Exhibit 10.4
Portions of this exhibit have been omitted pursuant to a request for
confidential treatment. The omitted portions, marked "[***]", have been
separately filed with the Securities and Exchange Commission.
SPONSORED RESEARCH AGREEMENT
Agreement, made this 23rd day of March, 1993, by and between THE
UNIVERSITY OF TEXAS M.D. XXXXXXXX CANCER CENTER (hereinafter referred to as
"CANCER CENTER"), a component institution of The University of Texas System
(hereinafter referred to as "SYSTEM"), located in Houston, Texas, and Ingenex,
Inc., a California corporation (hereinafter referred to as "SPONSOR"), located
at 000 Xxxxxx Xxxxx Xxxx, Xxxxx #000, Xxx Xxxxxxxxx, Xxxxxxxxxx 00000.
WITNESSETH:
WHEREAS, SPONSOR is the licensee of the technology described in the
patent applications shown in Exhibit III attached hereto, which has potential
utilization in patient care and treatment; and
WHEREAS, CANCER CENTER has research facilities and situations which
would allow investigation and study the technology for use in the treatment of
cancer as described in Exhibit I hereinafter referred to as ("Research"), a
copy of which is attached hereto and incorporated herein by reference; and
WHEREAS, both SPONSOR and CANCER CENTER consider it necessary and
desirable to perform the Research; and
WHEREAS, both SPONSOR and CANCER CENTER desire to cooperate in the
design construction, and use of a retroviral production facility to be located
at, and owned by CANCER CENTER to enable the clinical development of new
therapeutic pharmaceuticals;
NOW, THEREFORE, the parties agree as follows:
1. Evaluation. SPONSOR agrees to engage the services of CANCER CENTER
as an independent contractor to perform the Research. The Research will be
under the supervision of Xxxxxx X. Xxxxxxxxxx, M.D., Ph.D. (Principal
Investigator) at CANCER CENTER, with the assistance of appropriate associates
and colleagues at CANCER CENTER as may be required. In the event that Xxxxxx
X. Xxxxxxxxxx, M.D., Ph.D. is unable to act as Principal Investigator in the
STUDY for any reason, SPONSOR shall decide whether to terminate the STUDY,
subject to the provisions of Article 14, or whether to continue the STUDY
under the supervision of a substitute Principal Investigator who is mutually
acceptable to both CANCER CENTER and SPONSOR.
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2. Research.
a. CANCER CENTER agrees as an independent contractor to conduct the
Research. Such Research was originally approved by CANCER CENTER in accordance
with CANCER CENTER policy and may be subsequently amended only in accordance
with CANCER CENTER policy and the written agreement of CANCER CENTER and
SPONSOR as provided for in Article 16 herein below.
b. The Retroviral Production Facility referenced in Exhibit 1("FACILITY"),
will be provided and operated by CANCER CENTER with funding initially from
SPONSOR. SPONSOR will have the right to use the FACILITY for five (5) years,
subject to Article 14 herein, for the development and production of gene
therapy agents for clinical testing based on the human MDR1 gene, modified RB
genes, and other genetic therapy elements as may be evaluated in collaboration
with the Principal Investigator. CANCER CENTER agrees to limit access to the
FACILITY to persons directly involved in the performance of sponsored research
conducted on behalf of SPONSOR'S and to CANCER CENTER's own employees engaged
in academic research as permitted herein. CANCER CENTER will take the
necessary steps, including obtaining nondisclosure agreements from persons
having access to the FACILITY, to protect SPONSOR's proprietary information
and materials. CANCER CENTER may utilize the FACILITY for its own non-profit
educational and research purposes and agrees not to utilize the FACILITY to
conduct research directly or indirectly for the benefit of any third party
with rights in excess of the Federal Government without the prior written
approval of SPONSOR.
c. The purpose of the FACILITY will be to support the basic and clinical
research programs of the CANCER CENTER, and particularly of the Principal
Investigator, by providing a resource for the GMP (Good Manufacturing
Practices) production of clinical grade retroviral pharmaceutical reagents for
investigational use in humans to obtain United States Food and Drug
Administration (FDA) approval. The FACILITY will not be a commercial
enterprise.
3. Invention and Patents.
a. For all purposes herein, "Invention" shall man any discovery, concept
or idea whether or not patentable or copyrightable, which (i) arises
out of work performed pursuant to the obligations of, and funded
under, this Agreement; (ii) is conceived and reduced to practice
during the term of the Agreement as defined in Article 14 hereinbelow;
and (iii) includes but is not limited to processes, methods, software,
formulae, techniques, compositions of matter, devices, and
improvements thereof and know-how relating thereto.
Inventions made solely by the Principal Investigator and/or other
CANCER CENTER personnel as identified in Article 1 hereinabove, or
agents of CANCER CENTER, shall be the sole property of CANCER CENTER.
Inventions made jointly by employees or agents CANCER CENTER and
SPONSOR shall be jointly owned by CANCER CENTER and SPONSOR.
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b. In the event that an Invention is made, either solely by employees or
agents of CANCER CENTER or jointly by employees or agents of CANCER
CENTER and SPONSOR, CANCER CENTER and SPONSOR agree to give written
notice of such Invention to each other within thirty (30) days of the
identification of such Invention. Within thirty (30) days of notice of
Invention, CANCER CENTER and SPONSOR will thereupon exert their best
reasonable efforts in cooperation with each other to investigate,
evaluate and determine to the mutual satisfaction of both parties, the
disposition of rights to the Invention, including whether, by whom,
and where any patent applications are to be filed.
c. If, after consultation with SPONSOR, it is agreed by the parties that
a patent application should be filed, or if SPONSOR determines it
would like to file a patent application, SPONSOR will prepare and file
appropriate United States and foreign patent applications on
Inventions made under this Agreement, and pay the cost of preparing,
filing, prosecution and maintenance thereof. SPONSOR intends, and is
hereby authorized, to use the services of the law firm of Xxxxxx &
Xxxxxxx, of New York, in the preparing, filing and prosecution of all
such patent applications. SPONSOR agrees to consult with CANCER CENTER
in the preparation and prosecution of all such patent applications and
will provide CANCER CENTER'S a copy of all applications filed for
which SPONSOR has paid the cost of filing, as well as copies of any
documents received or filed during prosecution thereof. If SPONSOR
notifies CANCER CENTER that it does not intend to pay the cost of an
application, or if SPONSOR does not respond to CANCER CENTER,
notification that an invention has been made within thirty (30) days
after receipt of such notification, then CANCER CENTER may file such
application at its own expense, and with the attorneys of its choice,
and SPONSOR shall have no rights to such Invention. SPONSOR agrees to
maintain any such application in confidence until it is published by
CANCER CENTER or by the respective patent office. Any patent
application filed by SPONSOR which names a CANCER CENTER employee as
an inventor shall be filed at least in the name of BOARD, and CANCER
CENTER agrees to cooperate in the filing of all patent applications
filed pursuant to this agreement.
d. (i) CANCER CENTER agrees to grant SPONSOR an exclusive, worldwide,
royalty-bearing license to make, have made, use, and sell Inventions
(as well as patent applications, patents, and copyrights thereon) for
commercial purposes, under the terms and conditions set forth in the
form License Agreement attached hereto as Exhibit II (the "License
Agreement"), provided that SPONSOR shall pay all costs and expenses
associated with patent and copyright filing, prosecution, issuance,
and maintenance. The License Agreement shall become effective upon the
request of SPONSOR to activate the License Agreement, after the CANCER
CENTER has notified SPONSOR that an Invention has been made. SPONSOR
shall have thirty (30) days from the date of written notice of
Invention from CANCER CENTER pursuant to Article 3(b) hereinabove, to
give written
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notice to CANCER CENTER to activate the Agreement. The License
Agreement shall be activated and made effective by providing (1) the
description of the Invention to the licensed (including title,
inventors, patent application filing date, and serial number if
available), (2) the effective date, and (3) all of the required
signatures and approvals of both parties.
