CYSTEINE ENGINEERED ANTI-MUC16 ANTIBODIES AND ANTIBODY DRUG CONJUGATES

• US 20080311134A1
• Filed: 05/07/2008
• Published: 12/18/2008
• Est. Priority Date: 05/08/2007
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Mallet, William, Junutula, Jagath R.
• Granted Patent: US 7,723,485 B2
• Time in Patent Office: Days
• US Class Current: 424/178.1
• 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 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 51/1027 against receptors, cell-surface antigens or cell-surface determinants
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 2317/624 Disulfide-stabilized antibody (dsFv)
• CPC Class Codes: C07K 2317/71 Decreased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/72 Increased effector function due to an Fc-modification
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin

Abstract

Cysteine engineered anti-MUC16 antibodies are engineered by replacing one or more amino acids of a parent anti-MUC16 antibody with non cross-linked, reactive cysteine amino acids. Methods of design, preparation, screening, and selection of the cysteine engineered anti-MUC16 antibodies are provided. Cysteine engineered anti-MUC16 antibodies (Ab) are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered anti-MUC16 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 anti-MUC16 antibody which binds to a MUC16 polypeptide and comprising one or more free cysteine amino acids and a sequence selected from SEQ ID NOS:

9-40.

2. The cysteine engineered anti-MUC16 antibody of claim 1 prepared by a process comprising replacing one or more amino acid residues of a parent anti-MUC16 antibody by cysteine.

3. The cysteine engineered anti-MUC16 antibody of claim 1 wherein the one or more free cysteine amino acid residues are located in a light chain.

4. The cysteine engineered anti-MUC16 antibody of claim 3 comprising one or more sequences selected from:

5. The cysteine engineered anti-MUC16 antibody of claim 3 comprising one or more sequences selected from:

6. The cysteine engineered anti-MUC16 antibody of claim 1 wherein the one or more free cysteine amino acid residues are located in a heavy chain.

7. The cysteine engineered anti-MUC16 antibody of claim 6 comprising one or more sequences selected from:

8. The cysteine engineered anti-MUC16 antibody of claim 6 comprising one or more sequences selected from:

9. The cysteine engineered hu anti-MUC16 antibody of claim 1 comprising a heavy chain sequence comprising:

10. The cysteine engineered hu anti-MUC16 antibody of claim 1 comprising a light chain sequence comprising:

11. The cysteine engineered chimeric anti-MUC16 antibody of claim 1 comprising a heavy chain sequence comprising:

12. The cysteine engineered chimeric anti-MUC16 antibody of claim 1 comprising a light chain sequence comprising:

13. The cysteine engineered anti-MUC16 antibody of claim 1 wherein the parent anti-MUC16 antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody.

14. The cysteine engineered anti-MUC16 antibody of claim 1 which is a Fab fragment.

15. The cysteine engineered anti-MUC16 antibody of claim 1 which is produced in bacteria.

16. The cysteine engineered anti-MUC16 antibody of claim 1 which is produced in CHO cells.

20. The cysteine engineered anti-MUC16 antibody of claim 1 wherein the antibody is covalently attached to an auristatin drug moiety whereby an antibody-drug conjugate is formed.

17. A method of determining the presence of a MUC16 protein in a sample suspected of containing said protein, said method comprising the steps of:

exposing said sample to a cysteine engineered anti-MUC16 antibody having one or more free cysteine amino acids and a sequence selected from SEQ ID NOS;

9-40, and;

determining binding of said antibody to said MUC16 protein in said sample, wherein binding of the antibody to said protein is indicative of the presence of said protein in said sample.

18. The method of claim 17 wherein said cell is an ovarian, breast, lung, or pancreatic cancer cell.

19. The method of claim 17 wherein the antibody is covalently attached to a label selected from a fluorescent dye, a radioisotope, biotin, or a metal-complexing ligand.

21. A pharmaceutical formulation comprising a cysteine engineered anti-MUC16 antibody which binds to a MUC16 polypeptide and comprising one or more free cysteine amino acids and a sequence selected from SEQ ID NOS:

9-40, and a pharmaceutically acceptable diluent, carrier or excipient.

22. An antibody-drug conjugate comprising a cysteine engineered anti-MUC16 antibody (Ab) which binds to a MUC16 polypeptide and comprising one or more free cysteine amino acids, a sequence selected from SEQ ID NOS:

9-40, and an auristatin drug moiety (D) wherein the cysteine engineered anti-MUC16 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.

23. The antibody-drug conjugate compound of claim 22 wherein p is 2.

24. The antibody-drug conjugate compound of claim 22 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.

25. The antibody-drug conjugate compound of claim 24 having the formula:

26. The antibody-drug conjugate compound of claim 24 wherein W_w is valine-citrulline.

27. The antibody-drug conjugate compound of claim 24 wherein R¹⁷ is (CH₂)₅ or (CH₂)₂.

28. The antibody-drug conjugate compound of claim 24 having the formula:

29. The antibody-drug conjugate compound of claim 28 wherein R¹⁷ is (CH₂)₅ or (CH₂)₂.

30. The antibody-drug conjugate compound of claim 24 having the formula:

31. The antibody drug conjugate of claim 22 wherein L is MC-val-cit-PAB or MC.

32. The antibody-drug conjugate compound of claim 22 wherein L is a linker formed by linker reagents SMCC, BM(PEO)₂ or BM(PEO)₃.

