Cysteine engineered antibodies and conjugates

• US 7,521,541 B2
• Filed: 09/22/2005
• Issued: 04/21/2009
• Est. Priority Date: 09/23/2004
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genetec Incorporated (Noritsu Koki Co., Ltd.)
• Inventors: Raab, Helga E., Eigenbrot, Charles W., Vandlen, Richard, Lowman, Henry, Junutula, Jagath Reddy
• Primary Examiner(s): Helms; Larry R.
• Assistant Examiner(s): Natarajan; Meera
• Application Number:
  US11/233,258
• Publication Number:
  US 20070092940A1
• Time in Patent Office: 1,307 Days
• Field of Search: None
• US Class Current: 530/387.1
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/6817 Toxins
• CPC Class Codes: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• CPC Class Codes: A61K 47/6889 Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• 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 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/624 Disulfide-stabilized antibody (dsFv)

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.

Claims

1. A cysteine engineered antibody comprising a free cysteine amino acid having a thiol reactivity value in the range of 0.6 to 1.0;

and a sequence in the heavy chain selected from SEQ ID NOS;

11, 12, 13, and 15;

2. The cysteine engineered antibody of claim 1 wherein the cysteine engineered antibody is more reactive with a thiol-reactive reagent than the parent antibody.

3. The cysteine engineered antibody of claim 1 wherein the process further comprises determining the thiol reactivity of the cysteine engineered antibody by reacting the cysteine engineered antibody with a thiol-reactive reagent;

wherein the cysteine engineered antibody is more reactive with the thiol-reactive reagent than the parent antibody.

4. The cysteine engineered antibody of claim 1 wherein the thiol reactivity value is in the range of 0.7 to 1.0.

5. The cysteine engineered antibody of claim 1 wherein the thiol reactivity value is in the range of 0.8 to 1.0.

6. The cysteine engineered antibody of claim 1 prepared by a process comprising:

(i) mutagenizing a nucleic acid sequence encoding the cysteine engineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating and purifying the cysteine engineered antibody.

7. The cysteine engineered antibody of claim 6 wherein mutagenizing comprises site-directed mutagenesis.

8. The cysteine engineered antibody of claim 6 wherein the cysteine engineered antibody is expressed on a viral particle selected from a phage or a phagemid particle.

9. The cysteine engineered antibody of claim 6 further comprising:

(i) reacting the cysteine engineered antibody with a thiol-reactive affinity reagent to generate an affinity labelled, cysteine engineered antibody; and

(ii) measuring the binding of the affinity labelled, cysteine engineered antibody to a capture media.

10. The cysteine engineered antibody of claim 9 wherein the thiol-reactive affinity reagent comprises a biotin moiety.

11. The cysteine engineered antibody of claim 10 wherein the thiol-reactive reagent comprises a maleimide moiety.

12. The cysteine engineered antibody of claim 9 wherein the capture media comprises streptavidin.

13. The cysteine engineered antibody of claim 1 wherein the parent antibody is a fusion protein comprising the albumin-binding peptide (ABP).

14. The cysteine engineered antibody of claim 13 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.

15. The cysteine engineered antibody of claim 1 wherein the parent antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody.

16. The cysteine engineered antibody of claim 15 wherein the parent antibody is huMAb4D5-8 (trastuzumab).

17. The cysteine engineered antibody of claim 15 wherein the parent antibody is an anti-EphB2R antibody.

18. The cysteine engineered antibody of claim 15 wherein the parent antibody is an anti-MUC16 antibody.

19. The cysteine engineered antibody of claim 1 wherein the parent antibody is an intact antibody selected from IgA, IgD, IgE, IgG, and IgM.

20. The cysteine engineered antibody of claim 19 wherein the IgG is selected from subclasses IgG1, IgG2, IgG3, and IgG4.

21. The cysteine engineered antibody of claim 1 wherein the parent antibody is an antibody fragment.

22. The cysteine engineered antibody of claim 21 wherein the antibody fragment is a Fab fragment.

23. The cysteine engineered antibody of claim 22 wherein the Fab fragment is hu4D5Fabv8.

24. The cysteine engineered antibody of claim 1 wherein the cysteine engineered antibody or the parent 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, 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 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 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);

(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);

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes);

(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);

(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, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis);

(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);

(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); and

(36) TENB2 (putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin).

25. The cysteine engineered antibody of claim 1 wherein the antibody is covalently attached to a capture label, a detection label, or a solid support.

26. The cysteine engineered antibody of claim 25 wherein the antibody is covalently attached to a biotin capture label.

27. The cysteine engineered antibody of claim 25 wherein the antibody is covalently attached to a fluorescent dye detection label.

28. The cysteine engineered antibody of claim 27 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.

29. The cysteine engineered antibody of claim 25 wherein the antibody is covalently attached to a radionuclide detection label selected from ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, and ²¹³Bi.