(ii) CANCER CENTER grants SPONSOR an exclusive right of first refusal
to take a license under the License Agreement, pursuant to Section
1.3.2 and 4.1.1 (B) of the License Agreement, to inventions made with
funding from the federal Government (or from another source that does
not claim rights to intellectual property in excess of those of the
federal Government) and which were developed with the use of the
FACILITY. Within thirty (30) days after receipt of notice from the
CANCER CENTER that such an invention has been made, SPONSOR must
notify CANCER CENTER in writing of SPONSOR's intent to exercise this
right of first refusal. The right of first refusal becomes a
non-exclusive option if SPONSOR fails to exercise the right of first
refusal within the thirty (30) day period provided.
(iii) Subject to Article 2(b), above, CANCER CENTER grants SPONSOR a
non-exclusive option to negotiate a license to inventions made with
use of the FACILITY and with funding from a third party with
intellectual property rights that conflict with SPONSOR's rights under
this Agreement. This option, and any license granted to SPONSOR from
the CANCER CENTER pursuant to it, is expressly subject to the rights
of said third party. Within thirty (30) days after receipt of notice
from the CANCER CENTER in writing of SPONSOR's intent exercise this
option.
e. In the event Sponsor does not notify CANCER CENTER in writing of
SPONSOR's intent to exercise its rights under section 3(d), above,
within the thirty (30) day period provided, CANCER CENTER shall have
the right to enter into license agreements, including exclusive
license agreements, with third parties concerning the same Invention.
4. Confidentiality. Because CANCER CENTER and SPONSOR will be cooperating
with each other in this Research, and because each may reveal to the other
in the course of this Research certain confidential information, CANCER
CENTER and SPONSOR agree to hold any confidential information which (a) is
obtained during the course of this work and (b) is related thereto and (c)
is marked as "CONFIDENTIAL" in confidence, and each party will not
disclose same to any third party without the express written consent of
the other party to this Agreement. This requirement shall remain in force
for a period of five (5) years following completion of work under this
Agreement. Nothing in this paragraph shall in any way restrict the rights
of either CANCER CENTER or SPONSOR to use, disclose or otherwise deal with
any information which:
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a. Can be demonstrated to have been in public domain as of the effective
date of this Agreement or comes into the public domain through the
term of this Agreement through no act of the recipient; or
b. Can be demonstrated to have been known to the recipient prior to the
execution of this Agreement; or
c. Can be demonstrated to have been rightfully received by the recipient
after disclosure under this Agreement from a third party who did not
require the recipient to hold it in confidence or limit its use and
who did not acquire it, directly or indirectly, under obligation of
confidentiality to the disclosing party; or
d. Shall be required for disclosure to Federal regulatory agencies
pursuant to approval for use; or
e. Is independently invented by researchers of the recipient, which in
the case of CANCER CENTER includes SYSTEM, who have not had access to
the information provided to the recipient hereunder.
Nothing herein is intended to give either party the right to use for
any purpose pre-existing confidential information of the other party.
Notwithstanding the confidentiality obligations of this Agreement,
nothing herein shall prevent CANCER CENTER and any other component of
SYSTEM from using any information generated hereunder for ordinary
research and educational purposes of a university.
5. Publication Rights. Notwithstanding the provisions of Article 4 of this
Agreement, CANCER CENTER may publish scientific papers relating to the
collaborative research performed under this Agreement. In the event that
CANCER CENTER wishes to publish, CANCER CENTER shall notify SPONSOR of its
desire to publish at last sixty (60) days in advance of publication and
shall furnish to SPONSOR a written description of the subject matter of
the publication in order to permit SPONSOR to review and comment thereon.
6. Publicity. CANCER CENTER acknowledges SPONSOR's intention to distribute
periodically informational releases and announcements to the news media
regarding the progress of research hereunder. SPONSOR shall not release
such materials containing the name of CANCER CENTER or any of its
employees without prior written approval by an authorized representative
of CANCER CENTER, and said approval shall not be unreasonably withheld.
Should CANCER CENTER reject the news release, CANCER ENTER and SPONSOR
agree to discuss the reasons for CANCER CENTER's rejection, and every
effort shall be made to develop an appropriate informational news release
within the bounds of accepted academic practices. SPONSOR reserves the
same right in the event that CANCER CENTER desires to distribute a news
release concerning the
-5-
research program. Nothing herein shall be construed as prohibiting CANCER
CENTER or SPONSOR from reporting on this study to a governmental agency.
7. Responsibility. The parties each agree to assume individual responsibility
for the actions and omissions of their respective employees, agents and
assigns in conjunction with this evaluation.
8. Independent Contractor. SPONSOR will not have the right to direct or
control the activities of CANCER CENTER in performing the services
provided herein, and CANCER CENTER shall perform services hereunder only
as an independent contractor, and nothing herein contained shall be
construed to be inconsistent with this relationship or status. Under no
circumstances shall CANCER CENTER be considered to be an employee or agent
of SPONSOR. This Agreement shall not constitute, create or in any way be
interpreted as a joint venture, partnership or formal business
organization of any kind.
9. Title to Equipment. CANCER CENTER shall retain title to all equipment
purchased and/or fabricated by it with funds provided by SPONSOR under
this Agreement.
10. Survivorship. The provisions of Article 3, 4, 5, 6, and 12 shall survive
any expiration or termination of this Agreement.
11. Assignment. This Agreement may not be assigned by either party without the
prior written consent of the other party; provided, however, that SPONSOR
may assign this Agreement to any purchaser or transferee of all or
substantially all of SPONSOR's business upon prior written notice to
CANCER CENTER.
12. Indemnification. CANCER CENTER shall, to the extent authorized under the
Constitution and the laws of the State of Texas, hold SPONSOR harmless
from liability resulting from the negligent acts or omissions of CANCER
CENTER, its agents or employees pertaining to the activities to be carried
out pursuant to the obligations of this Agreement; provided, however, that
CANCER CENTER shall not hold SPONSOR harmless from claims arising out of
the negligence of SPONSOR, its officers, agents or any person or entity
not subject to CANCER CENTER's supervision or control.
SPONSOR shall indemnify and hold harmless SYSTEM, CANCER CENTER, their
regents, officers, agents and employees from any liability or loss
resulting from judgments or claims against them arising out of the
activities to be carried out pursuant to the obligations of this Agreement
or the use by SPONSOR of the results of the Research, provided, however,
that the following is excluded from SPONSOR's obligation to indemnify and
hold harmless:
a. the negligent failure of CANCER CENTER to comply with any applicable
governmental requirements; or
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b. the negligence or willful malfeasance by a regent, officer, agent or
employee of CANCER CENTER or SYSTEM.
13. Award. SPONSOR agrees to pay CANCER CENTER One Million four Hundred
Ninety-Nine Thousand Five Hundred Fifty-Eight and No/100 Dollars
($1,499,558.00) for expenses and other related costs incurred in
conjunction with the Research. This amount, as shown by approximate
category of expense in the attached Exhibit I which is attached hereto and
is incorporated herein by reference, for information only, shall be
payable according to the following schedule: one payment of two Hundred
Fifty Thousand Two Hundred Eighty-Seven and No/100 Dollars ($250,287.00)
shall be due within sixty (60) days of the date of execution of this
Agreement; a second payment of two Hundred Fifty Thousand Two Hundred
Eighty-Seven and No/100 Dollars ($250,287.00) shall be due January 31,
1994; upon the achievement of mutually agreeable milestones as set forth
in Appendix IV of this Agreement.