33. The antibody-drug conjugate compound of claim 22 wherein D is MMAE, having the structure:

34. The antibody-drug conjugate compound of claim 22 wherein D is MMAF, having the structure:

35. The antibody-drug conjugate compound of claim 22 wherein the cysteine engineered anti-MUC16 antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody.

36. The antibody-drug conjugate compound of claim 22 wherein the cysteine engineered anti-MUC16 antibody is a Fab fragment.

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

38. The antibody drug conjugate of claim 37 wherein Ab comprises SEQ ID NO:

1.

39. The antibody drug conjugate of claim 37 wherein Ab comprises SEQ ID NO:

2.

40. The antibody drug conjugate of claim 37 wherein Ab comprises SEQ ID NO:

3.

41. The antibody drug conjugate of claim 37 wherein Ab comprises SEQ ID NO:

4.

42. The antibody drug conjugate of claim 37 wherein Ab comprises SEQ ID NO:

1 and SEQ ID NO;

2.

43. The antibody drug conjugate of claim 42 wherein Ab comprises SEQ ID NO:

3 and SEQ ID NO;

4.

44. A mixture of antibody-drug conjugate compounds comprising a cysteine engineered anti-MUC16 antibody (Ab) which binds to a MUC16 polypeptide and comprising one or more free cysteine amino acids, a sequence selected from SEQ ID NOS:

9-40, and an auristatin drug moiety (D) wherein the cysteine engineered anti-MUC16 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 the average drug loading is from 1 to 2.

45. A pharmaceutical formulation comprising an antibody-drug conjugate comprising a cysteine engineered anti-MUC16 antibody which binds to a MUC16 polypeptide and comprising one or more free cysteine amino acids and a sequence selected from SEQ ID NOS:

9-40 and where the cysteine engineered anti-MUC16 antibody is covalently attached to an auristatin drug moiety, and a pharmaceutically acceptable diluent, carrier or excipient.

46. The pharmaceutical formulation of claim 45 further comprising a therapeutically effective amount of a chemotherapeutic agent selected from letrozole, oxaliplatin, docetaxel, 5-FU, lapatinib, capecitabine, leucovorin, erlotinib, pertuzumab, bevacizumab, and gemcitabine.

47. A method of treating cancer comprising administering to a patient a. pharmaceutical formulation comprising an antibody-drug conjugate comprising a cysteine engineered anti-MUC16 antibody which binds to a MUC16 polypeptide and having one or more free cysteine amino acids and a sequence selected from SEQ ID NOS:

9-40 and where the cysteine engineered anti-MUC16 antibody is covalently attached to an auristatin drug moiety, and a pharmaceutically acceptable diluent, carrier or excipient.

48. The method of claim 47 wherein the cancer is selected from the group consisting of ovarian cancer, prostate cancer, cancer of the urinary tract, pancreatic cancer, lung cancer, breast cancer, and colon cancer.

49. The method of claim 47 wherein the patient is administered a chemotherapeutic agent in combination with the antibody-drug conjugate compound, where the chemotherapeutic agent is selected from letrozole, cisplatin, carboplatin, taxol, paclitaxel, oxaliplatin, docetaxel, 5-FU, leucovorin, erlotinib, pertuzumab, bevacizumab, lapatinib, and gemcitabine.

50. An article of manufacture comprising

a pharmaceutical formulation comprising an antibody-drug conjugate comprising a cysteine engineered anti-MUC16 antibody which binds to a MUC16 polypeptide and having one or more free cysteine amino acids and a sequence selected from SEQ ID NOS:

9-40 and where the cysteine engineered anti-MUC16 antibody is covalently attached to an auristatin drug moiety, and a pharmaceutically acceptable diluent, carrier or excipient;

a container; and

a package insert or label indicating that the compound can be used to treat cancer characterized by the overexpression of a MUC16 polypeptide.

51. The article of manufacture of claim 50 wherein the cancer is ovarian cancer, prostate cancer, cancer of the urinary tract, pancreatic cancer, lung cancer, breast cancer, or colon cancer.

52. A method for making an antibody drug conjugate compound comprising a cysteine engineered anti-MUC16 antibody (Ab) which binds to a MUC16 polypeptide and having one or more free cysteine amino acids and a sequence selected from SEQ ID NOS:

9-40, and an auristatin 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 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 compound is formed;

or comprising the steps of;

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

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

whereby the antibody-drug conjugate compound is formed.

53. The method of claim 52 further comprising the step of expressing the cysteine engineered antibody in chinese hamster ovary (CHO) cells.

54. The method of claim 53 further comprising the step of treating the expressed cysteine engineered antibody with a reducing agent.

55. The method of claim 54 wherein the reducing agent is selected from TCEP and DTT.

56. The method of claim 55 further comprising the step of treating the expressed cysteine engineered antibody with an oxidizing agent, after treating with the reducing agent.

57. The method of claim 56 wherein the oxidizing agent is selected from copper sulfate, dehydroascorbic acid, and air.