Cysteine engineered antibodies and conjugates

• US 20070092940A1
• Filed: 09/22/2005
• Published: 04/26/2007
• Est. Priority Date: 09/23/2004
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Vandlen, Richard, Lowman, Henry, Raab, Helga, Eigenbrot, Charles, Junutula, Jagath
• Granted Patent: US 7,521,541 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• 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 47/68031 the drug being an auristatin
• CPC Class Codes: A61K 47/68033 the drug being a maytansine
• 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 one or more free cysteine amino acids having a thiol reactivity value in the range of 0.6 to 1.0,

wherein the cysteine engineered antibody is prepared by a process comprising replacing one or more amino acid residues of a parent antibody by cysteine.

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. 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 than the parent antibody with the thiol-reactive reagent.

4. The cysteine engineered antibody of claim 1 wherein the one or more free cysteine amino acid residues are located in a light chain.

5. The cysteine engineered antibody of claim 4 wherein the one or more free cysteine amino acid residues are located in the light chain in the ranges selected from:

L-10 to L-20;

L-38 to L-48;

L-105 to L-115;

L-139 to L-149; and

L-163 to L-173.

6. The cysteine engineered antibody of claim 1 comprising one or more sequences selected from:

7. The cysteine engineered antibody of claim 1 comprising one or more sequences selected from:

8. The cysteine engineered antibody of claim 1 comprising one or more sequences selected from:

9. The cysteine engineered antibody of claim 1 wherein the one or more free cysteine amino acid residues are located in a heavy chain.

10. The cysteine engineered antibody of claim 9 wherein the one or more free cysteine amino acid residues are located in the heavy chain in the ranges selected from:

H-35 to H-45;

H-83 to H-93;

H-114 to H-127; and

H-170 to H-184.

11. The cysteine engineered antibody of claim 1 comprising one or more sequences selected from:

12. The cysteine engineered antibody of claim 9 wherein the one or more free cysteine amino acid residues are located in the Fc region of the heavy chain in the ranges selected from H-268 to H-291;

H-319 to H-344;

H-370 to H-380; and

H-395 to H-405.

13. The cysteine engineered antibody of claim 1 comprising one or more sequences selected from:

14. The cysteine engineered antibody of claim 1 wherein the one or more free cysteine amino acid residues are selected from positions in the heavy chain or light chain of the variable region.

15. The cysteine engineered antibody of claim 1 wherein the one or more free cysteine amino acid residues are selected from positions in the constant region.

16. The antibody of claim 1 wherein the thiol reactivity value is in the range of 0.7 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. 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.

19. The cysteine engineered antibody of claim 18 wherein mutagenizing comprises site-directed mutagenesis.

20. The cysteine engineered antibody of claim 18 wherein the cysteine engineered antibody is expressed on a viral particle selected from a phage or a phagemid particle.

21. The cysteine engineered antibody of claim 18 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.

22. The cysteine engineered antibody of claim 21 wherein the thiol-reactive affinity reagent comprises a biotin moiety.

23. The cysteine engineered antibody of claim 22 wherein the thiol-reactive reagent comprises a maleimide moiety.

24. The cysteine engineered antibody of claim 21 wherein the capture media comprises streptavidin.

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

26. The cysteine engineered antibody of claim 25 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.

27. 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.

28. The cysteine engineered antibody of claim 27 wherein the parent antibody is huMAb4D5-8 (trastuzumab).

29. The cysteine engineered antibody of claim 27 wherein the parent antibody is an anti-EphB2R antibody.

30. The cysteine engineered antibody of claim 27 wherein the parent antibody is an anti-MUC16 antibody.

31. The cysteine engineered antibody of claim 1 comprising an amino acid sequence selected from SEQ ID NO:

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

28, and SEQ ID NO;

39.

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

33. The cysteine engineered antibody of claim 32 wherein the IgG is selected from subclasses IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

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

35. The cysteine engineered antibody of claim 34 wherein the antibody fragment is a Fab fragment.

36. The cysteine engineered antibody of claim 35 wherein the Fab fragment is hu4D5Fabv8.

37. The cysteine engineered antibody of claim 36 wherein the one or more of the amino acid residues of hu4D5Fabv8 replaced by cysteine are selected from L-V15, L-A43, L-V 110, L-A144, L-S168, H-A40, H-A88, H-S119, H-S120, H-A121, H-S 122, H-A175, and H-S179.

38. 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, 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 (CA 125, 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 I and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878);

(8) PSCA hlg (2700050C12R1k, C530008016R1k, 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-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. The cysteine engineered antibody of claim 1 wherein the antibody is covalently attached to a drug moiety.

