Claims for Patent: 10,077,318

Summary for Patent: 10,077,318

Title:	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.
Inventor(s):	Bhakta; Sunil (San Ramon, CA), Erickson; Hans (South San Francisco, CA), Junutula; Jagath R. (Fremont, CA), Kozak; Katherine (Half Moon Bay, CA), Ohri; Rachana (South San Francisco, CA), Pillow; Thomas (San Francisco, CA)
Assignee:	Genentech, Inc. (South San Francisco, CA)
Application Number:	14/851,348
Patent Claims:	1. A cysteine engineered antibody comprising an A140C engineered cysteine amino acid in the heavy chain according to EU numbering, and a K149C engineered cysteine amino acid in the light chain according to Kabat numbering. 2. The cysteine engineered antibody of claim 1, wherein the cysteine engineered antibody comprises the sequences: HC-A140C TSGGTCALGCL SEQ ID NO.:6 in the heavy chain, and LC-K149C AKVQWCVDNAL SEQ ID NO.:4 in the light chain. 3. 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. 4. 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. 5. The cysteine engineered antibody of claim 4, wherein the antibody fragment is a Fab fragment. 6. The cysteine engineered antibody of claim 1, wherein the cysteine engineered antibody is selected from an anti-HER2 antibody, an anti-Ly6E antibody, an anti-CD79b antibody, an anti-MUC16 antibody, an anti-STEAP1 antibody, an anti-NaPi2b antibody, an anti-CD22 antibody. 7. The cysteine engineered antibody of claim 6, wherein the cysteine engineered antibody is an anti-HER2 antibody, and wherein the anti-HER2 antibody is trastuzumab. 8. The cysteine engineered antibody of claim 6, wherein the cysteine engineered antibody is an anti-Ly6E antibody, and wherein the anti-Ly6E antibody comprises (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 179, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 180; an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 181; an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 176; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 177; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 178; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 175 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 174. 9. The cysteine engineered antibody of claim 6, wherein the cysteine engineered antibody is an anti-CD79b antibody, and wherein the anti-CD79b antibody comprises (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 186, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 187; an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 188; an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 189; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 190; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 191; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 184 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 185. 10. The cysteine engineered antibody of claim 6, wherein the cysteine engineered antibody is an anti-MUC16 antibody, and wherein the anti-MUC16 antibody comprises (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 152, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 153; an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 154; an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 149; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 150; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 151; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 156 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 157. 11. The cysteine engineered antibody of claim 6, wherein the cysteine engineered antibody is an anti-STEAP1 antibody, and wherein the anti-STEAP1 antibody comprises (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 157, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 158; an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 159; an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 160; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 161; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 162; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 164. 12. The cysteine engineered antibody of claim 6, wherein the cysteine engineered antibody is an anti-NaPi2b antibody, and wherein the anti-NaPi2b antibody comprises (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 165, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 167; an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 168; an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 169; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 170; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 171; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 172 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 173. 