[Federal Register Volume 64, Number 152 (Monday, August 9, 1999)]
[Notices]
[Pages 43200-43201]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 99-20455]


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DEPARTMENT OF HEALTH AND HUMAN SERVICES

National Institutes of Health


Government-Owned Invention; Availability for Licensing: ``Novel 
Method and Composition to Induce Apoptosis in Tumor Cells''

AGENCY: National Institutes of Health, Public Health Service, DHHS.

ACTION: Notice

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SUMMARY: The invention listed below is owned by an agency of the U.S. 
Government and is available for licensing in the U.S. in accordance 
with 35 U.S.C. 207 to achieve expeditious commercialization of results 
of federally funded research and development.

ADDRESSES: Licensing information and a copy of the U.S. patent 
application referenced below may be obtained by contacting J. R. Dixon, 
Ph.D., at the Office of Technology Transfer, National Institutes of 
Health, 6011 Executive Boulevard, Suite 325, Rockville, Maryland 20852-
3804 (telephone 301/496-7056 ext 206; fax 301/402-0220; E-Mail: 
[email protected]). A signed Confidential Disclosure Agreement is required 
to receive a copy of any patent application.

SUPPLEMENTARY INFORMATION: Invention Title: ``Apoptosis Inducing Agents 
and Methods'' Inventors: Drs. Lucio Miele (U.S.F.D.A.) and Leslie L. 
Shelly (NICHD) USPA SN: 60/102,816 [=DHHS Ref. No. E-176-98/0]--Filed 
with the U.S.P.T.O. October 2, 1998.
    Apoptosis or programmed cell death is caused by many anti-tumor 
drugs and by radiation therapy. These treatment modalities cause 
apoptosis in tumor cells and in many normal cells in the body. As 
cancer cells progress towards more aggressive forms, they often become 
highly resistant to drug- or radiation-induced apoptosis, generally 
through the loss of function p53, a gene which can trigger apoptosis in 
response to DNA damage. Thus, novel strategies to induce apoptosis in 
tumor cells, especially p53-deficient cells, is an attractive and an 
active area of research.
    An antisense molecule is a DNA or RNA which has the opposite 
beginning to end orientation compared to the ``normal'' gene. These 
molecules reduce the expression of the target gene by forming pairs 
with its ``normal'' DNA and RNA. Notch-1 is a gene which is known to be 
important in controlling cell differentiation in many organisms. Notch-
1 is expressed at high levels in several human tumors. However, its 
function in tumor cells has not been characterized. So far, its role in 
maintaining tumor cell survival has not been identified. Using a model 
constituted by a p53-deficient mouse leukemia cell line, NIH scientists 
found that: 1.) Antisense synthetic DNA oligonucleotides and stable 
incorporation of an antisense gene (a model for gene therapy) targeting 
notch-1, when given together with a differentiation-inducing antitumor 
drug, cause the cells to respond by massive apoptosis rather than 
differentiation; 2.) stable incorporation of an antisense notch-1 gene 
increases apoptosis in these cells even in the absence of any antitumor 
drugs. This suggests that antisense notch-1 treatment, by antisense 
oligonucleotides or by gene therapy, may be used alone or together with 
anti-cancer drugs to cause apoptosis in tumor cells.

[[Page 43201]]