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

41. The cysteine engineered antibody of claim 40 wherein the antibody is covalently attached to a biotin capture label.

42. The cysteine engineered antibody of claim 40 wherein the antibody is covalently attached to a fluorescent dye detection label.

43. The cysteine engineered antibody of claim 42 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.

44. The cysteine engineered antibody of claim 40 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, ¹³³ Xe ¹⁷⁷Lu ²¹¹At and ²¹³Bi.

45. The cysteine engineered antibody of claim 40 wherein the antibody is covalently attached to a detection label by a chelating ligand.

46. The cysteine engineered antibody of claim 45 wherein the chelating ligand is selected from DOTA, DOTP, DOTMA, DTPA and TETA.

47. A method of screening cysteine engineered antibodies for thiol reactivity comprising:

(a) introducing one or more cysteine amino acids into a parent antibody in order to generate a cysteine engineered antibody;

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

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

48. The method of screening of claim 47 further comprising determining the thiol reactivity of the cysteine engineered antibody with the thiol-reactive reagent.

49. The method of screening of claim 47 wherein step (a) of the process comprises:

(i) site-directed mutagenesis of a gene encoding the cysteine engineered antibody in a double-stranded (ds) plasmid; and

(ii) expressing the cysteine engineered antibody.

50. The method of screening of claim 49 further comprising isolating and purifying the expressed cysteine engineered antibody.

51. The method of screening of claim 47 wherein the thiol-reactive affinity reagent comprises a biotin moiety.

52. The method of screening of claim 47 wherein the thiol-reactive reagent comprises a maleimide moiety.

53. The method of screening of claim 47 wherein the capture media comprises streptavidin.

54. The method of screening of claim 47 wherein the parent antibody is a fusion protein comprising an albumin-binding peptide (ABP).

55. The method of screening of claim 54 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.

56. The method of screening of claim 47 wherein the parent antibody is an antibody fragment Fab.

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 prepared by a process comprising replacing one or more amino acid residues of a parent antibody by one or more free cysteine amino acids.

58. The antibody-drug conjugate compound of claim 57 wherein the cysteine engineered antibody is more reactive than the parent antibody with a thiol-reactive reagent.

59. The antibody-drug conjugate compound of claim 57 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.

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 or the parent 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 (2700050C12R1k, C530008016R1k, 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.

64. The antibody-drug conjugate compound of claim 57 wherein p is 1.

65. The antibody-drug conjugate compound of claim 57 wherein p is 2.

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:

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 R¹⁷ is (CH₂)₅ or (CH₂)₂.

70. The antibody-drug conjugate compound of claim 66 having the formula:

71. The antibody-drug conjugate compound of claim 70 wherein R¹⁷ is (CH₂)₅ or (CH₂)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.

74. The antibody-drug conjugate compound of claim 57 wherein L is BMPEO.

75. The antibody-drug conjugate compound of claim 57 wherein the drug moiety D is selected from a microtubulin inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, and a DNA intercalator.

76. The antibody-drug conjugate compound of claim 57 wherein the drug moiety D is selected from a maytansinoid, an auristatin, a dolastatin, and a calicheamicin.

77. The antibody-drug conjugate compound of claim 57 wherein D is MMAE, having the structure:

78. The antibody-drug conjugate compound of claim 57 wherein D is MMAF, having the structure:

79. The antibody-drug conjugate compound of claim 57 wherein D is DM1, having the structure:

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 parent 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 parent antibody is huMAb4D5-8 (trastuzumab).

83. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an anti-ErbB2 antibody.

84. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an anti-EphB2R antibody.

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

86. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an anti-MUC16 antibody.

87. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an intact antibody selected from IgA, IgD, IgE, IgG, and IgM.

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 parent antibody is an antibody fragment.

90. The antibody-drug conjugate compound of claim 89 wherein the antibody fragment is a Fab fragment.

91. The antibody-drug conjugate compound of claim 90 wherein the Fab fragment is hu4D5Fabv8.

92. The antibody-drug conjugate compound of claim 91 wherein the one or more of the amino acid residues of hu4D5Fabv8 replaced by cysteine are selected from L-V15, L-A43, L-V110, L-A144, L-S168, H-A40, H-A88, H-S119, H-A121, H-S122, H-A175, and H-S179.

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. A method for killing or inhibiting the proliferation of tumor cells or cancer cells comprising treating tumor cells or cancer cells with an amount of the antibody-drug conjugate compound of claim 57, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.

