Antibody-drug conjugates and methods

Abstract

The present invention relates to antibody-drug conjugate compounds of Formula I: Ab-(L-D) p   I where one or more maytansinoid drug moieties (D) are covalently linked by L to an antibody (Ab) which binds to an ErbB receptor, or which binds to one or more tumor-associated antigens or cell-surface receptors. These compounds may be used in methods of diagnosis or treatment of cancer, and other diseases and disorders.

1 . An antibody-drug conjugate compound comprising an antibody covalently attached by a linker to one or more maytansinoid drug moieties, the compound having Formula I:  
         Ab-(L-D) p   I  
       or a pharmaceutically acceptable salt or solvate thereof, wherein: 
 Ab is an antibody which binds to an ErbB receptor, or which binds to one or more tumor-associated antigens or cell-surface receptors selected from (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, C530008016Rik, 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, STAMP 1, 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, CD79β, 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-β-form, NP-001762.1);  
 (28) CD79a (CD79A, CD79a, 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;  
 provided that the antibody is not TA. 1;  
 L is a non-disulfide linker;  
 D is a maytansinoid drug moiety; and  
 p is 1 to 8.  
 
     
     
         2 . The antibody-drug conjugate compound of  claim 1  wherein Ab is an antibody which binds to one or more tumor-associated antigens or cell-surface receptors selected from (1)-(16) and (18)-(36).  
     
     
         3 . The antibody-drug conjugate compound of  claim 1  wherein L is a linker selected from the structures:  
       
         
           
           
               
               
           
         
       
       where the wavy lines indicate the covalent attachments to Ab and D; 
 X is:  
                     
 R is independently H or C 1 -C 6  alkyl; and n is 1 to 12.  
 
     
     
         4 . The antibody-drug conjugate compound of  claim 1  wherein D is selected from the structure:  
       
         
           
           
               
               
           
         
         where the wavy line indicates the covalent attachment to L,  
         R is independently H or C 1 -C 6  alkyl, and  
         m is 1, 2, or 3.  
       
     
     
         5 . The antibody-drug conjugate compound of  claim 4  wherein m is 2, and R is H.  
     
     
         6 . The antibody-drug conjugate compound of  claim 5  wherein the maytansinoid drug moiety is DM1, having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         7 . The antibody-drug conjugate compound of  claim 3  having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         8 . The antibody-drug conjugate compound of  claim 7  having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         9 . The antibody-drug conjugate compound of  claim 3  having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         10 . The antibody-drug conjugate compound of  claim 1  having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         11 . The antibody-drug conjugate of  claim 10  wherein Ab is trastuzumab, and p is 1, 2, 3, or 4.  
     
     
         12 . The antibody-drug conjugate compound of  claim 1  having the structure:  
       
         
           
           
               
               
           
         
         where Tr is trastuzumab, and p is 1, 2, 3, or 4.  
       
     
     
         13 . The antibody-drug conjugate compound of  claim 1  having the structure:  
       
         
           
           
               
               
           
         
         wherein n is 0, 1, or 2; and p is 1, 2, 3, or 4.  
       
     
     
         14 . The antibody-drug conjugate compound of  claim 13  wherein Ab is trastuzumab.  
     
     
         15 . The antibody-drug conjugate compound of  claim 1  wherein p is 1, 2, 3, or 4.  
     
     
         16 . The antibody-drug conjugate compound of  claim 1  wherein the antibody binds to a receptor encoded by an ErbB gene.  
     
     
         17 . The antibody-drug conjugate compound of  claim 16  wherein the receptor is selected from EGFR, HER2, HER3 and HER4.  
     
     
         18 . The antibody-drug conjugate compound of  claim 17  wherein the antibody specifically binds to a HER2 receptor.  
     
     
         19 . The antibody-drug conjugate compound of  claim 1  which specifically binds to the extracellular domain of the HER2 receptor and inhibits growth of tumor cells which overexpress HER2 receptor.  
     
     
         20 . The antibody-drug conjugate compound of  claim 1  wherein the antibody is selected from a monoclonal antibody, an antibody fragment, a chimeric antibody, and a humanized antibody.  
     
     
         21 . The antibody-drug conjugate compound of  claim 20  wherein the humanized antibody is selected from huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).  
     
     
         22 . The antibody-drug conjugate compound of  claim 21  wherein the antibody is huMAb4D5-8 (trastuzumab).  
     
