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. Certain highly reactive cysteine engineered antibodies were identified by the PHESELECTOR assay. Isolated cysteine engineered antibodies may be covalently attached to a capture label, a detection label, a drug moiety, or a solid support.

1 - 28 . (canceled) 
     
     
         29 . A method of preparing an antibody-drug conjugate comprising reacting at least one free cysteine of a cysteine engineered antibody (Ab) with a linker-drug (L-D) reagent to form an antibody-drug conjugate having Formula I:
   Ab-(L-D) p   I
   
       wherein Ab is the cysteine engineered antibody, 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 free cysteine amino acids, wherein at least one free cysteine amino acid is located at a position selected from heavy chain positions 4, 20, 23, 27, 32, 47, 68, 76, 78, 80, 81, 82, 94, 98, 108, 113, 154, 162, and 164 by Kabat numbering and light chain positions 49, 53, 73, 75, 80, 92, 95, 99, 101, 137, 138, 149, and 183 by Kabat numbering. 
     
     
         30 . The method of  claim 29 , wherein at least one free cysteine amino acid is located at a position selected from heavy chain positions 108, 113, 154, and 162 by Kabat numbering and light chain positions 101, 137, 138, and 149 by Kabat numbering 
     
     
         31 . The method of  claim 29 , 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. 
     
     
         32 . The method of  claim 31 , wherein the antibody fragment is a Fab fragment. 
     
     
         33 . The method of  claim 29 , wherein the cysteine engineered antibody is an anti-HER2 antibody. 
     
     
         34 . The method of  claim 29 , wherein the cysteine engineered antibody binds to one or more of receptors (1)-(36):
 (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 (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 F1120315);   (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, F1120041, 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) CXCR5 (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); and   (36) TENB2 (putative transmembrane proteoglycan).   
     
     
         35 . The method of  claim 29 , wherein the drug (D) is selected from a maytansinoid, an auristatin, a dolastatin, a trichothecene, CC1065, a calicheamicin, enediyne antibiotics, a taxane, and an anthracycline. 
     
     
         36 . The method of  claim 29 , wherein the antibody-drug conjugate has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         37 . The method of  claim 29 , wherein D is a maytansinoid having the structure: 
       
         
           
           
               
               
           
         
         wherein the wavy line indicates the covalent attachment of the sulfur atom of D to the linker;
 R is independently selected from H, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl; and 
 m is 1, 2, or 3. 
 
       
     
     
         38 . The method of  claim 29 , wherein D is selected from the structures: 
       
         
           
           
               
               
           
         
       
     
     
         39 . The method of  claim 29 , wherein the antibody drug conjugate has the structure: 
       
         
           
           
               
               
           
         
         wherein n is 0, 1, or 2. 
       
     
     
         40 . The method of  claim 29 , wherein D is a monomethylauristatin drug moiety MMAE or MMAF having the structures: 
       
         
           
           
               
               
           
         
       
     
     
         41 . The method of  claim 29 , wherein the antibody-drug conjugate is selected from the structures: 
       
         
           
           
               
               
           
         
       
       where Val is valine; Cit is citrulline; and p is 1, 2, 3, or 4. 
     
     
         42 . The method of  claim 29 , wherein the linker-drug (L-D) reagent comprises a thiol-reactive agent. 
     
     
         43 . The method of  claim 42 , wherein the thiol-reactive agent is selected from a maleimide, an iodoacetamide, and a pyridyl disulfide.