13. The cysteine engineered antibody of claim 6, wherein the cysteine engineered antibody is an anti-CD22 antibody, and wherein the anti-CD22 antibody comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 192, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 193; an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 194; an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 195; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 196; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 197. 14. 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, 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); (8) PSCA h1g (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, CD79.beta., 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) IL20R.alpha.; (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, CD79.alpha., 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); (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 1; Tomoregulin-1); (39) GDNF-Ra1 (GDNF family receptor alpha 1; 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; FLJ35226); (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; FLJ14627); (51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); (52) CD33; and (53) CLL-1 (CLEC12A, MICL, and DCAL2). 15. 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. 16. The cysteine engineered antibody of claim 15, wherein the antibody is covalently attached to a biotin capture label. 17. The cysteine engineered antibody of claim 15, wherein the antibody is covalently attached to a fluorescent dye detection label. 18. The cysteine engineered antibody of claim 17, wherein the fluorescent dye is selected from a fluorescein type, a rhodamine type, dansyl, lissamine, a cyanine, a phycoerythrin, and an analog thereof. 19. The cysteine engineered antibody of claim 15, wherein the antibody is covalently attached to a radionuclide detection label selected from .sup.3H, .sup.11C, .sup.14C, .sup.18F, .sup.32P, .sup.35S, .sup.64Cu, .sup.68Ga, .sup.86Y, .sup.89Zr, .sup.99Tc, .sup.111In, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.133Xe, .sup.177Lu, .sup.211At, and .sup.213Bi. 20. The cysteine engineered antibody of claim 15, wherein the antibody is covalently attached to a detection label by a chelating ligand. 21. The cysteine engineered antibody of claim 20 wherein the chelating ligand is selected from DOTA, DOTP, DOTMA, DTPA and TETA. 22. An antibody-drug conjugate comprising the cysteine engineered antibody of claim 1, wherein the antibody-drug conjugate has Formula I: Ab-(L-D).sub.p I where 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 drug moiety is conjugated to the engineered cysteine amino acid. 23. The antibody drug conjugate of claim 22, wherein L comprises a group selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit (-vc)), alanine-phenylalanine (ala-phe), and p-aminobenzyloxycarbonyl (PAB). 24. The antibody-drug conjugate of claim 22 prepared from a linker reagent selected from N-Succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), 4-(2-Pyridyldithio)butyric acid-N-hydroxysuccinimide ester (SPDB), and N-Succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB). 25. The antibody-drug conjugate of claim 22, prepared from a linker reagent comprising maleimide, iodoacetamide, bromoacetamide or disulfide. 26. The antibody-drug conjugate of claim 22, wherein L forms a disulfide linker. 27. The antibody-drug conjugate of claim 25, wherein the linker reagent comprises a pyridyl disulfide (PDS). 28. The antibody-drug conjugate of claim 22, wherein the drug moiety (D) is a maytansinoid, an auristatin, a dolastatin, a trichothecene, CC1065, a calicheamicin, enediyne antibiotics, a taxane, a pyrrolobenzodiazepine (PBD) dimer, a 1-(Chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer, a CBI-PBD heterodimer, or an anthracycline. 29. The antibody-drug conjugate of claim 28 wherein D is a monomethylauristatin drug moiety MMAE having the structure: ##STR00079## wherein the wavy line indicates the covalent attachment site to the linker. 30. The antibody-drug conjugate of claim 28 wherein the antibody-drug conjugate is selected from the structures: ##STR00080## where Val is valine; Cit is citrulline; and p is 1, 2, 3, or 4. 