     
         23 . The antibody-drug conjugate of  claim 20  wherein the antibody is a Fab fragment.  
     
     
         24 . The antibody-drug conjugate compound of  claim 1  wherein the antibody is attached to the linker through a cysteine thiol of the antibody.  
     
     
         25 . The antibody-drug conjugate compound of  claim 24  wherein p is 1, 2, 3, or 4.  
     
     
         26 . A pharmaceutical composition comprising the antibody-drug conjugate compound of  claim 1 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient.  
     
     
         27 . The pharmaceutical composition of  claim 26  further comprising a therapeutically effective amount of a chemotherapeutic agent selected from Erlotinib, Bortezomib, Fulvestrant, Sutent, Letrozole, Imatinib mesylate, PTK787/ZK 222584, Oxaliplatin, 5-FU, Leucovorin, Rapamycin, Lapatinib, Lonafarnib, Sorafenib, and Gefitinib.  
     
     
         28 . The pharmaceutical composition of  claim 26  further comprising a therapeutically effective amount of a anti-angiogenic agent.  
     
     
         29 . The pharmaceutical composition of  claim 26  further comprising a therapeutically effective amount of bevacizumab.  
     
     
         30 . A method of inhibiting cellular proliferation comprising treating mammalian cells in a cell culture medium with an antibody-drug conjugate compound of  claim 1 , whereby proliferation of the cells is inhibited.  
     
     
         31 . The method of  claim 30  wherein the mammalian cells have HER2 receptor proteins to the antibody-drug conjugate compound.  
     
     
         32 . The method of  claim 31  wherein the mammalian cells are breast tumor cells.  
     
     
         33 . The method of  claim 30  wherein the antibody-drug conjugate compound is more cytotoxic than a maytansinoid compound comprising the maytansinoid moiety of the antibody-drug conjugate compound.  
     
     
         34 . The method of  claim 30  wherein the antibody-drug conjugate induces apoptosis.  
     
     
         35 . A method of treating cancer comprising administering to a patient a formulation of an antibody-drug conjugate compound of  claim 1  and a pharmaceutically acceptable diluent, carrier or excipient.  
     
     
         36 . The method of  claim 35  wherein the cancer is selected from the group consisting of breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, colorectal, thyroid, pancreatic, prostate and bladder cancer.  
     
     
         37 . The method of  claim 36  wherein the cancer is breast cancer which overexpresses ErbB2 at a 2+ level or more.  
     
     
         38 . The method of  claim 35  wherein the amount of antibody-drug conjugate compound administered to the patient is in the range of about 0.1 to about 10 mg/kg of patient weight per dose.  
     
     
         39 . The method of  claim 35  wherein the antibody-drug conjugate is administered at about three week intervals.  
     
     
         40 . The method of  claim 35  wherein the antibody-drug conjugate is administered by infusion.  
     
     
         41 . The method of  claim 35  wherein the antibody-drug conjugate is formulated with a pharmaceutically acceptable parenteral vehicle.  
     
     
         42 . The method of  claim 41  wherein the antibody-drug conjugate is formulated in a unit dosage injectable form.  
     
     
         43 . The method of  claim 42  wherein the antibody-drug conjugate is administered intravenously.  
     
     
         44 . The method of  claim 35  wherein the patient is administered a growth inhibitory antibody, in combination with the antibody-drug conjugate compound.  
     
     
         45 . The method of  claim 35  wherein the patient is administered a second antibody which binds ErbB2 and blocks ligand activation of an ErbB receptor, in combination with the antibody-drug conjugate compound.  
     
     
         46 . The method of  claim 45  wherein the second antibody comprises monoclonal antibody 2C4 or humanized 2C4.  
     
     
         47 . The method of  claim 46  wherein said second antibody is conjugated with a cytotoxic agent.  
     
     
         48 . The method of  claim 35  wherein the patient is administered a chemotherapeutic agent, in combination with the antibody-drug conjugate compound, where the chemotherapeutic agent is selected from Erlotinib, Bortezomib, Fulvestrant, Sutent, Letrozole, Imatinib mesylate, PTK787/ZK 222584, Oxaliplatin, 5-FU, Leucovorin, Rapamycin, Lapatinib, Lonafarnib, Sorafenib, and Gefitinib.  
     
     
         49 . The method of  claim 35  wherein the patient is administered an anti-angiogenic agent in combination with the antibody-drug conjugate compound where the anti-angiogenic agent is selected from bevacizumab.  
     
