Cell penetrating anti-guanosine antibody based therapy for cancers with Ras mutations

Patent metadata

Field	Value
Publication number	US-10040867-B2
Application number	US-201515123195-A
Country	US
Kind code	B2
Filing date	Mar 4, 2015
Priority date	Mar 4, 2014
Publication date	Aug 7, 2018
Grant date	Aug 7, 2018

Abstract

It has been established that cancer cells with oncogenic mutants in the small GTPase K-Ras are susceptible to antibodies that bind intracellular guanosine, but delivery of antibodies into cells can be challenging. A subset of lupus autoantibodies is associated with anti-guanosine activity, and is capable of cellular penetration. These antibodies have potential as therapeutic agents targeted towards K-Ras associated malignancies.

First claim

We claim:

1. A method of treating cancer comprising administering to a subject with a cancer comprising cells with one or more K-Ras gene mutations an effective amount of a cell-penetrating anti-guanosine antibody or an antigen binding fragment thereof or fusion protein comprising
a heavy chain variable region comprising sequentially the first, second, and third complementarity determining regions (CDRs) of SEQ ID NO:5, and
a light chain variable region comprising sequentially the first, second, and third CDRs of SEQ ID NO:1.

2. The method of claim 1 , wherein the antibody or antigen binding fragment thereof or fusion protein is in a pharmaceutical composition comprising a sterile pharmaceutically acceptable excipient for administration to a human.

3. The method of claim 2 , wherein the antibody or antigen binding fragment thereof or fusion protein is present in a dosage unit in an amount from about 0.01 to about 100 mg/kg body weight of a human.

4. The method of claim 1 , wherein the antibody or antigen binding fragment thereof or fusion protein inhibits or reduces phosphorylation of ERK and/or Akt in a cell as compared to an untreated control cell.

5. The method of claim 1 , wherein the antibody or antigen binding fragment thereof or fusion protein causes cytotoxicity to cells having one or more amino acid mutations in the small GTPase K-Ras.

6. The method of claim 1 , wherein the cell-penetrating anti-guanosine antibody is monoclonal antibody 4H2 or an antigen binding fragment thereof.

7. The method of claim 1 , further comprising administering the subject one or more antineoplastic or radiosensitizing agents selected from the group consisting of cisplatin, cytoxan, doxorubicin, methotrexate, mitomycin c, nitrogen mustard, hydroxyurea, bevacizumab, cetuximab, rituximab, trastuzumab tirapazamine, temozolomide, camptothecin, cisplatin, gemcitabine, 5-fluorouracil, hydroxyurea, pentoxifylline, vinorelbine, and combinations thereof.

8. The method of claim 2 , wherein the pharmaceutical composition is in a unit dosage form for intravenous injection or intra-tumoral injection.

9. The method of claim 1 , wherein the antibody or antigen binding fragment or fusion protein is a humanized form of monoclonal antibody 4H2 or an antigen binding fragment thereof.

10. The method of claim 6 , wherein the antigen binding fragment of monoclonal antibody 4H2 comprises a single chain variable fragment (scFv).

11. The method of claim 9 , wherein the antigen binding fragment binds to the same epitope as monoclonal antibody 4H2.

12. The method of claim 1 , wherein the antibody, or antigen binding fragment thereof or fusion protein is administered to the subject in an amount effective to inhibit or reduce one or more symptoms of the cancer.

13. The method of claim 1 , wherein the cancer is selected from the group consisting of pancreatic ductal adenocarcinoma, colorectal carcinoma, nonsmall cell lung carcinoma (NSCLCS), small cell lung cancer, malignant melanoma, urinary bladder carcinoma, thyroid carcinomas, hematopoietic malignancies, breast cancer, hepatocellular carcinomas, prostate cancer, biliary tract adenocarcinomas, angiosarcomas, malignant fibrous histiocytoma, neuroblastomas, cervix adenocarcinomas, stomach cancers and neck and head cancer, bowel cancer and pediatric cancers.