Sponsor shall also provide payment of $999,608. for establishment and
operation of the Retroviral Production Facility. This payment shall occur
as follows: one payment of $300,000 due within thirty (30) days after
receipt of notice by Sponsor from the Recombinant Advisory Committee that
Cancer Center has completed the animal experiment necessary for RAC
approval; a second payment of $100,000 due ninety (90) days after the
first payment; a third payment of $100,000 due one hundred and eighty
(180) days after the first payment, a fourth payment of $250,000 due one
year after the first payment, and $249,608 due eighteen (18) months after
the first payment.
14. Alternative Production Facility. Sponsor and Cancer Center agree that in
the event Sponsor is able to provide a retroviral production facility
acceptable to the Principal Investigator through Sponsor's lease or
purchase of such a facility, but which is not located at or owned by
Cancer Center, Sponsor may choose to conduct the retroviral production
research and development under this agreement at such alternative
facility. In such event Sponsor shall not provide payment of $999,608 as
described in Article 13 above, but Sponsor shall provide all operating
expenses necessary to provide for the same scope, extent, and timely
performance of work to be performed at the Retroviral Production Facility.
These total operating expenses are currently estimated at $740,000, but
may be adjusted by mutual agreement of Sponsor and Cancer Center. The
alternative production facility would be operated or owned by Sponsor, and
Sponsor would provide all access necessary to the facility for Principal
Investigator and Cancer Center employees directly involved in the
Sponsored Research. Sponsor would also make the alternate facility
available for the non-profit educational and research purposes of the
Cancer Center. Inventions and patents made by Cancer Center employees and
its agents utilizing the alternate facility would be governed by Article 3
of this agreement. Inventions made by Sponsor's employees and its agents
in the alternate facility would be the sole property of Sponsor and not
governed or subject to Article 3 of this agreement. Inventions made by
both Cancer Center employees or its agents and sponsor, or its agents in
the alternate facility, will be governed by Article 3 of this agreement.
Sponsor's rights under Article 3
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shall continue, in the event of utilization of this alternate facility,
for the duration of collaboration with and utilization of the alternate
facility by Cancer Center, and shall not expire on the sixth anniversary
of this agreement, as described in Article 15 below. Sponsor would have
the full right to use such alternate facility for its own research and
development purposes, and Sponsor's duration of use, occupancy and/or
ownership of such alternate facility will not be limited by Article 2b,
Article 14, or any other part of this Agreement.
15. Basic Term. (i) This Agreement shall become effective as of the date first
hereinabove written and, unless earlier terminated as hereinafter
provided, shall continue in force for a period of two (2) years after the
same, provided, however, that the term of this Agreement may be extended,
contingent upon continued collaboration between SPONSOR and the Principal
Investigator, at the option of SPONSOR (exercised by written notice to
CANCER CENTER) for one (1) year periods, for three (3) consecutive years
after the second anniversary of this Agreement. (ii) SPONSOR's rights
under Article 3 (d) (ii) and (iii), above, shall terminate on the sixth
anniversary of the effective date of this Agreement, regardless of whether
this Agreement is otherwise extended beyond that date.
16. Default and Termination. In the event that either party to this Agreement
shall be in default of any of its material obligations hereunder and shall
fail to remedy such default within thirty (30) days after receipt of
written notice thereof, the party not in default shall have the option of
terminating this Agreement by giving written notice thereof,
notwithstanding anything to the contrary contained in this Agreement.
SPONSOR shall have the right to termination of this Agreement, with proper
notice as described above, if the milestones, as outlined in Exhibit III
attached hereto, are not achieved, unless CANCER CENTER justifies to the
satisfaction of SPONSOR, the failure to achieve the milestone, or
otherwise cures the breach. Termination of this Agreement shall not affect
the rights and obligations of the parties which accrued prior to the
effective date of termination. SPONSOR shall pay CANCER CENTER for all
reasonable expenses incurred or committed to be expended as of the
effective termination date, subject to the maximum amount as specified in
Article 13.
17. Entire Agreement. The parties acknowledge that this Agreement and the
attached Exhibits hereto represent the sole and entire Agreement between
the parties hereto pertaining to the Research and that such supersedes all
prior Agreements, understandings, negotiations and discussions between the
parties regarding same, whether oral or written. There are no warranties,
representations or other Agreements between the parties in connection with
the subject matter hereof except as specifically set forth herein. No
supplement, amendment, alteration, modification, waiver or termination of
this Agreement shall be binding unless executed in writing by the parties
hereto.
18. Reform of Agreement. If any provision of this Agreement is, becomes or is
deemed invalid, illegal or unenforceable in any United States
jurisdiction, such provision shall be deemed amended to conform to
applicable laws so as to be valid and enforceable; or if it
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cannot be so amended without materially altering the intention of the
parties, it shall be stricken, and the remainder of this Agreement shall
remain in full force and effect.
19. Notices. Any notices, statements, payments, or reports required by this
Agreement shall be considered given if sent by United States Certified
Mail, postage prepaid and addressed as follows:
If to CANCER CENTER:
Xxxxxxx X. Best
Chief Financial Officer
The University of Texas M.D. Xxxxxxxx Cancer Center
0000 Xxxxxxxx Xxxx.
Xxxxxxx, Xxxxx 00000
(000) 000-0000
If to SPONSOR:
Xx. Xxx Xxxxxx
Ingenex, Inc.
000 Xxxxxx Xxxxx Xxxxxxxxx, Xxxxx 000
Xxx Xxxxxxxxx, Xxxxxxxxxx 00000
(000) 000-0000
20. Captions. The captions in this Agreement are for convenience only and
shall not be considered a part of or affect the construction or
interpretation of any provision of this Agreement.
21. Governing Law. This Agreement shall be governed and interpreted in
accordance with the substantive laws of the State of Texas and with
applicable laws of the United States of America.
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IN WITNESS WHEREOF, CANCER CENTER and SPONSOR entered into this
Agreement effective as of the date first hereinabove written and have executed
three (3) originals each of which are of equal dignity.
INGENEX, INC. THE UNIVERSITY OF TEXAS
M.D. XXXXXXXX CANCER CENTER
BY: /s/ Xxxxx X. Xxxxxx BY: /s/ Xxxxxxx X. Best
---------------------- ------------------------
Xxxxxxx X. Best
TITLE: President Chief Financial Officer
-------------------
I have read this agreement and
understand my obligations
hereunder: CONTENT APPROVED:
BY: /s/ Xxxxxx X. Xxxxxxxxxx BY: /s/ Xxxxx X. Xxxxxxx
---------------------------------- -----------------------------
Xxxxxx X. Xxxxxxxxxx, M.D, Ph.D. Xxxxx X. Xxxxxxx, CPA
Principal Investigator Manager, Sponsored Agreements
FORM APPROVED:
BY: /s/ Xxxxx X. Xxxxxxx BY: /s/ Xxxxxxx X. Xxxx
----------------------------- ----------------------
Xxxxx X. Xxxxxxx, M.D. Xxxxxxx X. Xxxx, X.X.
Head, Division of Medicine Legal Services Officer
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EXHIBIT I
PROPOSAL TO INGENEX, INC.
PRINCIPAL INVESTIGATOR: XXXXXX XXXXXXXXXX, M.D.,
PH.D.