    The notch gene belongs to a family of epidermal growth factor 
(``EGF'') like homeotic genes, which encode transmembrane proteins with 
a variable number of cystgeine-rich EGF-like repeats in the 
extracellular region. Four notch genes have been described in mammals, 
which include notch-1, notch-2, notch-3, and notch-4 (Int-3), which 
have been implicated in the differentiation of the nervous system and 
other structures. The EGF-like proteins Delta and Serrate have been 
identified as ligands of notch-1.
    Mature notch proteins are heterodimeric receptors derived from the 
cleavage of notch pre-proteins into an extracellular subunit 
(NEC) containing multiple EGF-Like repeats and a 
transmembrane subunit including intracellular region (Ntm). 
Notch activation results from the binding of ligands expressed by 
neighboring cells, and signaling from activated notch involves a 
network of transcription regulators.
    Alteration of notch-1 signaling or expression may contribute to 
tumorigenesis. Deletions of the extracellular portion of human notch-1 
are associated with about 10% of the cases of T-Cell acute 
lymphoblastic leukemia. Truncated forms of notch-1 cause T-Cell 
lymphomas when introduced into mouse bone marrow stem cells and are 
onogenic in rat kidney cells. The human notch-1 gene is in a 
chromosomal region (9q34) associated with hematopoietic malignancies of 
lymphoid, myeloid, and erythroid lineage. Additionally, strikingly 
increased expression of notch-1 has been documented in a number of 
human tumors including cervical cancer, colon tumors, lung tumors, and 
pre-neoplastic lesions of the uterine cervix.
    Notch antisense oligonucleotides (or other molecules that interfere 
with the expression or function of notch) could be therapeutically 
administered to treat or prevent tumors. It has not been found that 
administration of notch antisense oligonucleotide alone is ineffective 
as an anti-neoplastic treatment. The present invention has overcome 
this problem by combining the administration of a cell differentiation 
agent with a molecule that interferes with the expression or function 
of a notch protein (such as the notch-1 protein). This combination of 
approaches has unexpectedly been found to induce apoptosis in 
neoplastic cells, and provide a useful therapeutic application of this 
technology. The method of the present invention includes inducing 
apoptosis in a target cell by inhibiting a cell fate determining 
function of a notch protein in the target cell at a time when the cell 
is undergoing differentiation. In particular, the target cell is 
induced to differentiate and upregulate notch expression, so that 
interference with notch expression or function causes the target cell 
to commit to an apoptotic pathway. Inhibition of notch expression or 
interference with its function can include exposing the cell to a notch 
protein antisense oligonucleotide that includes at least six 
nucleotides that comprise a sequence complementary to at least a 
portion of the RNA transcript of a notch gene (such as the notch-1 
gene), and is hybridizable to the RNA transcript. Although the 
antisense oligonucleotide can be hybridizable to any region of the RNA 
transcript, particular oligonucleotides that have been found to be 
useful are antisense oligonucleotides to the notch-1 EGF repeat region, 
Lin/notch region, or ankyrin region. Alternatively the molecule can be 
a monoclonal antibody that antagonizes the function of a notch protein 
in the cell.
    In particular the tumor cell is one that is characterized by 
increased activity or increased expression of a notch protein, such as 
a notch-1 or notch-2 protein. Examples of tumor types that over express 
notch-1 include cervical cancer, breast cancer, colon cancer, melanoma, 
seminoma, lung cancer and hematopoietic malignancies, such as erythroid 
leukemia, myeloid leukemia, (such as chronic or acute myelogenous 
leukemia), neuroblastoma and medulloblastoma. The differentiation 
inducing agent to which the cell is exposed can be selected from a 
broad variety of agents, including retinoids, polar compounds (such as 
hexamethylene bisacetanmide), short chain fatty acids, organic acids, 
Vitamin D derivatives, cyclooxygenase inhibitors, arachidonate 
metabolism inhibitors, ceramides, diacylglycerol, cyclic nucleotide 
derivatives, hormones, hormone antagonists, biologic promoters of 
differentiation, and derivatives of any of these agents.

Technology

    This invention provides a method and pharmaceutical composition for 
treating a tumor by causing apoptosis in tumor cells that expresses 
notch-1 protein, and in particular cells that exhibit increased 
expression of notch-1. Hence, this technology discloses methods and 
compositions to induce apoptosis in cells that over express the notch 
proteins. A cell fate determining function of notch is specifically 
disrupted at a time when the cell is undergoing differentiation, which 
causes the cell to undergo apoptosis. The invention includes therapies 
for tumors that over express a notch protein (such as notch-1) by 
inducing differentiation of the cells in the tumor with a 
differentiation inducing agent such as hexamethylene bisacetamide and 
other such differentiation agents. At a time during which 
differentiation has been promoted, and the cell is susceptible to 
interference with the anti-apoptosis effect of notch, the function of 
the notch protein is disrupted. Disruption of notch function can be 
achieved, for example, by the expression of antisense oligonucleotides 
that specifically interfere with expression of the notch protein on the 
cell, or by monoclonal antibodies that specifically bind to notch and 
inactivate it. This technology represents a novel method to induce 
apoptosis in tumor cells.
    The above mentioned invention is available, including any available 
foreign intellectual property rights, for licensing.

    Dated: August 3, 1999.
Jack Spiegel,
Director, Division of Technology Development and Transfer, Office of 
Technology Transfer, National Institutes of Health.
[FR Doc. 99-20455 Filed 8-6-99; 8:45 am]
BILLING CODE 4140-01-P