     
         50 . A method of inhibiting the growth of tumor cells that overexpress a growth factor receptor, comprising administering to a patient an antibody-drug conjugate of  claim 1  which binds specifically to a growth factor receptor selected from HER2 or EGF and a chemotherapeutic agent wherein said antibody-drug conjugate and said chemotherapeutic agent are each administered in amounts effective to inhibit growth of tumor cells in the patient.  
     
     
         51 . A method for the treatment of a human patient susceptible to or diagnosed with a disorder characterized by overexpression of ErbB2 receptor, comprising administering a combination of an antibody-drug conjugate of  claim 1  and a chemotherapeutic agent or a growth inhibitory agent.  
     
     
         52 . The method of  claim 51  wherein the antibody of the antibody-drug conjugate is an anti-ErbB2 antibody.  
     
     
         53 . The method of  claim 52  wherein the anti-ErbB2 antibody has a biological characteristic of a 4D5 monoclonal antibody.  
     
     
         54 . The method of  claim 52  wherein the anti-ErbB2 antibody binds essentially the same epitope as a 4D5 monoclonal antibody.  
     
     
         55 . The method of  claim 52  wherein the anti-ErbB2 antibody is selected from the group consisting of humanized antibodies huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).  
     
     
         56 . An assay for detecting cancer cells comprising: 
 (a) exposing cells to an antibody-drug conjugate compound of  claim 1;  and    (b) determining the extent of binding of the antibody-drug conjugate compound to the cells.    
     
     
         57 . The assay of  claim 56  wherein the cells are breast tumor cells.  
     
     
         58 . The assay of  claim 56  wherein the extent of binding is determined by measuring levels of ErbB-encoding nucleic acid by fluorescent in situ hybridization (FISH).  
     
     
         59 . The assay of  claim 56  wherein the extent of binding is determined by immunohistochemistry (IHC).  
     
     
         60 . An article of manufacture comprising 
 an antibody-drug conjugate compound of  claim 1;     a container; and    a package insert or label indicating that the compound can be used to treat cancer characterized by the overexpression of an ErbB receptor.    
     
     
         61 . The article of manufacture of  claim 60  wherein said package insert of label indicates that the compound can be used to treat cancer characterized by the overexpression of an ErbB2 receptor.  
     
     
         62 . The article of manufacture of  claim 61  wherein the cancer is breast cancer.  
     
     
         63 . The article of manufacture of  claim 62  wherein the cancer is characterized by the overexpression of an ErbB2 receptor at a 2+ level or above.  
     
     
         64 . A method of making an antibody-drug conjugate compound comprising an antibody covalently attached by a linker to one or more maytansinoid drug moieties, the compound having Formula I:  
         Ab-(L-D) p   I  
       or a pharmaceutically acceptable salt or solvate thereof, wherein: 
 Ab is an antibody which binds to an ErbB receptor, or which binds to one or more tumor-associated antigens or cell-surface receptors selected from (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, C530008016Rik, 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, CD79β, 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-β-form, 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;  
 provided that the antibody is not TA.1;  
 L is a linker selected from the structures;  
                     
 where the wavy lines indicates the covalent attachments to Ab and D;  
 X is:  
                     
 where R is independently H or C 1 -C 6  alkyl; and n is 1 to 12;  
 D is a maytansinoid drug moiety selected from the structure:  
                     
 where the wavy line indicates the covalent attachment to L; R is independently H or C 1-6  alkyl; m is 1, 2, or 3; and p is 1 to 8;  
 wherein the method comprises:  
 reacting Ab with a linker reagent to form antibody-linker intermediate Ab-L, and then reacting Ab-L with a drug moiety D to form the antibody-drug conjugate; or  
 reacting a drug moiety D with a linker reagent to form a drug-linker intermediate D-L, and then reacting D-L with Ab to form the antibody-drug conjugate.  
 
     
     
         65 . The method of  claim 64  wherein Ab is an antibody which binds to one or more tumor-associated antigens or cell-surface receptors selected from (1)-(16) and (18)-(36).  
     
     
         66 . The method of  claim 64  wherein the linker reagent is SMCC.  
     
     
         67 . The method of  claim 64  wherein the linker reagent is a bis-maleimide reagent selected from DTME, BMB, BMDB, BMH, BMOE, BM(PEO) 3 , and BM(PEO) 4 .