THE U.T. M.D. XXXXXXXX CANCER CENTER, HOUSTON,
TEXAS
OCTOBER 1, 1992
ABSTRACT:
Fifty-percent of the one out of eight American women diagnosed with breast
cancer are destined to die following the diagnosis of the disease. An
additional 50% of patients diagnosed with ovarian cancer will also die. If one
delays treatment until the time of evolution of advanced disease, only 15% of
these patients can be rescued even with the most intensive therapy. The doses
of therapy which can be used to eradicate this disease, either in the adjuvant
setting for poor prognosis patients or in the advanced disease setting, is
limited by myelosuppression. In resistant gestational malignancies, as well as
in ovarian cancer, depletion of the marrow reserve by therapy leads to
inability to deliver the full doses of therapy which are nenessary to cure the
disease. In order to protect marrow from chemotherapy-induced toxicity, we are
proposing to introduce the multidrug resistance cDNA into the normal
hematopoietic stem cells of patients with carcinomas of the breast and ovary
as well as gestational malignancies and to transplant them before they are
exposed to combination chemotherapy. We will use safety modified retroviruses
which contain the MDR cDNA to modify hematopoietic progenitor cells. We will
then use in vivo selection of the cells which are transduced and therefore
resistant to chemotherapy. This will permit the delivery of effective doses of
combination chemotherapy to which the disease will be sensitive. Project 1A
will deal with the development of preclinical data for retroviruses which
carry MDR-1 for use in genetic modification of hematopoietic cells. Projects
1B and 1C contain proposals for clinical implementation of the programs for
chemoprotection of the carcinoma of the breast and ovary. Project 1D contains
a proposal for setting up a production facility for clinical grade
safety-modified retroviruses to enable the U.T. M.D. Xxxxxxxx Cancer Center in
partnership with Ingenex, Inc. to quickly take laboratory grade materials from
the lab to the clinic. Finally, Project IE contains a proposal for clinical
implementation for the prevention of recurrence of human papilloma virus
positive carcinoma of the cervix by regional exposure to safety-modified
retroviruses which contain the Rb and p53 cDNAs.
A. SPECIFIC AIMS
We are proposing a partnership with Ingenex, Inc. to establish programs
for the introduction of chemotherapy resistance genes into the normal
hematopoietic stem cells of poor prognosis patients with carcinomas of the
ovary, breast, and trophoblastic disease of pregnancy. This is designed to
result in the less myelosuppression following chemotherapy and to permit the
safe delivery of higher doses of chemotherapy than are usually permissible
over prolonged periods of time in settings in which the tolerance of the
marrow to chemotherapy is limited: after autologous transplant or in the
adjuvant setting. In addition, we will apply this genetic chemoprotection to
primary refractory patients with trophoblastic disease of pregnancy. In poor
prognosis advanced ovarian cancer, and in the trophoblastic disease of
pregnancy, the chemotherapy needed to induce cures often results in reduction
of the marrow reserve which then results in a prolongation of the intervals
between successive doses of chemotherapy. This is often not correctable by the
administration of growth factors. The longer intervals between chemotherapy
provide more time for recovery of the abnormal cells, leading to relapse and
escape from curative therapy. Although advanced disease patients with relapsed
carcinoma of the breast often exhibit striking responses to a single dose of
autologous transplantation, the patients often relapse and cannot receive
therapy
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The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
following transplant to prevent relapse due to the sensitivity of the graft to
the suppressive effects of chemotherapy. The high dose adjuvant therapy also
may induce a reduction of the marrow stem cell reserve.
[ ***
]
SPECIFIC AIM 1: Development of Vectors: Construction of vectors and
producer cell lines which contain the MDR-1 cDNA in a safety-modified
retrovirus (Deisseroth, Xxxxxxx and Fu).
SPECIFIC AIM 2: Testing of the virus in cells to determine its
transduction frequency and the stability of the change in phenotype
(chemotherapy resistance) induced by its transduction in cells following
transplantation. MDR viruses will be tested.
SPECIFIC AIM 3: Testing of the significance of the change in phenotype
induced in the cells transduced by this retrovirus in animal models. The MDR
vectors will be tested by Drs. Deisseroth, Xxxxxxx and Fu in a breast cancer
mouse model.
SPECIFIC AIM 4: Evaluation of the producer cell lines and retroviral
supernatants so as to produce a clinical grade retroviral supermatant.
SPECIFIC AIM 5: Development of a clinical trial for the in vivo selection
of genetically modified hematopoietic cells and the testing of the clinical
impact of genetic modification of the resistance of sensitivity of cells to
chemotherapy in carcinomas of the breast and ovary.
SPECIFIC AIM 6: Presentation of the clinical programs for review through
the regulatory process to the local IRB, the NIH RAC, and the FDA. The time
line for implementation of these specific aims is reviewed in Table II.
[ ***
]
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B. BACKGROUND
SECTION ON RATIONALE FOR MDR CHEMOPREVENTION IN CARCINOMAS OF THE OVARY,
BREAST, AND TROPHOBLASTIC DISEASE OF PREGNANCY:
During the past 40 years, two factors have limited the safe delivery of
doses of chemotherapy which can eradicate breast cancer or ovarian cancer in
poor prognosis patients: (1) Resistance of the carcinoma cells to the doses of
chemotherapy which are non-toxic to the normal tissues of most patients; and
(2) the sensitivity of the bone marrow to the doses of chemotherapy which are
necessary to eradicate the neoplastic disease.
Several strategies have been attempted to circumvent these two
limitations. One strategy involved drugs which will inhibit the molecules
which contribute to drug resistance. These studies have involved primarily
drugs which may inhibit the efflux drug pumps MDR-1, which has been found to
be present in the neoplastic cells of relapsed patients. Unfortunately, since
the normal hematopoietic cells depend upon one of these molecules, MDR1, for
whatever resistance they have to chemotherapy (1), and the level of clinical
resistance in the tumor is not always due to MDR-1, these trials have been
unsuccessful.
Another method which has been studied extensively at M.D. Xxxxxxxx, as
well as at other centers, is the use of dose escalation of the therapy, and
rescue of the patient from hematopoietic toxicity by infusion of the
autologous marrow which is kept out of the body of the patients during the
administration of the intensive chemotherapy (2-5). Our center as well as
workers at the Xxxxxx Xxxxxx Cancer Center, Duke University, and at other
institutions have observed striking responses at the higher dose levels (2-5).
However, only 20% of the advanced disease patients have exhibited durable
responses (2-5).
Since these patients usually relapse in the sites of bulky disease which
were present at diagnosis, most experts have concluded that the remissions
induced by a single dose of chemotherapy decrease the tumor burden below the
level which is clinically detectable, but do not eradicate it, and this
residual disease simply regrows. It is uniformly recognized that most patients
who receive intensive systemic therapy followed by an autologous bone marrow
transplantation are extremely sensitive to even conventional dose
chemotherapy, which excludes the administration of continuation cycles of
therapy at conventional doses following transplant to eradicate the residual
disease and prevent tumor regrowth.
Another strategy which has been used to circumvent the propensity of
breast cancer to regrow in the adjuvant setting after regional therapy
(radiation and/or surgery) is the administration of escalating doses of
adjuvant chemotherapy in poor prognosis patients (Stage II or III breast
cancer). Studies at our institution (6-8) have shown in this setting that the
disease-free survival has been directly related to the doses of cytotoxic
therapy given, and the response rate has correlated with the dose intensity in
the advanced disease setting. In this setting, the administration of intensive
FAC chemotherapy (which has a 5 year disease-free survival of 66% for Stage II
and 47% in Stage II disease delivered at conventional doses), has resulted in
higher initial response rates. However,
-4-
therapists have been unable to continue to deliver these intensive doses of
FAC therapy after three cycles of this therapy. Thus, this initial increased
response rate does not translate into increased survival. In this latter
setting, both the duration and depth of the granulocytopenia as well as the
thrombocytopenia become more profound with each successive dose of therapy,
suggesting that the use of hematopoietic growth factors will not rescue
patients from this problem (6-8).
