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 free cysteine amino acids, wherein at least one free cysteine amino acid is located at a position selected from heavy chain positions 112, 113, 114, and 168 by Kabat numbering and light chain positions 15, 43, 110, 114, 121, 127, 144, 153, 158, 168, and 205 by Kabat numbering. 
     
     
         2 . The cysteine engineered antibody of  claim 1  wherein the cysteine engineered antibody is more reactive than the parent antibody with a thiol-reactive reagent. 
     
     
         3 . (canceled) 
     
     
         4 . The cysteine engineered antibody of  claim 1  wherein the antibody comprises at least one free cysteine amino acid in a light chain. 
     
     
         5 .- 8 . (canceled) 
     
     
         9 . The cysteine engineered antibody of  claim 1  wherein the antibody comprises at least one free cysteine amino acid in a heavy chain. 
     
     
         10 .- 15 . (canceled) 
     
     
         16 . The antibody of  claim 1  wherein the thiol reactivity value of at least one free cysteine amino acid is in the range of 0.6 to 1.0. 
     
     
         17 . The cysteine engineered antibody of  claim 1  wherein the thiol reactivity value is in the range of 0.8 to 1.0. 
     
     
         18 .- 26 . (canceled) 
     
     
         27 . The cysteine engineered antibody of  claim 1 , which is a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, or a humanized antibody. 
     
     
         28 . The cysteine engineered antibody of  claim 27  wherein the variable regions are from an antibody selected from huMAb4D5-8 (trastuzumab), an anti-EphB2R antibody, and an anti-MUC16 antibody. 
     
     
         29 .- 31 . (canceled) 
     
     
         32 . The cysteine engineered antibody of  claim 27 , which is an IgA, IgD, IgE, IgG, or IgM antibody. 
     
     
         33 . The cysteine engineered antibody of  claim 32  wherein the IgG is selected from subclasses IgG1, IgG2, IgG3, and IgG4. 
     
     
         34 . The cysteine engineered antibody of  claim 1  which is an antibody fragment. 
     
     
         35 . The cysteine engineered antibody of  claim 34  wherein the antibody fragment is a Fab fragment. 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . The cysteine engineered antibody of  claim 1  wherein the cysteine engineered antibody binds to one or more of receptors (1)-(36):
 (1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203); 
 (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486); 
 (3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449); 
 (4) 0772P (CA125, MUC16, Genbank accession no. AF361486); 
 (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823); 
 (6)  Napi 3b ( NAPI -3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424); 
 (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™ and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878); 
 (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628); 
 (9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463); 
 (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763); 
 (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, Genbank accession no. AF455138); 
 (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636); 
 (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212); 
 (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004); 
 (15) CD79b (CD79B, CD79(3, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626); 
 (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764); 
 (17) HER2 (Genbank accession no. M11730); 
 (18) NCA (Genbank accession no. M18728); 
 (19) MDP (Genbank accession no. BC017023); 
 (20) IL20Rα (Genbank accession no. AF184971); 
 (21) Brevican (Genbank accession no. AF229053); 
 (22) EphB2R (Genbank accession no. NM_004442); 
 (23) ASLG659 (Genbank accession no. AX092328); 
 (24) PSCA (Genbank accession no. AJ297436); 
 (25) GEDA (Genbank accession no. AY260763; 
 (26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3, NP_443177.1); 
 (27) CD22 (B-cell receptor CD22-B isoform, NP-001762.1); 
 (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with IgM molecules, transduces a signal involved in B-cell differentiation, Genbank accession No. NP_001774.1); 
 (29) CXCR5(Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1); 
 (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+T lymphocytes, Genbank accession No. NP_002111.1); 
 (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2); 
 (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank accession No. NP_001773.1); 
 (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_005573.1); 
 (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); 
 (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies, Genbank accession No. NP_112571.1); and 
 (36) TENB2 (putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin, Genbank accession No. AF179274. 
 
     
     
         39 . (canceled) 
     
     
         40 . The cysteine engineered antibody of  claim 1  wherein at least one free cysteine is covalently attached to a capture label, a detection label, or a solid support. 
     
     
         41 . The cysteine engineered antibody of  claim 40  wherein the capture label is a biotin capture label; the detection label is a fluorescent dye detection label selected from a fluorescein type, a rhodamine type, dansyl, Lissamine, a cyanine, a phycoerythrin, Texas Red, and an analog thereof; or the detection label is a a radionuclide detection label selected from  3 H,  11 C,  14 C,  18 F,  32 P,  35 S,  64 Cu,  68 Ga,  86 Y,  99 Tc,  111 In,  123 I,  124 I,  125 I,  131 I,  133 xe,  177 Lu,  211 At, and  213 Bi. 
     
     
         42 .- 44 . (canceled) 
     
     
         45 . The cysteine engineered antibody of  claim 40  wherein at least one free cysteine is covalently attached to a detection label by a chelating ligand selected from DOTA, DOTP, DOTMA, DTPA and TETA. 
     
     
         46 .- 56 . (canceled) 
     
     
         57 . An antibody-drug conjugate compound comprising a cysteine engineered antibody (Ab), and a drug moiety (D) wherein the cysteine engineered 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; and wherein the cysteine engineered antibody is an antibody of  claim 1 .   
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . The antibody-drug conjugate compound of  claim 57  further comprising an albumin-binding peptide (ABP) sequence. 
     
     
         61 . The antibody-drug conjugate compound of  claim 60  wherein the ABP comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. 
     
