CYSTEINE ENGINEERED ANTIBODIES AND CONJUGATES

• US 20160130358A1
• Filed: 09/11/2015
• Published: 05/12/2016
• Est. Priority Date: 09/12/2014
• Status: Active Grant

• Current Assignee: F Hoffmann-La Roche AG (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: BHAKTA, Sunil, ERICKSON, Hans, JUNUTULA, Jagath R., KOZAK, Katherine, OHRI, Rachana, PILLOW, Thomas
• Granted Patent: US 10,077,318 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/68031 the drug being an auristatin
• CPC Class Codes: A61K 47/68035 the drug being a pyrrolobenzodiazepine
• CPC Class Codes: A61K 47/6809 Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
• CPC Class Codes: A61K 47/6817 Toxins
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 47/6869 the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
• CPC Class Codes: A61K 51/10 Antibodies or immunoglobulins; Fragments thereof , the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/3069 Reproductive system, e.g. ovaria, uterus, testes, prostate
• CPC Class Codes: C07K 16/3092 against tumour-associated mucins
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: G01N 33/582 : with fluorescent label

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.

Claims

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 I106C, 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:

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.

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 13, wherein the antibody fragment is a Fab fragment.

15. 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.

16. The cysteine engineered antibody of claim 15, wherein the cysteine engineered antibody is an anti-HER2 antibody, and wherein the anti-HER2 antibody is trastuzumab.

18. The cysteine engineered antibody of claim 15, 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.

20. The cysteine engineered antibody of claim 15, 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.

22. The cysteine engineered antibody of claim 15, 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.

24. The cysteine engineered antibody of claim 15, 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.

26. The cysteine engineered antibody of claim 15, 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.

28. The cysteine engineered antibody of claim 15, 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.

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) 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).

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. The cysteine engineered antibody of claim 30, wherein the antibody is covalently attached to a biotin capture label.

32. The cysteine engineered antibody of claim 30, wherein the antibody is covalently attached to a fluorescent dye detection label.

33. The cysteine engineered antibody of claim 32, wherein the fluorescent dye is selected from a fluorescein type, a rhodamine type, dansyl, Lissamine, a cyanine, a phycoerythrin, Texas Red, and an analog thereof.

34. The cysteine engineered antibody of claim 30, wherein the antibody is covalently attached to a radionuclide detection label selected from ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, and ²¹³Bi.

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. An antibody-drug conjugate comprising the cysteine engineered antibody of claim 1, wherein the antibody-drug conjugate has Formula I:

Ab-(L-D)_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.

38. The antibody drug conjugate of claim 37, 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).

39. The antibody-drug conjugate of claim 37 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).

40. The antibody-drug conjugate of claim 37, prepared from a linker reagent comprising maleimide, iodoacetamide, bromoacetamide or disulfide.

41. The antibody-drug conjugate of claim 37, wherein L forms a disulfide linker.

42. The antibody-drug conjugate of claim 40, wherein the linker reagent comprises a pyridyl disulfide (PDS).

44. The antibody-drug conjugate of claim 37, 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.

45. The antibody-drug conjugate of claim 44 wherein D is a monomethylauristatin drug moiety MMAE having the structure:

46. The antibody-drug conjugate of claim 44 wherein the antibody-drug conjugate is selected from the structures:

47. The antibody drug conjugate of claim 44 wherein D is a PBD dimer drug having the structure:

48. The antibody drug conjugate of claim 47 wherein the structure of the PBD dimer is:

49. The antibody drug conjugate of claim 47 selected from:

50. The cysteine engineered antibody of claim 44 wherein D is a CBI dimer drug having the structure:

51. The antibody drug conjugate of claim 50 selected from:

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.

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. The antibody drug conjugate of claim 59 wherein the D is the CBI-PBD heterodimer:

61. The antibody drug conjugate of claim 37 wherein the cysteine engineered antibody has a light chain mutation selected from the group consisting of R142C and K149C according to EU numbering and L comprises a pyridyl disulfide (PDS).

62. The antibody drug conjugate of claim 37 wherein the cysteine engineered antibody has a heavy chain mutation selected from the group consisting of A140C, L174C, L179C, G371C, Y373C, and S424C according to EU numbering and L comprises a pyridyl disulfide (PDS).

63. The antibody drug conjugate of claim 37 wherein the cysteine engineered antibody has a light chain mutation selected from the group consisting of L106C and R108C according to EU numbering and L is a −

vc linker.

64. The antibody drug conjugate of claim 37 wherein the cysteine engineered antibody has a heavy chain mutation selected from the group consisting of T114C, T187C, T209C, V262C, G371C, E382C, and N434C according to EU numbering and L is a −

vc linker.

66. A pharmaceutical composition comprising an antibody drug conjugate of claim 37.

3-7. -7. (canceled)

9-12. -12. (canceled)

17. (canceled)

19. (canceled)

21. (canceled)

23. (canceled)

25. (canceled)

27. (canceled)

43. (canceled)

53-57. -57. (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.