These data, plus the absence of clinically significant elevations of the
MDR pump in the untreated breast or ovarian cancer patients (9) as well as the
presence of only minimal elevations of MDR in the cells of patients with
resistant carcinoma of the ovary and breast (10), have suggested that the
introduction of the MDR-1 cDNA into the normal hematopoietic cells of the
marrow might permit the use of cyclical administration of relatively higher
doses of combination chemotherapy, either in the adjuvant setting in poor
prognosis disease (ovary or breast), or following bone marrow transplantation
in the minimal disease setting of advanced disease (breast or ovarian cancer)
patients who have received intensive therapy.
What is the evidence that chemoprotection will work? Pastan, Xxxxxxxxx and
their colleagues have reported that transgenic mice overexpressing the human
MDR1 cDNA, which codes for a 107 kDa glycoprotein, a drug efflux pump in the
plasma membrane of cells, exhibit normal patterns of hematopoiesis, expression
of levels of MDR glycoprotein which are 3-fold higher than most MDR resistant
fresh tumor cells or tumor cell lines, and exhibit a 10-fold increase in the
resistance of hematopoietic cells to drugs like Taxol (9-12). Retroviral
vectors have also been used to infect mammalian cells and in populations
selected by FACS for the modified cells, the resistance to MDR drugs increased
up to 26-fold (13). It is clear, however, that in vivo or in vitro selection
is necessary following retroviral transduction to maintain the resistant
phenotype in mammalian cells. Several mouse and large animal model studies
have also been recently reported (14-16), which document the feasibility of
introduction of resistance genes into human marrow.
Clinical trials of chemoprotection have been proposed in carcinomas of the
breast and ovary. It is clear, from published studies, that the human MDR1 is
not naturally expressed in normal breast or ovarian tissue, and that all of
the fresh specimens of breast or ovarian cancer do not exhibit elevated levels
of MDR providing they were not previously exposed to chemotherapy (17-19). The
levels of MDR in ovarian and breast cancers following therapy in the advanced
disease setting are elevated by 2 to 3-fold. Thus, the introduction of MDR1
into the normal hematopoietic cells of patients with poor prognosis disease,
would make possible the testing of therapeutic questions that would not be
possible with drugs like Taxol. Animal model data suggests that the levels of
chemotherapy resistance, which can be achieved with MDR chemoprotection in
marrow stem cells using selection for the modified cells, is 3-fold higher
than that seen in resistant tumors which have been previously exposed to MDR
drugs (5).
In our institutional experience (2-21) and that of others, the toxicity of
Taxol is primarily myelosuppression, when the continuous infusion schedule of
Taxol alone is used. The clinical programs proposed for breast, ovary and a
third model, trophoblastic disease of pregnancy, are summarized in detail in
this project (Projects 1B and 1C). The presence of marrow contamination with
neoplastic cells, which is a problem with breast cancer, is not a significant
risk in ovary or
-5-
trophoblastic disease. The technical details of this program, including marrow
purging before retroviral transduction, will be dealt with in these projects.
In Projects lA-lC, we are proposing to develop preclinical and clinical
models for the testing of the hypothesis that the transduction of marrow cells
by safety-modified retroviruses which contain the MDR-1 cDNA, which would
protect individuals against the higher doses of anthracyclines and Taxol.
Project 1A will deal with the development of the vectors, preclinical, in
vitro and animal models, as well as developing the clinical grade retroviral
supernatants as outlined in Tables I-II, while Projects 1B and 1C will deal
with the clinical programs.
MDR PUMP PROTEINS AND THE MDR DRUGS (PROJECT 1A)
The MDR-1 gene protects cells against the toxic effects of chemotherapy
since it codes for a plasma membrane ATP-dependent efflux pump. This pump
promotes the secretion of the following drugs: actinomycin D, doxorubicin,
vincristine, vinblastine, VP-16, and Taxol. The M.D. Xxxxxxxx Cancer Center
Breast Section has developed and studied an adjuvant combination chemotherapy
program for carcinoma of the breast which has been associated with a 60%
long-term disease-free survival rate (6-8). As outlined above, patients are
unable to sustain the high doses required to control advanced or poor
prognosis disease in the adjuvant setting (8).
Recently, the plant product Taxol has exhibited significant activity in
previously untreated advanced disease setting of breast cancer (22), and in
those breast cancer patients who have exhibited resistance to adriamycin. This
drug exhibits myelosuppression as its dose-limiting toxicity in eight of the
ten continuous infusion trials which are available for analysis in which Taxol
was used as the only agent (23-32). The dose intervals between the onset of
limiting myelosuppression and other non-hematopoietic toxicities such as
neuropathy or mucositis depends on the schedule of administration and whether
it is used with other drugs such as platinum. The toxic effect of clinically
significant bradycardia is seen only in the pulse schedule. Several studies
have indicated that Taxol is subject to the MDR-1 pumping mechanism and that
the exposure of cells to Taxol may select for cells which exhibit higher
levels of the MDR protein (33). XxXxxxx has shown that ovarian cancer patients
previously treated with platinum are also very sensitive to the
myelosuppressive effect of Taxol and the sensitivity of hematopoietic cells to
Taxol increases with successive cycles of administration (34). Similarly,
previous exposure of patients to radiation therapy produces prohibitive levels
of sensitivity to the myelosuppressive effects of Taxol. The M.D. Xxxxxxxx
experience with Taxol (DM 106 and NCI T86-0270) has suggested that
myelosuppression is the major dose limiting toxicity in all but two patients
at the 250 m1/m2 per day continuous infusion trial. All the non-hematological
toxicities in the continuous infusion trials were mild, except for a rare
patient with myalgias and bradycardia (22). These data have suggested that the
use of retroviral modification for chemoprotection will be an important
approach to therapy.
The laboratory of Xx. Xxx of M.D. Xxxxxxxx have shown that the presence of
MDR-1 in human cells and the presence of MDR-3 in mouse cells leads to
protection of the cells from the toxic effects of chemotherapy. Several groups
have used retroviruses to introduce the MDR cDNA into the bone marrow cells
and have shown that this confers upon these cells resistance to daunomycin
-6-
and protects these cells to myelosuppression (35-38). It is clear that the MDR
is expressed in the most primitive hematopoietic precursor cells (1), and that
the level of expression is normally reduced at later stages of myeloid
maturation. Unregulated expression of the MDR cDNA at all stages of myeloid
and erythroid and megakaryocytoid maturation protects against
chemotherapy-induced myelosuppression (39). In fact, transgenic animal model
studies show that the presence of the MDR protects the hematopoietic cells of
the mice from doses of drugs which are 10-fold from those which usually
suppress hematopoiesis (39). Since the primary dose-limiting toxicity of the
MDR drugs is myelosuppression, the use of the genetic modification
chemoprotection strategy is a strategy of great potential importance to breast
cancer patients.
PREVIOUS WORK WITH ANIMAL MODELS WHICH INVOLVES INTRODUCTION OF RESISTANCE
GENES INTO NORMAL HEMATOPOIETIC GENES:
As outlined above, we will use the mouse animal model to test strategies
for the introduction of cDNAs for MDR chemotherapy into the normal marrow
early progenitor cells of a mouse following transplant. I will now review what
experience has been developed for introduction of retroviral vectors carrying
resistance genes into normal marrow cells as part of bone marrow
transplantation.
