attached to the free cysteine; and 
 (ii) measuring the binding of the affinity labelled, cysteine engineered antibody to a capture media. 
 
     
     
         11 . The cysteine engineered antibody of  claim 10  wherein the thiol-reactive reagent comprises a maleimide moiety. 
     
     
         12 . The cysteine engineered antibody of  claim 10  wherein the thiol-reactive reagent comprises a biotin moiety and the capture media comprises streptavidin. 
     
     
         13 . The cysteine engineered antibody of  claim 1 , wherein the cysteine engineered antibody is selected from a monoclonal antibody, an antibody fragment, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody. 
     
     
         14 . The cysteine engineered antibody of  claim 1 , wherein the antibody fragment is a Fab fragment. 
     
     
         15 . The cysteine engineered antibody of  claim 1 , wherein the cysteine engineered antibody is an anti-HER2 antibody, an anti-Ly6E antibody, an anti-CD79b antibody, an anti-MUC16 antibody, an anti-STEAP1 antibody, an anti-NaPi2b antibody, or an anti-CD22 antibody. 
     
     
         16 .- 28 . (canceled) 
     
     
         29 . The cysteine engineered antibody of  claim 1 , wherein the cysteine engineered antibody binds to one or more of receptors (1)-(53):
 (1) BMPR1B (bone morphogenetic protein receptor-type IB);   (2) E16 (LAT1, SLC7A5);   (3) STEAP1 (six transmembrane epithelial antigen of prostate);   (4) 0772P (CA125, MUC16);   (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);   (6) Napi3b (NaPi2b, NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b);   (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMASB, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B);   (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene);   (9) ETBR (Endothelin type B receptor);   (10) MSG783 (RNF124, hypothetical protein FLJ20315);   (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein);   (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4);   (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor);   (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792);   (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29);   (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C);   (17) HER2;   (18) NCA;   (19) MDP;   (20) IL20Rα;   (21) Brevican;   (22) EphB2R;   (23) ASLG659;   (24) PSCA;   (25) GEDA;   (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3;   (27) CD22 (B-cell receptor CD22-B isoform);   (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B cell-specific protein);   (29) CXCRS (Burkitt's lymphoma receptor 1, a G protein-coupled receptor);   (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen);   (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5);   (32) CD72 (B-cell differentiation antigen CD72, Lyb-2);   (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family);   (34) FcRH1 (Fc receptor-like protein 1);   (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2);   (36) TENB2 (putative transmembrane proteoglycan);   (37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL);   (38) TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains I; Tomoregulin-1);   (39) GDNF-Ra1 (GDNF family receptor alpha I; GFRA1; GDNFR; GDNFRA; RETL1;   TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1);   (40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67,RIG-E,SCA-2,TSA-1)   (41) TMEM46 (shisa homolog 2);   (42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1);   (43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67);   (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1);   (45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; F1135226);   (46) GPR19 (G protein-coupled receptor 19; Mm.4787);   (47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12);   (48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982);   (49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3);   (50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; F1114627);   (51) GPR172A (G protein-coupled receptor 172A; GPCR41; F1111856; D15Ertd747e);   (52) CD33; and   (53) CLL-1 (CLEC12A, MICL, and DCAL2).   
     
     
         30 . The cysteine engineered antibody of  claim 1 , wherein the antibody is covalently attached to a capture label, a detection label, a drug moiety, or a solid support. 
     
     
         31 .- 34 . (canceled) 
     
     
         35 . The cysteine engineered antibody of  claim 30 , wherein the antibody is covalently attached to a detection label by a chelating ligand. 
     
     
         36 . The cysteine engineered antibody of  claim 35  wherein the chelating ligand is selected from DOTA, DOTP, DOTMA, DTPA and TETA. 
     
     
         37 . The cysteine engineered antibody of  claim 1 , wherein the antibody is covalently attached to a drug moiety to form an antibody-drug conjugate having Formula I:
   Ab-(L-D) p      where Ab is the antibody, L is a linker, D is the drug moiety, and p is 1, 2, 3, or 4, and wherein the drug moiety is conjugated to the engineered cysteine amino acid.   
     
     
         38 .- 51 . (canceled) 
     
     
         52 . A method of preparing an antibody-drug conjugate comprising reacting at least one cysteine of a cysteine engineered antibody (Ab) with a linker-drug intermediate to form an antibody-drug conjugate having Formula I:
   Ab-(L-D) p   I
   wherein Ab is the cysteine engineered antibody of  claim 1 , L is a linker, D is a drug moiety, and p is 1, 2, 3, or 4; and wherein the cysteine engineered antibody comprises one or more cysteine amino acids.   
     
     
         53 . The method of preparing an antibody-drug conjugate of  claim 52 , wherein the cysteine mutation is selected from the group consisting of HC-L174C, HC-A140C, and HC-Y373C according to EU numbering. 
     
     
         54 .- 57 . (canceled) 
     
     
         58 . The antibody drug conjugate of  claim 37  wherein the cysteine engineered antibody has a heavy chain mutation of A140C according to EU numbering, L comprises a pyridyl disulfide (PDS), and D is selected from the group consisting of a CBI-PBD heterodimer, cryptophycin, a taxoid, and tubulysin M. 
     
     
         59 . The antibody drug conjugate of  claim 37  wherein the cysteine engineered antibody has a heavy chain mutation of A140C according to EU numbering, L comprises a -vc linker, and D is selected from the group consisting of a CBI-PBD heterodimer, cryptophycin, a taxoid, and tubulysin M. 
     
     
         60 .- 64 . (canceled) 
     
     
         65 . An antibody drug conjugate, wherein the antibody drug conjugate is selected from one of the antibody drug conjugates recited in Table 18 or Table 19. 
     
     
         66 . A pharmaceutical composition comprising the cysteine engineered antibody of  claim 1 .