14. The method of claim 13 , wherein the cancer is resistant to radiotherapy.

15. The method of claim 13 , wherein the cancer is resistant to chemotherapy.

16. The method of claim 1 , wherein the cancer is characterized by an activating mutation in the small GTPase K-RAS.

17. The method of claim 16 , wherein the subject has been diagnosed with one or more mutations in the small GTPase K-Ras gene causing changes in amino acids at position 12, 13, 61, or a combination thereof in the K-Ras protein.

18. The method of claim 1 , further comprising treating the subject with radiation therapy or chemotherapy, wherein the pharmaceutical composition increases the cell's sensitivity to radiation therapy or chemotherapy.

19. The method of claim 18 , wherein the antibody, or an antigen binding fragment thereof or fusion protein is administered to the subject at least 24 hours before the radiation therapy or chemotherapy, is administered to the subject concurrently with radiation therapy or chemotherapy, or is administered to the subject within 24 hours after radiation therapy or chemotherapy.

20. The method of claim 1 , wherein the cell-penetrating anti-guanosine antibody is transported into the cytoplasm of the cell without the aid of a carrier or conjugate.

21. The method of claim 1 , wherein the
light chain variable region comprises SEQ ID NO:1 or a humanized form thereof; and
the heavy chain variable region comprises SEQ ID NO:5 or a humanized form thereof.

22. The method of claim 21 , wherein the light chain variable region comprises SEQ ID NOS:2, 3, and 4 and the heavy chain variable region comprises SEQ ID NOS:6, 7, and 8.

23. The method of claim 22 , wherein the antibody or antigen binding fragment thereof is a humanized antibody or antigen binding fragment thereof comprising SEQ ID NOS:2, 3, and 4 and SEQ ID NOS:6, 7, and 8.

24. The method of claim 22 , wherein the antibody or antigen binding fragment thereof is a single chain variable fragment (scFv).

25. The method of claim 1 , wherein the antibody or antigen binding fragment thereof or fusion protein is administered to the subject in an amount effective to reduce or inhibit the growth or proliferation of cells having one or more amino acid mutations in the small GTPase K-Ras.

26. The method of claim 1 , wherein the light chain variable region comprises SEQ ID NO:1 and the heavy chain variable region comprises SEQ ID NO:5.

27. The method of claim 1 , wherein the antibody or antigen binding fragment thereof or fusion protein is a humanized antibody or an antigen binding fragment thereof.

28. The method of claim 1 , wherein the antibody or antigen binding fragment thereof, or fusion protein is a chimeric antibody.

29. The method of claim 28 , wherein
the first light chain CDR comprises the amino acid sequence of SEQ ID NO:2;
the second light chain CDR comprises the amino acid sequence of SEQ ID NO:3;
the third light chain CDR comprises the amino acid sequence of SEQ ID NO:4;
the first heavy chain CDR comprises the amino acid sequence of SEQ ID NO:6;
the second heavy chain CDR comprises the amino acid sequence of SEQ ID NO:7; and
the third heavy chain CDR comprises the amino acid sequence of SEQ ID NO:8.

30. A method of treating cancer, comprising administering to a subject with a cancer comprising cells with one or more K-Ras gene mutations an effective amount of a cell-penetrating anti-guanosine antibody or an antigen binding fragment thereof or fusion protein comprising
a heavy chain variable region comprising sequentially the first, second, and third complementarity determining regions (CDRs) and an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:5, and
a light chain variable region comprising sequentially the first, second, and third CDRs and an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:1.

31. The method of claim 30 , wherein the antibody or antigen binding fragment thereof or fusion protein is a humanize

Assignees

• Univ Yale
• Us Veterans Affairs
• Us Veterans Affairs

Inventors

• Hansen James E
• Weisbart Richard H
• Young Melissa
• Noble Philip W

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C07K2317/77

  Internalization into the cell · CPC title

• C07K2317/73

  Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation · CPC title

• A61K39/39558

  against tumor tissues, cells, antigens · CPC title

• C07K2317/82

  functional in the cytoplasm, the inner aspect of the cell membrane, the nucleus or the mitochondria · CPC title