30. The cysteine engineered antibody of claim 25 wherein the antibody is covalently attached to a detection label by a chelating ligand.

31. The cysteine engineered antibody of claim 30 wherein the chelating ligand is selected from DOTA, DOTP, DOTMA, DTPA and TETA.

32. An antibody-drug conjugate compound comprising a cysteine engineered antibody (Ab) comprising a free cysteine amino acid having a thiol reactivity value in the range of 0.6 to 1.0;

and a sequence in the heavy chain selected from SEQ ID NOS;

11, 12, 13, and 15;

33. The antibody-drug conjugate compound of claim 32 wherein the cysteine engineered antibody is prepared by a process comprising:

(a) replacing one or more amino acid residues of a parent antibody by cysteine; and

(b) determining the thiol reactivity of the cysteine engineered antibody by reacting the cysteine engineered antibody with a thiol-reactive reagent;

wherein the cysteine engineered antibody is more reactive than the parent antibody with the thiol-reactive reagent.

34. The antibody-drug conjugate compound of claim 32 further comprising an albumin-binding peptide (ABP) sequence.

35. The antibody-drug conjugate compound of claim 34 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.

36. The antibody-drug conjugate compound of claim 32 wherein the cysteine engineered antibody binds to an ErbB receptor selected from EGFR, HER2, HER3, and HER4.

37. The antibody-drug conjugate compound of claim 32 wherein the cysteine engineered antibody or the parent 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, 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 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 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);

(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);

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes);

(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);

(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, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis);

(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);

(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); and

(36) TENB2 (putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin).

38. The antibody-drug conjugate compound of claim 32 wherein p is 1.

39. The antibody-drug conjugate compound of claim 32 wherein p is 2.

40. The antibody-drug conjugate compound of claim 32 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.

41. The antibody-drug conjugate compound of claim 40 having the formula:

42. The antibody-drug conjugate compound of claim 41 wherein W_w is valine-citrulline.

43. The antibody-drug conjugate compound of claim 41 wherein R¹⁷ is (CH₂)₅ or (CH₂)₂.

44. The antibody-drug conjugate compound of claim 40 having the formula:

45. The antibody-drug conjugate compound of claim 44 wherein R¹⁷ is (CH₂)₅ or (CH₂)₂.

46. The antibody-drug conjugate compound of claim 40 having the formula:

47. The antibody-drug conjugate compound of claim 32 wherein L is SMCC.

48. The antibody-drug conjugate compound of claim 32 wherein L is BMPEO.

49. The antibody-drug conjugate compound of claim 32 wherein D is MMAE, having the structure:

50. The antibody-drug conjugate compound of claim 32 wherein D is MMAF, having the structure:

51. The antibody-drug conjugate compound of claim 32 wherein D is DM1, having the structure:

52. The antibody-drug conjugate compound of claim 32 wherein the parent antibody is a fusion protein comprising the albumin-binding peptide (ABP).

53. The antibody-drug conjugate compound of claim 32 wherein the parent antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody.

54. The antibody-drug conjugate compound of claim 32 wherein the parent antibody is huMAb4D5-8 (trastuzumab).

55. The antibody-drug conjugate compound of claim 32 wherein the parent antibody is an anti-ErbB2 antibody.

56. The antibody-drug conjugate compound of claim 32 wherein the parent antibody is an anti-EphB2R antibody.

57. The antibody-drug conjugate compound of claim 32 wherein the parent antibody is an anti-CD22 antibody.

58. The antibody-drug conjugate compound of claim 32 wherein the parent antibody is an anti-MUC16 antibody.

59. The antibody-drug conjugate compound of claim 32 wherein the parent antibody is an intact antibody selected from IgA, IgD, IgE, IgG, and IgM.

60. The antibody-drug conjugate compound of claim 59 wherein the IgG is selected from subclasses:

IgG1, IgG2, IgG3, and IgG4.

61. The antibody-drug conjugate compound of claim 32 wherein the parent antibody is an antibody fragment.

62. The antibody-drug conjugate compound of claim 61 wherein the antibody fragment is a Fab fragment.

63. The antibody-drug conjugate compound of claim 62 wherein the Fab fragment is hu4D5Fabv8.

64. The antibody-drug conjugate compound of claim 32 having the structure:

65. A pharmaceutical composition comprising the antibody-drug conjugate compound of claim 32 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient.

66. The pharmaceutical composition of claim 65 further comprising a therapeutically effective amount of an additional chemotherapeutic agent.

67. An article of manufacture comprising

an antibody-drug conjugate compound of claim 32,

a container, and

a package insert or label indicating that the compound can be used to treat cancer.

68. The antibody-drug conjugate compound of claim 32 selected from the structures: