[Federal Register Volume 66, Number 78 (Monday, April 23, 2001)]
[Notices]
[Pages 20470-20472]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 01-9924]


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DEPARTMENT OF HEALTH AND HUMAN SERVICES

National Institutes of Health


National Cancer Institute: Opportunity for a Cooperative Research 
and Development Agreement (CRADA) for the Clinical Development of a 
Biologically Active, Epitope-Tagged Transforming Growth Factor Beta 
(TGF-[) Protein

    The National Cancer Institute's Laboratory of Cell Regulation and 
Carcinogenesis (LCRC) has created and characterized a recombinant 
transforming growth factor-beta (TGF-) ligand that contains 
the FLAG epitope tag and yet retains full biological activity.
AGENCY: National Institutes of Health, PHS, DHHS.

ACTION: Notice.

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SUMMARY: The National Cancer Institute (NCI) seeks a Cooperative 
Research and Development Agreement (CRADA) Collaborator to aid NCI in 
the preclinical and clinical development of a tagged form of the TGF-
 protein. Initial studies from LCRC demonstrate that a 
specific, eight-amino acid tag (known as FLAG) can be inserted in at 
least three different sites in the TGF-1 molecule, without 
interfering with its biological activity. LCRC has made three FLAG-
tagged porcine TGF-1 constructs and the identical murine FLAG-
TGF-1 cDNAs. Each construct differs only in the location of 
insertion of the FLAG tag, and these include either insertion 
immediately following the cleavage site N-terminal in the mature, 
processed TGF- molecule, or between amino acids 4 and 5 or 11 
and 12 of the mature TGF- molecule. The tagged molecule can 
detected by using a number of different techniques, including: 
Immunohistochemistry, immunoprecipitation, flow cytometry, 
immunofluorescence microscopy, ELISA, immunoblotting (``western''), and 
affinity chromatography.

DATES: Interested parties should notify NCI in writing of their 
interest in filing a formal proposal no later than June 22, 2001. 
Potential CRADA Collaborators will then have an additional thirty (30) 
days to submit a formal proposal. Additional proposals will be 
considered after the posted deadline in the event that a CRADA partner 
is not found during the initial posted timeperiod.

ADDRESSES: Inquiries and proposals regarding this opportunity should be 
addressed to Holly Symonds Clark, PhD., Technology Development 
Specialist (Tel. # 301-496-0477, FAX # 301-402-2117), Technology 
Development and Commercialization Branch, National Cancer Institute, 
6120 Executive Blvd., Suite 450, Rockville, MD 20852. Inquiries 
directed to obtaining a patent license(s) needed for participation in 
the CRADA opportunity should be addressed to John Rambosek, PhD., 
Technology Licensing Specialist, Office of Technology Transfer, 
National Institutes of Health, 6011 Executive Blvd., Suite 325, 
Rockville, MD 20852, (Tel. 301-496-7056, ext. 270; FAX 301-402-0220).

SUPPLEMENTARY INFORMATION: A Cooperative Research and Development 
Agreement (CRADA) is the anticipated joint agreement to be entered into 
with NCI pursuant to the Federal Technology Act of 1986, NCI seeks a 
CRADA Collaborator to aid LCRC in the preclinical and clinical studies 
of a tagged form of the TGF- protein. The expected duration of 
the CRADA would be from one (1) to five (5) years.

Background Information

    NCI's LCRC has produced the first epitope-tagged, biologically 
active version of a member of the transforming growth factor- 
(TGF-) family of proteins. Transforming growth factor-
1 (TGF-1) is the prototype for a large family of 
secreted polypeptides including the three mammalian TGF- 
isoforms (TGF-1, TGF-2, TGF-3), the bone 
morphogenetic proteins (BMPs), the activins and several more distantly 
related factors that regulate cell growth and function. The various 
members of the TGF- superfamily play roles in development, 
immune homeostasis, cancer progression, autoimmune disorders and wound 
repair. TGF-s are produced and secreted from the cell as large 
latent (inactive) molecules (pro-proteins). The Latency Associated 
Peptide (LAP) encompasses amino acids 1 through 279 (porcine TGF-
1) of the pro-protein. Association of LAP through disulfide 
bonds with the mature TGF-1 sequence keeps TGF- in a 
biologically inactive form. Conversion of this pro-peptide to a 
biologically active form can be achieved in several ways. These include 
disruption of the disulfide bonds (for example, mutation of two 
cysteine residues involved in forming this bond), cleavage of the 
protein to release the smaller biologically active TGF- (amino 
acids 280 through 391 in TGF-1), or denaturation of the 
associated LAP by acidification or heat.
    The ability to track the distribution of any exogenously 
administered, recombinant forms of these proteins has been restricted 
by the inability to distinguish between the endogenous forms of the 
protein produced in treated cells or tissues, and because most 
available antibodies exhibit some degree of cross-reactivity with 
related family members. LCRC's invention demonstrates a successful 
approach to adding an opitope tag to the mature TGF-1 
molecule. Epitope tags are short stretches of amino acids to which a 
specific antibody can be raised, allowing one to directly identify and 
track the tagged protein that has been added to a living organism or to 
cultured cells. Examples of useful epitope tags include FLAG, HA 
(hemagglutinin) and myc. In principle, any of these epitope tags could 
be used to tag TGF- family members, but NCI's LCRC has been 
the first to identify a way to retain biological function of the 
molecule following addition of the tag. Thus, it will be possible to 
track LCRC's tagged TGF- molecule when used in

[[Page 20471]]

preclinical studies or when administered to patients in a clinical 
setting.
    The FLAG epitope tag is short amino acid sequence of just eight 
amino acids (DYKDDDDK). Because of its small size, LCRC hypothesized 
that addition of this sequence to a region of the mature TGF-1 
molecule where structural constraints were minimal would result in no 
loss of biological activity. LCRC chose to add the FLAG tag 
independently in two different locations, creating two FLAG-tagged 
versions of the molecule. The mature, cleaved TGF- molecule 
should thus possess the FLAG epitope tag. Evaluation of the biological 
activities of the FLAG-tagged versions of TGF-1 indicate that 
there is no loss of biological activity as a consequence of inserting 
the FLAG epitope tag. In principle, the FLAG epitope tag can be added 
in an analogous way to any of the other TGF- isoforms or to 
closely related family members (e.g. the BMPs).
    The described methods are the subject of a U.S. provisional patent 
application filed on October 20, 2000 by the Public Health Service on 
behalf of the Federal Government.
    Under the present proposal, the goal of the CRADA will be to 
further characterize and develop the flag-tagged TGF- molecule 
in the following areas:
    1. Additional pre-clinical studies.
    (a). These studies would include, but not be limited to analyses of 
functionality of the epitope-tagged TGF-s. Of particular 
interest are the relative effects of different sites of tag insertion 
on the processing of the latent TGF- precursor, on receptor 
affinity, and on the dose-dependent effects of TGF- in a 
variety of functional assays, such as in vitro assays of leukocyte 
chemotaxis, epithelial and hematopoietic cell growth and 
differentiation, and lymphocyte activation. In addition, the epitope-
tagged TGF-s offer the unique potential to use 
immunofluorescence and confocal microscopy techniques to follow 
receptor trafficking and disposition following ligand binding.
    (b). The availability of the epitope-tagged TGF- also 
allows for a more quantitative evaluation of the pharmacodynamics of 
the peptide ligand. In vivo studies of plasma half-life, tissue 
distribution, CNS penetration, and elimination will be pursued. These 
studies will include assessment of both enteral and perenteral 
administration of TGF-. Other studies currently proposed 
include the generation of a ``knock-in'' mouse, in which the epitope-
tagged TGF- is used to replace the endogenous TGF- 
gene in mice, to confirm the absence of a deleterious effect of the 
epitope tag on normal function throughout development and in the adult 
organism. Such studies will allow for assessment of the transplacental 
transfer and distribution of maternal TGF- during gestation.
    (c). LCRC investigations also plan to focus on the development of 
diagnostic tools and screens based on the epitope-tagged TGF-
1. These experiments will include assays to provide a 
quantitative assessment of the level of functional TGF- 
receptor expression in both normal and tumor tissues by quantitative 
histochemical techniques and flow cytometric analysis. ELISA-based 
screens for the epitope tag will be developed as tools for 
pharmacokinetic studies of the epitope-tagged TGF-1 following 
either systemic, enteral, or local administration.
    2. Clinical trials focused on the applications of functional 
epitope-tagged TGF- ligands in the treatment of cancer, wound 
healing, and immune disorders.
    There are several important implications of this discovery for the 
development and application of TGF- family members for 
therapeutic purposes. To date, TGF- family members have been 
studied in clinical trials in the treatment of rheumatoid arthritis, 
multiple sclerosis, wound healing, and in the prevention of 
chemotherapy-induced mucositis. Preclinical studies in animal models 
predict that a number of applications will be tested in the near 
future. These include the evaluation of TGF- in the treatment 
of bronchial asthma, uveitis, osteoinduction following irradiation, 
management of inflammatory bowel disease, and a variety of autoimmune 
disorders and neuropathies having an autoimmune etiology. The ability 
to administer an epitope-tagged TGF- to a patient in a 
clinical setting would allow the researcher to accurately follow drug 
(FLAG-TGF-) distribution, half-life, elimination, and 
circulating levels without the complication of detecting the endogenous 
TGF- that is already present in the patient's tissues. The 
epitope-tagged molecule now provides a unique and novel tool to detect 
receptors for the TGF- proteins in tissues. The progression of 
many pathologic conditions, including cancer and immune system 
disorders, is often associated with loss of expression of these 
receptors.
    Following the completion of the preclinical studies described in 
section 1 above, LCRC plans to test the epitope-tagged TGF- in 
the following settings in the clinic:
    (a). LCRC plans to study the utility of epitope-tagged TGF-
 as a diagnostic tool for the quantitative, real-time measure 
of receptor expression in diseases in which altered expression of TGF-
 receptors and binding proteins have been described, such as 
cancer and immune disorders. These studies will include, but not be 
limited to the following:
    (i). Malignancies including lymphoid and those of the 
gastrointestinal tract, in which loss of receptor expression has been 
linked to disease pathogenesis.
    (ii). Autoimmune disorders, such as Systemic Lupus Erthematosis 
(SLE) and Sjogren's Syndrome, where altered production and 
responsiveness to TGF- may play a role in disease progression.
    (iii). In patients with Hereditary Hemorrhagic Telangiectasia 
(HHT), a disorder that exists in two forms and results from mutational 
inactivation of specific cell surface receptors. FLAG-TGF- 1 
can serve as the basis for a number of new diagnostic tools that may be 
useful in determining disease severity and prognosis in each of these 
disorders.
    (b). LCRC's tagged TGF- provides an example of how the 
members of the extended TGF- family might be tagged. Thus, an 
additional goal of the clinical phase of the research is to determine 
whether or not adding an epitope tag to other members of the extended 
TGF- family will allow for efficient tracking of the tagged 
molecule.
    (c). In the future, LCRC intends to examine the feasibility of 
inserting tag sequences that will not only allow for tracking, but will 
also enhance the delivery of these proteins by ferrying them into 
target tissues without loss of function. The latter represents an 
important area of ongoing research at NCI that will aim to reduce the 
incidence of adverse effects associated with the systemic 
administration of TGF- and related proteins. This avenue of 
research is of primary importance, as the widespread expression of TGF-
 receptors throughout vascular endothelia, and the rapid 
clearance of either active or latent forms from the circulation make 
the development of new delivery strategies essential.
    (d). LCRC intends to test the therapeutic application of the above 
functionalized, tagged TGF- molecules in the treatment of 
disorders, including, but not limited to the following:
     Disorders of chronic/delayed wound healing,
     Lymphoproliferative and autoimmune syndromes,
     Inflammatory disorders of the gastrointestinal tract, and