31. The antibody drug conjugate of claim 28 wherein D is a PBD dimer drug having the structure: ##STR00081## and salts and solvates thereof, wherein: the wavy line indicates the covalent attachment site to the linker; the dotted lines indicate the optional presence of a double bond between C1 and C2 or C2 and C3; R.sup.2 is independently selected from H, OH, .dbd.O, .dbd.CH.sub.2, CN, R, OR, .dbd.CH--R.sup.D, .dbd.C(R.sup.D).sub.2, O--SO.sub.2--R, CO.sub.2R and COR, and optionally further selected from halo or dihalo, wherein R.sup.D is independently selected from R, CO.sub.2R, COR, CHO, CO.sub.2H, and halo; R.sup.6 and R.sup.9 are independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR', NO.sub.2, Me.sub.3Sn and halo; R.sup.7 is independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR', NO.sub.2, Me.sub.3Sn and halo; Q is independently selected from O, S and NH; R.sup.11 is either H, or R or, where Q is O, SO.sub.3M, where M is a metal cation; R and R' are each independently selected from optionally substituted C.sub.1-8 alkyl, C.sub.1-12 alkyl, C.sub.3-8 heterocyclyl, C.sub.3-20 heterocycle, and C.sub.5-20 aryl groups, and optionally in relation to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring; R.sup.12, R.sup.16, R.sup.19 and R.sup.17 are as defined for R.sup.2, R.sup.6, R.sup.9 and R.sup.7 respectively; R'' is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatom; e.g. O, S, N(H), NMe and/or aromatic rings, e.g. benzene or pyridine, which rings are optionally substituted; and X and X' are independently selected from O, S and N(H). 32. The antibody drug conjugate of claim 31 wherein the structure of the PBD dimer is: ##STR00082## including salts and solvates thereof, wherein the wavy line indicates the covalent attachment site to the linker; the wavy line connected to the OH indicates the S or R configuration; R.sup.V1 and R.sup.V2 are independently selected from H, methyl, ethyl and phenyl (which phenyl may be optionally substituted with fluoro, particularly in the 4 position) and C.sub.5-6 heterocyclyl; wherein R.sup.V1 and R.sup.V2 may be the same or different; and n is 0 or 1. 33. The antibody drug conjugate of claim 31 selected from: ##STR00083## 34. The cysteine engineered antibody of claim 28 wherein D is a CBI dimer drug having the structure: ##STR00084## where R.sup.1 is selected from H, P(O).sub.3H.sub.2, C(O)NR.sup.aR.sup.b, or a bond to a linker (L); R.sup.2 is selected from H, P(O).sub.3H.sub.2, C(O)NR.sup.aR.sup.b, or a bond to a linker (L); R.sup.a and R.sup.b are independently selected from H and C.sub.1-C.sub.6 alkyl optionally substituted with one or more F, or R.sup.a and R.sup.b form a five or six membered heterocyclyl group; T is a tether group selected from C.sub.3-C.sub.12 alkylene, Y, (C.sub.1-C.sub.6 alkylene)-Y--(C.sub.1-C.sub.6 alkylene), (C.sub.1-C.sub.6 alkylene)-Y--(C.sub.1-C.sub.6 alkylene)-Y--(C.sub.1-C.sub.6 alkylene), (C.sub.2-C.sub.6 alkenylene)-Y--(C.sub.2-C.sub.6 alkenylene), and (C.sub.2-C.sub.6 alkynylene)-Y--(C.sub.2-C.sub.6 alkynylene); where Y is independently selected from O, S, NR.sup.1, aryl, and heteroaryl; where alkylene, alkenylene, aryl, and heteroaryl are independently and optionally substituted with F, OH, O(C.sub.1-C.sub.6 alkyl), NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OP(O).sub.3H.sub.2, and C.sub.1-C.sub.6 alkyl, where alkyl is optionally substituted with one or more F; or alkylene, alkenylene, aryl, and heteroaryl are independently and optionally substituted with a bond to L; D' is a drug moiety selected from: ##STR00085## where the wavy line indicates the site of attachment to T; X.sup.1 and X.sup.2 are independently selected from O and NR.sup.3, where R.sup.3 is selected from H and C.sub.1-C.sub.6 alkyl optionally substituted with one or more F; R.sup.4 is H, CO.sub.2R, or a bond to a linker (L), where R is C.sub.1-C.sub.6 alkyl or benzyl; and R.sup.5 is H or C.sub.1-C.sub.6 alkyl. 35. The antibody drug conjugate of claim 34 selected from: ##STR00086## ##STR00087## ##STR00088## 36. 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).sub.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. 37. The antibody drug conjugate of claim 22 wherein 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. 38. The antibody drug conjugate of claim 22 wherein L comprises a -vc linker, and D is selected from the group consisting of a CBI-PBD heterodimer, cryptophycin, a taxoid, and tubulysin M. 39. The antibody drug conjugate of claim 38 wherein the D is the CBI-PBD heterodimer: ##STR00089## 40. The antibody drug conjugate of claim 22 wherein L comprises a pyridyl disulfide (PDS). 41. A pharmaceutical composition comprising an antibody drug conjugate of claim 22.

Details for Patent 10,077,318

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	09/25/1998		2034-09-12
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	02/10/2017		2034-09-12
Genentech, Inc.	HERCEPTIN HYLECTA	trastuzumab and hyaluronidase-oysk	Injection	761106	02/28/2019		2034-09-12