National Cancer Institute: Opportunity for a Cooperative Research and Development Agreement (CRADA) for the Further Development and Commercialization of Methods Designed To Screen and Use Modulators of Nitric Oxide Synthase 2 (NOS2) Activity for the Diagnosis and Treatment of Cancer

A Cooperative Research and Development Agreement (CRADA) is the anticipated joint agreement to be entered into with NCI pursuant to the Federal Technology Transfer Act of 1986 and Executive Order 12591 of April 10, 1987 as amended by the National Technology Transfer Advancement Act of 1995. NCI is looking for a CRADA partner to collaborate with NCI in the further development and commercialization of screening assays and methods relating to the analysis of NOS2 activity in cancers exhibiting a nonfunctional p53 and to the use of NOS2 inhibitors in the treatment of such cancers. The expected duration of the CRADA would be from one (1) to five (5) years. Increased expression of inducible nitric oxide synthase (NOS2) has been found in a variety of human cancers (3-6). NOS2 is an inducible enzyme that produces nitric oxide (NO), a mutagenic and angiogenic molecule (1,2). NO is an activator of the p53 tumor suppressor gene function (7-9), however, p53 will repress the expression of NOS2, both in vitro and in vivo (8,10,11). To investigate the role of NO in tumor progression, NCI created genetically engineered human carcinoma cell lines that constitutively produce endogenous NO. Using these cell lines, NCI has found that the effect of NO on tumor growth is p53- dependent due to its ability to repress the expression of NOS2, and that endogenously produced NO accelerates tumor growth by inducing expression of the vascular endothelial growth factor (VEGF) and neovascularization. The NCI's data indicates that, in the presence of wild-type p53, constitutive expression of NOS2 in tumors could lead to a p53-mediated growth arrest in the epithelial cells closely surrounding the source of NO production. The growth inhibition of such cells would provide a strong selective pressure for a mutation to occur in the p53 gene. Indeed, tumors of the breast, brain, head and neck and colon that overexpress NOS2 have a high frequency of p53 mutations (3, 4, 6, 11, 12). Tumor growth would then be further supported by the NO-mediated induction of VEGF and angiogenesis. Since p53 has been shown to regulate the production of NO by altering the activity of NOS2, NO production would remain unchecked, supporting the growth of the tumor (8, 10, 11). The loss of p53 function in p53 deficient or mutant cells would permit both the growth of tumor cells in the presence of moderate NO concentrations and the release of angiogenic factors such as VEGF. However, NCI suggests that such tumors with mutant p53 function could be therapeutically and prophylactically treated with NOS2 inhibitors. To address this possibility, NCI has developed a series of methods aimed at screening modulators of NOS2 activity using p53 mutant cells that express NOS2. In one method, modulators of NOS2 activity are screened in vitro, using p53 mutant cells that constitutively or endogenously express NOS2. The cells are exposed to potential NOS2 inhibitors, and the level of VEGF expression is determined by various methods. In addition, the level of nitrate versus nitrite produced is measured to determine the level of NOS2 activity in the presence of the potential inhibitor compounds. To further assess the therapeutic benefit of a potential NOS2 inhibitor, NCI has developed an in vivo method of screening modulators of NOS2 activity. In such method, p53 mutant cells that constitutively or endogenously express NOS2 are implanted into an immune deficient athymic nude mouse model and then are treated with potential NOS2 inhibitor compounds. Modulation of NOS2 activity can be determined as above and also by measuring tumor growth in the treated animals as compared to untreated control animals. NCI suggests that the benefit of administering potential NOS2 inhibitors to cancer patients may be assessed by determining the p53 status and NOS2 expression pattern of the tumors. If a patient has a cancer that expresses NOS2 and is deficient in normal p53 activity, then the patient may be a candidate for treatment with NOS2 inhibitors. NCI has developed methods to both assess the benefit of such treatment and to administer potential NOS2 inhibitors to cancer patients in a clinical setting. NCI is seeking one or more CRADA Collaborators to further develop the above methods for preclinical, diagnostic and clinical uses. Specifically, NCI believes the methods could be applied to a drug screening protocol in which potential modulators of NOS2 could be identified and characterized. Furthermore, NCI predicts that the methods could be applied to a diagnostic kit for use in a

clinical setting to determine whether or not a particular cancer patient is a candidate for such treatment with regards to the p53 status of the tumor. Once identified and characterized, novel NOS2 inhibitors may be administered to candidate cancer patients and evaluated in their ability to treat various tumors. The described methods are the subject of U.S. provisional patent application, USSN 60/109,563, filed on November 23, 1998 by the Public Health Service on behalf of the Federal Government. Furthermore, the initial report and characterization of the invention is described in: Ambs et al, Nature Medicine (1998) vol. 4, no.12:1371-1376.