[[Page 20472]]

     Hematopoietic and epithelial malignancies.

Party Contributions

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 development and characterization of the epitope-tagged 
TGF- ligands, in vitro and in vivo assays of TGF- 
function, techniques for the detection and quantitation of epitope-
tagged TGF- proteins in biological specimens.
    3. Planning research studies and interpreting research results.
    4. Carrying out research to evaluate the pharmacokinetics and 
toxicity profiles of epitope-tagged TGF- ligands.
    5. Publishing research results.
    6. Developing additional potential applications of the FLAG-tagged 
TGF- molecule.

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. Providing technical and/or financial support to facilitate 
scientific goals and for further design of applications of the 
technology outlined in the agreement.
    4. Publishing research results.
    5. Providing resources and support for production and purification 
of the recombinant, epitope-tagged TGF- ligands.

Selection Criteria for Choosing the CRADA Collaborator Will Include

    1. A demonstrated record of success in the areas of cytokine 
expression systems, large scale purification of recombinant proteins 
and the evaluation of cytokine function.
    2. A demonstrated background and expertise in the preclinical 
development of biological response modifiers and in the design and 
execution of clinical trials.
    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 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 or joint 
inventor.

    Dated: April 12, 2001.
Kathleen Sybert,
Chief, Technology Development and Commercialization Branch, National 
Cancer Institute, National Institutes of Health.
[FR Doc. 01-9924 Filed 4-20-01; 8:45 am]
BILLING CODE 4140-01-M