MOUSE MODEL FOR GENETIC CHEMOPROTECTION:
Xxxxxx Bank of Columbia University in New York City has attempted genetic
modification of mice using several resistance genes (51). The protocol he has
chosen resembles that previously developed by Xxxxxxx Xxxxx of Memorial
Xxxxx-Xxxxxxxxx Cancer Center. On day 1, 5FU is administered in order to
destroy the late progenitor cells which do not contribute to the
reconstituting capability of the marrow. On day 3, an engrafting dose of
marrow is harvested. The marrow is cocultivated with viral producer cell
lines. The producer cell lines are the triply mutated cell lines in which the
probability of reconstituting a replication competent virus is very low. In
fact, no replication competent helper virus has been detected with these cell
lines in over 250 different laboratories which have used these cell lines. On
day 5, the cells are placed in two mg/ml of G418. On day 6, these cells are
transplanted intravenously into a mouse which has received 1100 rads of
radiotherapy.
In order to assess the percentage of cells which are modified, and to
establish if the earliest cells have been modified by the virus, the animal is
subjected to a laparotomy, and the spleen colonies are collected sterilely,
and transplanted into a new mouse which has been exposed to 1100 rads of total
body irradiation. These spleen colonies are also taken for culture in
methylcellulose and are also analyzed directly for the presence of MDR mRNA
and protein by PCR and FACS analysis.
This cycle is repeated once more and the spleen colonies are harvested and
analyzed for the presence of MDR mRNA and protein. This protocol can be used
for the generation of genetically modified mice. However, several parameters
need to be examined in order to optimize the transduction frequency and the
stability of the retroviral transgenome in the recipient mice.
-7-
DOG MODEL DATA FOR GENETIC MODIFICATION OF NORMAL HEMATOPOIETIC CELLS:
Xxxxxx Xxxxx of the Xxxx Xxxxxxxxxx Cancer Center used Ficoll hypaque
density gradient separated cells and co-cultivated them in a 2/1 ratio with
irradiated producer cell lines (52). CFUGM assay in selective media (G418 for
neo and methotrexate for DHFR) were 6% and 12%, respectively. However, five
weeks following transplant, only 2-5 % of CFUGM were viral genome positive,
whereas at 10 weeks, none of the 2/6 colonies studied were positive,
suggesting that only the late myeloid progenitors had been modified.
Transduction protocols which utilized long-term bone marrow culture as an
element of the transduction system showed an increase of the level of
positivity to 43%. This program was organized as follows: on day -12, the dogs
were given therapy with GCSF in combination with kit ligand for seven days. On
day 5, the cells were harvested and co-cultivated in a Dexter culture in the
presence of the vector supernatant. Fresh vector positive medium was added on
day 2. On day 0, 9.2 xxxx of TBI was administered followed by infusion of the
transduced marrow.
A third protocol involved administration of cyclophosphamide at a dose of
40 mg/kg I.V. on day -8, marrow aspiration and co-cultivation of the Ficoll
hypaque separated marrow for 24 hours on vector producer cell lines for 24
hours, and transplantation following delivery of 9.2 xxxx of TBI to the
animals on day 0. Between 1-50% of the cells were seen to be positive 50-100
weeks following transplantation when the neo gene was used. No growth factors
or preselection were used. Xx. Xxxxx has indicated that only 25% of the marrow
bearing areas needed to be irradiated, to provide space for the engrafting
marrow. Both of these investigators were using safety modified retroviruses
but were using growth factors and co-cultivation intermittently (52).
MOUSE AND PRIMATE MODELS FOR TRANSFER OF MDR TO NORMAL HEMATOPOIETIC CELLS:
Xxxxx Xxxxxx and Xxxxxx Xxxxxxxx of the NIH used a safety modified
retrovirus with the packaging cell lines of Xxxxxx Bank to modify the normal
marrow cells of mice, using a protocol similar to that described above in the
work of Dr. Bank (53). Forty-eight hours following 5FU pretreatment, bone
marrow was harvested and modified by incubation in the supernatant of a Bank
producer cell lines which produced virus containing Xxxxxxxxx'x MDR cDNA.
Following transplantation of the modified cells, Xxxxxx and Xxxxxxxx
administered Taxol intraperitoneally at high doses to select for growth of the
modified marrow cells. The marrow cells of the mice were still positive at
eight months after transplant for MDR. These workers could not establish a
dose and schedule of Taxol which would have permitted them to gradually select
for MDR positive marrow cells due to toxicity and treatment deaths generated
in the mice by the treatment schedule they used.
GENETIC THERAPY OF BLADDER CANCER:
Carcinoma of the bladder affects 50,000 individuals every year.
Eventually, two-thirds of these individuals will relapse. Once relapse occurs,
chemotherapy and cystectomy are required.
-8-
Molecular analysis of the primary bladder cancer cells at diagnosis shows that
absence of the retinoblastoma (RB) protein in the primary bladder cancer is
predictive of relapse. The survival of bladder cancer patients whose initial
tumor is negative for the RB protein is 18%, and the survival of patients
whose cells are positive for the RB protein is 55% in at least two studies
(Cordon Cardo et al at Memorial Xxxxx-Xxxxxxxxx and Xxxxxxxxxx et al at the
U.T. M.D. Xxxxxxxx Cancer Center, JNCI, 1992). This data has suggested that
replacement of the RB might be of therapeutic value and reduce the probability
of recurrence.
Later on in this grant request, we are proposing to use safety-modified
retroviruses which contain the RB cDNA in a transcription unit. Since the
normal cells on the luminal surface of the bladder are not proliferating but
cancer cells which contribute to relapse are dividing, it is possible that the
RB virus would modify the cancer cells and not the normal luminal cells in the
bladder. This program could result in a decreased incidence of recurrence and
ultimately to organ preservation (no cystectomy). This proposed work will be
described later on in this grant.
CERVICAL CANCER:
Carcinoma of the cervix is a public health problem of world-wide
dimensions. Despite introduction of effective screening procedures, the
development of surgically resectable carcinoma of the cervix occurs all too
frequently. It is estimated that the frequency of frankly invasive carcinoma
of the cervix following resection of carcinoma in situ, or frankly invasive
carcinoma of the cervix which is unresectable in individuals who do not
participate in follow-up pap smear screening following detection of dysplasia
at initial pap smear screening, is estimated at 20,000 cases/year in the
United States. In principle, all of these cases are preventable if the
recurrence rate after diagnosis of initial dysplasia could be reduced to zero.
Carcinoma of the cervix is a disease in which a viral etiological agent
has been established as the human papilloma virus. The mechanism through which
this virus transforms cells is attributed to the production of two virally
transforming proteins, E6 and E7, which bind and functionally inactivate the
p53 and retinoblastoma antioncogene tumor suppressor proteins. Normally,
functioning p53 and Rb restricts the precession of the normal cells through
the G1/S interface in the absence of extracellular growth stimulatory
proteins. Although the exposure of cells to growth factors will increase the
levels of both the p53 and Rb proteins, the regulatory environment of the
proliferating cell functionally inactivates the Rb and p53 antioncogenes, thus
relieving the cell from their growth inhibitory influence.
The presence of the E6 and E7 HPG proteins produces the same result as the
growth factor exposure as they inactivate the Rb and p53 proteins, thus
resulting in disorganized and unregulated cell growth.