References

1. Nguyen et al (1992) PNAS 89:3030-3034. 2. Jenkins et al (1995) PNAS 92:4392-4396. 3. Thomsen et al (1995) Br. J. Cancer 72:41-44. 4. Ellie et al (1995) Neuroreport 7:294-296. 5. Ambs et al (1998) Cancer Res. 58:334-341. 6. Gallo et al (1998) J. Natl. Cancer Inst. 90:587-596. 7. Messmer et al (1996) Biochem J. 319:299-305. 8. Forrester, K. et al (1996) PNAS 93:2442-2447. 9. Calmels et al (1997) Cancer Res. 57:3365-3369. 10. Ambs et al (1997) Faseb J. 11:443-448. 11. Ambs et al (1998) PNAS 95:8823-8828. 12. Thomsen et al (1997) Cancer Res. 57:3300-3304.

Under the present proposal, the overall goal of the CRADA collaboration will involve the following: 1. Use of the genetically engineered cells lines and assays in preclinical screening assays of potential NOS2 inhibitors; and 2. Use of the cell lines and candidate NOS2 inhibitors in diagnostic, preclinical and clinical settings.

Party Contributiions:

The role of the NCI in the CRADA may include, but not be limited to: 1. Providing intellectual, scientific, and technical expertise and experience to the research project. 2. Providing the CRADA Collaborator with information and data relating to the methods developed to assess the activity of p53 and NOS2 and to screen for potential modulators of NOS2 activity. 3. Planning research studies and interpreting research results. 4. Carrying out research to validate the use of the NOS2-related methods and candidate NOS2 inhibitors in preclinical, diagnostic and clinical settings. 5. Publishing research results. 6. Developing additional potential applications of the methods.

The role of the CRADA Collaborator may include, but not be limited to: 1. Providing significant intellectual, scientific, and technical expertise or experience to the research project. 2. Planning research studies and interpreting research results. 3. Producing candidate NOS2 inhibitors under cGMP conditions in sufficient quantities to support the CRADA studies. 4. Carrying out research to validate the use of the NOS2-related methods and candidate NOS2 inhibitors in preclinical, diagnositc and clinical settings, including toxicologic and pharmacologic assays, as appropriate. 5. Providing technical and/or financial support to facilitate scientific goals and for futher design of applications of the technology outlined in the agreement. 6. Publishing research results.

Selection criteria for choosing the CRADA Collaborator may include, but not be limited to: 1. A demonstrated record of success in the screening of chemotherapeutic agents. 2. A demonstrated background and expertise in cancer research and treatment. 3. The ability to collaborate with NCI on further research and development of this technology. This ability will be demonstrated through experience and expertise in this or related areas of technology indicating the ability to contribute intellectually to ongoing research and development. 4. The demonstration of adequate resources to perform the research and development of this technology (e.g. facilities, personnel and expertise) and to accomplish objectives according to an appropriate timetable to be outlined in the CRADA Collaborator's proposal. 5. The willingness to commit best effort and demonstrated resources to the research and development of this technology, as outlined in the CRADA Collaborator's proposal. 6. The demonstration of expertise in the commercial development and production of products related to this area of technology. 7. The level of financial support the CRADA Collaborator will provide for CRADA-related Government activities. 8. The willingness to cooperate with the National Cancer Institute in the timely publication of research results. 9. The agreement to be bound by the appropriate DHHS regulations relating to human subjects and to all PHS policies relating to the use and care of laboratory animals. 10. The willingness to accept the legal provisions and language of the CRADA with only minor modifications, if any. These provisions govern the distribution of future patent rights to CRADA inventions. Generally, the rights of ownership are retained by the organization that is the employer of the inventor with (1) the grant of a license for research and other Government purposes to the Government when the CRADA Collaborator's employee is the sole inventor, or (2) the grant of an option to elect an exclusive or nonexclusive license to the CRADA Collaborator when the Government employee is the sole inventor.

Dated: October 8, 1999. Kathleen Sybert, Chief, Technology Development and Commercialization Branch, National Cancer Institute, National Institutes of Health. [FR Doc. 99-32138 Filed 12-10-99; 8:45 am] BILLING CODE 4140-01-P