     
         62 . The antibody-drug conjugate compound of  claim 57  wherein the cysteine engineered antibody binds to an ErbB receptor selected from EGFR, HER2, HER3, and HER4. 
     
     
         63 . The antibody-drug conjugate compound of  claim 57  wherein the cysteine engineered antibody binds to one or more of receptors (1)-(36):
 (1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203); 
 (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486); 
 (3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449); 
 (4) 0772P (CA125, MUC16, Genbank accession no. AF361486); 
 (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823); 
 (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424); 
 (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878); 
 (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628); 
 (9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463); 
 (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763); 
 (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, Genbank accession no. AF455138); 
 (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636); 
 (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212); 
 (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004); 
 (15) CD79b (CD79B, CD79(3, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626); 
 (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764); 
 (17) HER2 (Genbank accession no. M11730); 
 (18) NCA (Genbank accession no. M18728); 
 (19) MDP (Genbank accession no. BC017023); 
 (20) IL20Rα (Genbank accession no. AF184971); 
 (21) Brevican (Genbank accession no. AF229053); 
 (22) EphB2R (Genbank accession no. NM_004442); 
 (23) ASLG659 (Genbank accession no. AX092328); 
 (24) PSCA (Genbank accession no. AJ297436); 
 (25) GEDA (Genbank accession no. AY260763; 
 (26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3, NP_443177.1); 
 (27) CD22 (B-cell receptor CD22-B isoform, NP-001762.1); 
 (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation, Genbank accession No. NP_001774.1); 
 (29) CXCR5(Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1); 
 (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+T lymphocytes, Genbank accession No. NP_002111.1); 
 (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2); 
 (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank accession No. NP_001773.1); 
 (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_005573.1); 
 (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); 
 (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies, Genbank accession No. NP_112571.1); and 
 (36) TENB2 (putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin, Genbank accession No. AF179274. 
 
     
     
         64 . The antibody-drug conjugate compound of  claim 57  wherein p is 1 to 2. 
     
     
         65 . (canceled) 
     
     
         66 . The antibody-drug conjugate compound of  claim 57  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. 
 
 
     
     
         67 . The antibody-drug conjugate compound of  claim 66  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. 
       
     
     
         68 . The antibody-drug conjugate compound of  claim 67  wherein W w  is valine-citrulline. 
     
     
         69 . The antibody-drug conjugate compound of  claim 67  wherein R 17  is (CH 2 ) 5  or (CH 2 ) 2 . 
     
     
         70 . The antibody-drug conjugate compound of  claim 66  having the formula: 
       
         
           
           
               
               
           
         
       
     
     
         71 . The antibody-drug conjugate compound of  claim 70  wherein R 17  is (CH 2 ) 5  or (CH 2 ) 2 . 
     
     
         72 . The antibody-drug conjugate compound of  claim 66  having the formula: 
       
         
           
           
               
               
           
         
       
     
     
         73 . The antibody-drug conjugate compound of  claim 57  wherein L is SMCC or BMPEO. 
     
     
         74 .- 76 . (canceled) 
     
     
         77 . The antibody-drug conjugate compound of  claim 57  wherein D is MMAE, having the structure: 
       
         
           
           
               
               
           
         
         where the wavy line indicates the attachment site to the linker L. 
       
     
     
         78 . The antibody-drug conjugate compound of  claim 57  wherein D is MMAF, having the structure: 
       
         
           
           
               
               
           
         
         where the wavy line indicates the attachment site to the linker L. 
       
     
     
         79 . The antibody-drug conjugate compound of  claim 57  wherein D is DM1, having the structure: 
       
         
           
           
               
               
           
         
         where the wavy line indicates the attachment site to the linker L. 
       
     
     
         80 . The antibody-drug conjugate compound of  claim 57  wherein the parent antibody is a fusion protein comprising the albumin-binding peptide (ABP). 
     
     
         81 . The antibody-drug conjugate compound of  claim 57  wherein the antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody. 
     
     
         82 . The antibody-drug conjugate compound of  claim 57  wherein the variable regions are from an antibody selected from huMAb4D5-8 (trastuzumab), an anti-ErbB2 antibody, an anti-EphB2R antibody, an anti-CD22 antibody, and an anti-MUC16 antibody. 
     
     
         83 .- 86 . (canceled) 
     
     
         87 . The antibody-drug conjugate compound of  claim 57  wherein the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. 
     
     
         88 . The antibody-drug conjugate compound of  claim 87  wherein the IgG is selected from subclasses: IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. 
     
     
         89 . The antibody-drug conjugate compound of  claim 57  wherein the antibody is an antibody fragment. 
     
     
         90 . The antibody-drug conjugate compound of  claim 89  wherein the antibody fragment is a Fab fragment. 
     
     
         91 . (canceled) 
     
     
         92 . (canceled) 
     
     
         93 . The antibody-drug conjugate of  claim 57 , which has a structure selected from: 
       
         
           
           
               
               
           
         
         wherein Val is valine; and Cit is citrulline. 
       
     
     
         94 . The antibody-drug conjugate of  claim 57 , having the structure: 
       
         
           
           
               
               
           
         
         wherein n is 0, 1, or 2. 
       
     
     
         95 . A pharmaceutical composition comprising the antibody-drug conjugate compound of  claim 57  or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient. 
     
     
         96 . The pharmaceutical composition of  claim 95  further comprising a therapeutically effective amount of an additional chemotherapeutic agent. 
     
     
         97 .- 126 . (canceled)