In an attempt to develop a molecular approach to the prevention of
recurrence in cervical carcinoma, we are proposing to construct or acquire
safety-modified retroviruses with cDNAs for the normal p53 and Rb antioncogene
proteins. These viruses will be applied topically to the abnormal surface of
the dysplastic cervix to prevent recurrence. Patients eligible for this
program
-9-
will be those not approachable with case resection. The cell-free retroviral
supernatant will be applied weekly for one year in eligible patients whose
disease has been shown to be HPG positive. This program will be conducted with
the U.T. M.D. Xxxxxxxx surgical GYN Oncology group in collaboration with Xx.
Xxxxxxxxxx.
C. PRELIMINARY DATA FROM OUR OWN LABORATORY
(I) LEVELS OF MDR IN MOUSE MARROW CELLS AND IN BREAST CANCER CELL LINES (X.
XXXXXXX, X. XXXXX, AND X. XXXXXXXXXX)
In order to work out the genetic modification of early hematopoietic cells
and to optimize the conditions under which these genetically modified cells
can be selected in vivo with conventional dose chemotherapy, we are proposing
to work with a mouse transplantation model which is described in this section.
This section will describe the insertion of a multidrug resistance (MDR) cDNA
vector into the early progenitor cells of the mouse.
These are two MDR genes in human cells: MDR-1, which confers upon cells
the expression of an efflux pump which reduces intracellular levels of the
following chemotherapeutic agents: actinomycin D, VP-16, vinblastine,
vincristine, anthracyclines and the taxol family of drugs. This pump was
discovered in early hematopoietic progenitor xxxXx by its ability to pump
rhodamine out of cells (1). The presence of this enzyme in these early cells
suggests that it is playing a role in the protection of these cells from the
effects of toxic compounds such as chemotherapeutic agents, as has been shown
to be the case. MDR-2 does not confer upon the cell an efflux pump. The MDR-1
in man corresponds to MDR-3 in the mouse and glycoprotein 1 in the hamster,
while MDR-2 in man corresponds to MDR-2 in the mouse and glycoprotein 2 in the
hamster. Xxxxxxxxx has shown that the microinjection of the MDR3 into mouse
blastocytes generate mice in which the resistance to chemotherapy is above
present in the marrow stem cell of the normal mouse (39).
We conducted experiments to establish if normal hematopoietic cells of the
mouse are more sensitive to chemotherapy than the established breast cancer
cell lines. If the MDR levels in the mouse breast cancer were equal to that
present in the hematopoietic cells, then the delivery of the chemotherapy
based on MDR drugs would kill the marrow cells to a degree which is equal to
that seen with the breast cancer cells. In contrast, if the marrow cells were
modified with the MDR cDNA, then the level of resistance of these cells might
be elevated to a level above that which is present in the breast cancer cell.
Then the killing of breast cancer cells would be greater than that seen in the
normal marrow cells. The fact that MDR resistance has not been reported to
play a major role in breast cancer suggests that the introduction of MDR genes
into hematopoietic cells may be of value therapeutically, since if the breast
cancer cell lines were very high with respect to the MDR phenotype, then there
would be very little benefit from modifying the normal marrow cells. In fact,
the incidence of MDR resistance in untreated breast or ovarian cells is very
rare, thus suggesting that the transduction with MDR cDNA containing viruses
would be of help.
In order to test these concepts, we exposed the normal marrow cells, or
breast cancer cell lines from man or mouse to the effects of various
concentrations of adriamycin. As can be seen in
-10-
Figure 1 or in Table III, the breast cancer cell lines vary in their
propensity to grow in the different concentrations of adriamycin. For these
experiments, we used 24 well plates and exposed the cells to the various
concentrations of the cells, and determined the number of colonies which grew
in each well in two weeks in the presence and absence of the drug. The percent
cell growth was obtained by calculating the ratio of the cell colony number in
the presence of the drug divided by the colony number in the absence of the
drug.
As seen in Figure 2, there are two cell lines which are more resistant
than the rest of the cell lines and more resistant than the normal bone marrow
cell lines. However, most of the established cell lines do not exhibit any
high levels of MDR, as judged by chemotherapy resistance, than do normal
cells.
In order to directly test if the varying levels of resistance and
sensitivity were attributable to the presence or absence of the MDR mRNA in
the cells which are resistance or sensitive, Xx. Xxxxxxx Xxxxxxxx of our group
used PCR to determine if there was MDR cDNA in the resistant but not sensitive
cells. As shown in lanes 8 and 9 of Figure 3, the PCR assay detected the
presence of the MDR specific signal in the PCR assay in the two cell lines
which were the most resistant to the chemotherapy, whereas all of the rest of
the breast cancer cell lines and the normal marrow were devoid of detectable
levels of MDR mRNA. This data suggests that the use of the MDR viruses will be
helpful in protecting the normal hematopoietic cells from the effects of
chemotherapy.
We have developed vectors in our laboratory which are safety-modified and
which contain a transcription unit competent to promote the expression of
functional levels of MDR-1 sufficient to confer chemotherapy resistance upon
hematopoietic cells following transplantation with marrow cells which have
been transduced with these viruses.
In order to evaluate these viruses, we harvested marrow cells from BALB C
mice 48 hours following treatment with 150 mg/kg of 5-Fluorouracil. The marrow
was transduced with the supernatant from producer cell lines which contained
our own laboratory MDR-1 virus. Some animals were transduced with a virus
constructed by Xxxxxxx Xxxxxxxxx. The latter virus has been published. 0.8 to
1 million of these cells were infused into each mouse following treatment of
that mouse with total body irradiation.
Mice were given chemotherapy following hematopoietic recovery (this
requires 2-3 weeks). The chemotherapy was Taxol, freshly reconstituted at 7,
10, 14, 20, and 30 mg/kg.
The time course of recovery from Taxol-induced myelosuppression was
measured following two or three doses of chemotherapy. The data from these
experiments are shown in Figure 3 in which the following experimental groups
are outlined:
Marrow containing Xxxxxxxxx virus following:
Curve 1: Two courses of 10 mg/kg Taxol;
Curve 2: Three courses of 7 mg/kg Taxol;
-11-
Curve 3: Three courses of 14 mg/kg Taxol;
Marrow containing our laboratory virus following:
Curve 4: Three courses of 14 mg/kg Taxol;
Curve 5: Three courses of 7 mg/kg Taxol;
Curve 6: Two courses of 10 mg/kg Taxol;
Marrow containing no virus following:
Curve 7: One course of 10 mg/kg Taxol;
Curve 8: One course of 14 mg/kg Taxol;
Curve 9: One course of 20 mg/kg Taxol;
Curve 10: One course of 20 mg/kg Taxol.
The mice died following 20 and 30 mg/kg without virus.
This data shows a faster recovery and a reduction in the degree of
myelosuppression following virus when given our laboratory virus and two or
three courses of Taxol at 10 and 14 mg/kg. The degree of protection given by
the Xxxxxxxxx virus was similar to that observed with our own laboratory
virus.
D. WORK PROPOSED
SPECIFIC AIMS 1-2: CONSTRUCTION AND DEVELOPMENT OF CLINICAL GRADE RETROVIRUSES
FOR INTRODUCTION OF MDR cDNA IN A SAFETY-MODIFIED VIRUS FOR OVARIAN CANCER
(PROJECT 1A)
We are working in collaboration with Ingenex, Inc. and Targeted Genetics,
Inc. of Seattle, Washington to develop these programs. We have obtained the
MDR cDNA from Xxxxxxx Xxxxxxxxx to produce vectors which can be developed into
a clinical grade reagent for testing in clinical programs for treatment of
carcinomas of the breast, ovary, and trophoblastic disease of pregnancy
(Projects 1B and 1C).
We have obtained the MDR producer cell line used by Xxxxxxx Xxxxxxxxx and
X. Xxxxxx in the mouse studies which he has conducted and has published
(58-59). The Xxxxxxxxx plasmid was digested to release the MDR fragment. The
MDR-1 cDNA was then introduced into the HyTK vector from Targeted Genetics.