98. A method of treating cancer comprising administering to a patient a formulation of an antibody-drug conjugate compound of claim 57 and a pharmaceutically acceptable diluent, vehicle, carrier or excipient.

99. The method of claim 98 further comprising administering an effective amount of an additional chemotherapeutic agent.

100. The method of claim 98 wherein the antibody-drug conjugate compound binds to a receptor encoded by an ErbB gene.

101. The method of claim 98 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.

102. The method of claim 98 wherein said antibody-drug conjugate compound is administered by infusion.

103. The method of claim 98 wherein said antibody-drug conjugate compound is formulated in a unit dosage injectable form.

104. The method of claim 98 further comprising the administration of a second antibody which binds ErbB2 and blocks antibody activation of an ErbB receptor.

105. The method of claim 98 further comprising the administration of a cytotoxic agent or chemotherapeutic agent.

106. A method of inhibiting cellular proliferation comprising:

(a) exposing mammalian cells in a cell culture medium to the antibody-drug conjugate compound of claim 57, and

(b) measuring a cytotoxic or cytostatic activity of the antibody-drug conjugate compound, whereby proliferation of the cells is inhibited

107. A method of inhibiting the growth of tumor cells that overexpress a growth factor receptor selected from a HER2 receptor and an EGF receptor comprising administering to a patient an antibody-drug conjugate compound of claim 57 which binds specifically to said growth factor receptor, 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.

108. The method of claim 107 wherein said antibody-drug conjugate compound sensitizes the tumor cells to said chemotherapeutic agent.

109. A method for the treatment of a human patient susceptible to, or diagnosed with, a disorder characterized by overexpression of ErbB2 receptor, comprising administering an effective amount of a combination of an antibody-drug conjugate compound of claim 57 and a chemotherapeutic agent.

110. A method for the treatment of cancer in a mammal, wherein the cancer is characterized by the overexpression of an ErbB receptor and does not respond, or responds poorly, to treatment with an unconjugated anti-ErbB antibody, comprising administering to the mammal a therapeutically effective amount of an antibody-drug conjugate compound of claim 57.

111. The method of claim 110 wherein the mammal is human.

112. The method of claim 110 wherein the ErbB receptor is selected from the group consisting of ErbB1 (EGFR), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4).

113. The method of claim 110 wherein the antibody of the antibody-drug conjugate compound is an anti-ErbB2 antibody.

114. The method of claim 110 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.

115. The method of claim 114 wherein the cancer is breast cancer and the breast cancer overexpresses ErbB2 at a 2+ level or more.

116. A method of treating an autoimmune disease in a patient comprising administration of a formulation of an antibody-drug conjugate compound of claim 57 and a pharmaceutically acceptable diluent, vehicle, carrier or excipient.

117. An article of manufacture comprising

an antibody-drug conjugate compound of claim 57;

a container, and

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

93. An antibody-drug conjugate compound selected from the structures:

94. An antibody-drug conjugate compound having the structure:

118. A method for making an antibody drug conjugate compound comprising a cysteine engineered antibody (Ab), and a drug moiety (D) wherein the cysteine engineered antibody is attached through the one or more engineered 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;

the method comprising replacing one or more amino acid residues of a parent antibody by cysteine to prepare the cysteine engineered antibody.

119. The method of claim 118 further comprising the steps of:

(a) reacting an engineered cysteine group of the cysteine engineered antibody with a linker reagent to form antibody-linker intermediate Ab-L; and

(b) reacting Ab-L with an activated drug moiety D;

whereby the antibody-drug conjugate is formed.

120. The method of claim 118 further comprising the steps of:

(a) reacting a nucleophilic group of a drug moiety with a linker reagent to form drug-linker intermediate D-L; and

(b) reacting D-L with an engineered cysteine group of the cysteine engineered antibody;

whereby the antibody-drug conjugate is formed.

121. The method of claim 118 further comprising the step of expressing the cysteine engineered antibody in chinese hamster ovary (CHO) cells.

122. The method of claim 121 wherein the expressed cysteine engineered antibody is an IgG antibody.

123. The method of claim 121 further comprising the step of treating the expressed cysteine engineered antibody with a reducing agent.

124. The method of claim 123 wherein the reducing agent is selected from TCEP and DTT.

125. The method of claim 123 further comprising the step of treating the expressed cysteine engineered antibody with an oxidizing agent, after treating with the reducing agent.

126. The method of claim 125 wherein the oxidizing agent is copper sulfate.