Cell penetrating anti-guanosine antibody based therapy for cancers with Ras mutations
Inventors
Hansen, James E. • Weisbart, Richard H. • Young, Melissa • Noble, Philip W.
Assignees
United States Government As Represented By Dpartment Of Veterans Affairs • Yale University • US Department of Veterans Affairs
Publication Number
US-10040867-B2
Publication Date
2018-08-07
Expiration Date
2035-03-04
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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.
Core Innovation
The invention relates to cell-penetrating autoantibodies capable of binding to guanosine, termed anti-guanosine antibodies. These antibodies are more toxic to cells harboring mutant small GTPase K-Ras genes than cells with wild-type K-Ras. The exemplary monoclonal antibody 4H2, a lupus autoantibody, penetrates cells and selectively targets cancer cells with activating K-Ras mutations by inhibiting phosphorylation of intracellular signaling molecules ERK and Akt, leading to cytotoxicity in mutant K-Ras cells.
The problem addressed is the difficulty in delivering therapeutic agents intracellularly to target oncogenic K-Ras mutations, which are present in approximately 30% of human malignancies. Existing therapeutic antibodies cannot penetrate cancer cells and are limited to attacking extracellular targets. Small-molecule inhibitors lack specificity and can cause significant toxicity, and K-Ras has been historically considered undruggable due to its intracellular location. There is a need for agents that selectively target malignant cells with K-Ras mutations while minimizing toxicity to normal cells.
The invention provides pharmaceutical compositions containing cell-penetrating anti-guanosine antibodies, such as monoclonal antibody 4H2 or its variants including single chain variable fragments (scFv), capable of entering cells without carriers. These antibodies inhibit or reduce ERK and/or Akt phosphorylation and cause selective cytotoxicity to cancer cells with K-Ras mutations. The compositions can be administered alone or in combination with additional therapeutic agents including chemotherapeutics and radiosensitizers, and can be formulated for intravenous, intratumoral, or oral administration. The methods disclosed include treating or preventing cancers characterized by activating mutations in K-Ras, with improved specificity and reduced side effects compared to existing therapies.
Claims Coverage
The patent includes one independent claim detailing a method of treating cancer using cell-penetrating anti-guanosine antibodies or antigen binding fragments or fusion proteins. Several inventive features are recited describing the antibody characteristics, therapeutic compositions, and combination therapies.
Use of cell-penetrating anti-guanosine antibodies targeting K-Ras mutations
Administering to a subject with cancer containing cells bearing one or more K-Ras gene mutations an effective amount of a cell-penetrating anti-guanosine antibody, antigen binding fragment, or fusion protein comprising heavy and light chain variable regions with defined complementarity determining regions (CDRs) as set forth in SEQ ID NO:5 and SEQ ID NO:1, respectively.
Pharmaceutical compositions and dosage forms for human administration
The antibodies or fragments are provided in sterile pharmaceutically acceptable compositions suitable for human administration with dosage units ranging approximately from 0.01 to 100 mg/kg body weight, including unit dosage forms for intravenous or intra-tumoral injection.
Inhibition of phosphorylation of ERK and Akt as mechanism of action
The antibody or fragment inhibits or reduces phosphorylation levels of ERK and/or Akt in treated cells compared to untreated controls, contributing to selective cytotoxicity against mutant K-Ras cells.
Selective cytotoxicity to mutant K-Ras cells
The antibody or fragment causes cytotoxicity preferentially in cells harboring amino acid mutations in small GTPase K-Ras, sparing cells with wild-type K-Ras.
Use of monoclonal antibody 4H2 and its variants
The cell-penetrating anti-guanosine antibody used can be monoclonal antibody 4H2 or humanized forms, antigen binding fragments including single chain variable fragments (scFv), which bind the same epitope as 4H2.
Combination with antineoplastic or radiosensitizing agents
The method can further comprise administering one or more additional agents selected from cisplatin, cytoxan, doxorubicin, methotrexate, mitomycin c, nitrogen mustard, hydroxyurea, bevacizumab, cetuximab, rituximab, trastuzumab, tirapazamine, temozolomide, camptothecin, gemcitabine, 5-fluorouracil, pentoxifylline, vinorelbine, or combinations thereof.
Treatment of multiple cancer types associated with K-Ras mutations
The method targets cancers including pancreatic ductal adenocarcinoma, colorectal carcinoma, non-small cell lung carcinoma, small cell lung cancer, malignant melanoma, urinary bladder carcinoma, thyroid carcinomas, hematopoietic malignancies, breast cancer, hepatocellular carcinoma, prostate cancer, biliary tract adenocarcinomas, angiosarcomas, malignant fibrous histiocytoma, neuroblastomas, cervix adenocarcinomas, stomach and head and neck cancers, bowel cancer, and pediatric cancers.
Enhancement of sensitivity to radiotherapy and chemotherapy
Administering the antibody or fragment increases cancer cell sensitivity to radiation or chemotherapy by at least 10%, with administration possible at least 24 hours before, concurrently, or within 24 hours after radiation or chemotherapy.
Antibody transport without carrier or conjugate
The cell-penetrating anti-guanosine antibody can enter the cytoplasm of cells without the aid of a carrier or conjugate, enabling direct intracellular targeting.
Use of variants and humanized or chimeric forms
The antibody or fragment can be a humanized or chimeric antibody that retains binding specificity to the 4H2 epitope as defined by specified CDR sequences, with at least 85% amino acid sequence identity to SEQ ID NO:1 and SEQ ID NO:5.
The claims cover methods of treating cancers bearing K-Ras mutations by administering cell-penetrating anti-guanosine antibodies, specifically monoclonal antibody 4H2 or its variants, in pharmaceutical compositions. The inventive features include selective targeting of mutant K-Ras cells via inhibition of ERK and Akt phosphorylation, formulations for human use, combination with other cancer therapies, and improved intracellular delivery independent of carriers.
Stated Advantages
Selective cytotoxicity to cancer cells harboring K-Ras mutations with minimal toxicity to wild-type K-Ras cells.
Ability to penetrate living cells and target intracellular oncogenic K-Ras, overcoming limitations of existing therapeutic antibodies that act only extracellularly.
Reduction of phosphorylation of key intracellular signaling molecules (ERK and Akt), thereby inhibiting cancer cell proliferation and survival pathways.
Potential to increase sensitivity of cancer cells to radiotherapy and chemotherapy, enhancing the efficacy of combination treatments.
Documented Applications
Treatment of cancers characterized by cells harboring activating mutations in the small GTPase K-Ras gene, including pancreatic ductal adenocarcinoma, colorectal carcinoma, lung cancers, malignant melanoma, bladder carcinoma, thyroid carcinomas, hematopoietic malignancies, breast cancer, hepatocellular carcinoma, prostate cancer, biliary tract adenocarcinomas, angiosarcomas, malignant fibrous histiocytoma, neuroblastomas, cervical adenocarcinomas, stomach cancers, head and neck cancers, bowel cancer, and pediatric cancers.
Use as a radiosensitizing agent to increase cancer cell sensitivity to radiation therapy.
Use to enhance chemosensitivity of cancer cells to chemotherapeutic agents.
Potential treatment for cancers resistant to radiotherapy or chemotherapy.
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