The producer cell lines for the MDR-1 virus will produce titres of virus
which are suitable for the transduction experiments (at least 100,000 pfu/cc)
and which are replication incompetent. In order to test the potential for the
virus to modify the chemotherapy sensitivity of the human target cells, we
will incubate the supernatant of the virus in the presence of the CD34 cells
isolated from normal individuals or individuals who do not have any evidence
of involvement of the marrow with
-12-
neoplastic cells. The CD34 positive cells will be isolated with CellPro
columns. These cells will be subjected to transduction by the viral
transduction protocol which is summarized in Table IV. This protocol has been
shown by our laboratory and our collaborators to generate levels of
transduction of 60% of the earlier progenitor cells (60).
We will analyze the cells for the integration of the retrovirus by the PCR
assays which are specific for the MDR cDNA. These assays will be used to
identify the presence of the retroviruses in the normal cells. The PCR assay
for the housekeeping sequence actin will be used as an internal control for
these PCR assays. As shown in Figures 4-5, my laboratory has used these assays
in combination to analyze colonies of cells which contain 40 cells (61-62).
Thus, we can identify the integrants bearing the functional retroviral
transgenome in the normal population.
The functional change on the phenotype of the transduced marrow cell will
be tested by the chemotherapy sensitivity assays. Xx. Xxxxxxx Xxxxxxxx and Xx.
Xxxxxxxx Xxxxx will assist us with the implementation of these assays. The
cells will be incubated in a concentration series of chemotherapy agents, some
of which are MDR drugs, and some of which are not MDR drugs. Cells before and
after transduction will be tested. The LD 50 will be determined for the cells.
Colony counts will be determined for these two populations as well. Functional
assays of the transduced cells will also be carried out to show the efflux of
the MDR drugs is greater in the cells in which introduction of the MDR drug
has occurred. FACS analysis will also be used for this analysis. Thus, the
relative transduction and chemotherapy resistance of the transduced and
untransduced cells will be determined. When functional resistance to MDR drugs
such as Taxol is established, we will proceed to the production of the
clinical grade retrovirus, as outlined in Table I for time lines for this
program.
SPECIFIC AIMS 3-4: STUDY OF THE TRANSPLANTATION MODEL IN A MOUSE MODEL
USING MOUSE CELLS
The mouse model will be used by X. Xxxxxxx of my laboratory to analyze
chemotherapy resistance of the marrow cells before and after transduction. Xx.
Xxxxxxx is a Ph.D. who received his degree for a project focused on the use of
a mouse model for characterizing molecular changes in the evolution of breast
cancer. Xx. Xxxxx has extensive experience with the mouse transplantation and
chemotherapy model. Marrow cells will be isolated from mice 48 hours following
administration of 5-FU. The 5-FU is administered to ablate the late myeloid
precursors from the marrow and to permit the isolation of the early
progenitors from the marrow. The marrow from four mice will be exposed to the
supernatant of the PA317-MDR producer cell line following the protocol
outlined in Table IV. The cells will then be incubated for 48 hours in
non-selective medium before imposition of selection.
The transduced cells of four 5-FU treated mice which survive 48 hours of
the selection in suspension culture will be transplanted into the one mouse
which has been exposed to 1100 rads of irradiation. We will sacrifice some
animals at day 12 and analyze the spleen colonies for the presence of cells
which are positive for the MDR retrovirus. We will explant the cells in
methylcellulose assay, pick colonies at day 14 and analyze the cells by PCR
assay for the presence
-13-
of the MDR retrovirus. We have already evaluated our ability to transduce the
cells from mouse marrow with the safety modified MDR virus.
Mice will be treated with 5-FU to destroy the mature marrow stem cells,
which will be collected from the mouse 48 hours following the exposure to the
drug. The femurs and the tibia from 4 mice so treated will be removed and
flushed with PBS to remove the marrow stem cells and placed immediately into
DMEM and 15% fetal calf serum.
The cells will be modified with the Gla MDR vector, into which has been
placed the human MDR cDNA of Xxxxxxxxx. The 5-FU treated cells will be infused
into a single mouse immediately following completion of 1100 rads of TBI. The
mice will be followed for hematopoietic recovery, and the appearance of the
MDR cDNA and the retroviral transgenome using PCR analysis of the peripheral
blood cells of the mouse, obtained by tail vein phlebotomy. In vitro selection
can be used before infusion to increase the percentage of cells which are
modified and expressing the retroviral transgenome, but in this event,
increased numbers of cells must be collected by treating additional numbers of
mice with the 5-FU. These mice will be followed immediately following
hematopoietic recovery, and at three weekly intervals following
transplantation, in order to determine the percentage of cells in each lineage
which express the retroviral transgenome. These mice will be followed with and
without administration of low dose chemotherapy so as to monitor and establish
the effect of the in vivo selection with chemotherapy on the persistence of
the modified cells. The doses of chemotherapy (doxorubicin), which will not
ablate the transplanted marrow but will result in selection, will be
determined empirically.
-14-
SPECIFIC AIMS 5-6:
PROJECT 1B
"DEVELOPMENT OF CLINICAL PROGRAM FOR CREMOPROTECTION OF
OVARIAN CANCER AND TROPHOBLASTIC XXXXX"
-00-
XXXXXXXX
Patients with poor prognosis ovarian cancer (epithelial and germ cell
disease) have been shown to have an 80% probability of relapse. In addition,
the therapies available frequently have a cumulative suppressive effect on the
normal early progenitor cells of the marrow. The results in longer and longer
intervals between successive of chemotherapy due to reduce numbers of
progenitor cells. This results in successive prolongation in the time required
for hematopoietic recovery before successive cycles of chemotherapy can be
administered.`A similar problem is encountered in the patients who do not
respond to therapy for trophoblastic disease of pregnancy. This results in a
situation in which the neoplastic population has longer and longer periods of
time to recover, and the marrow stem cells have fewer and fewer numbers of
cells with which to reconstitute the marrow. This prolongation of the
intervals between successive cycles of chemotherapy cannot be reversed by the
growth factors GMCSF or GCSF. This is due to the fact that the slow
hematopoietic recovery is not due to fewer numbers of late progenitors, which
are.sensitive to the late myeloid growth factors, like GMCSF, but is due to
the loss of the early progenitor cells. Thus, these patients are ideal for the
chemoprotective therapy. The proposed program is designed to apply safety
modified retroviruses to the genetic modification of normal stem cells of
patients with ovarian cancer and trophoblastic disease of pregnancy for
chemoprotection.
-16-
PROGRAM IN OVARIAN CANCER
A. SPECIFIC AIMS:
MDR drugs like Taxol show significant activity in refractory ovarian
cancer but are dose limited by neutropenic myelosuppression.(1,2,3,4) A dose
response phenomena is probably present and the drug is known to be extruded
from cells by the MDR1 transporter (P-Glycoprotein). Mice transfected with MDR
show a quantitative increase in MDR positive stem cells and gene copies per
cell after exposure to Taxol.(20) Thus increasing the presence of MDR in a
patient's stem cells may allow greater dose intensity with Taxol treatment.
1.1 To determine the efficacy of autologous bone marrow transfected
with MDR to prevent myelosuppression in patients with epithelial
ovarian cancer treated with high dose Taxol.
1.2 To assess the safety and toxicities of this therapy.
1.3 To study the efficiency of the gene transfer to the autologous
marrow.
B-C. BACKGROIUND AND PRELIMINARY DATA:
Systemic chemotherapy yields a significant number of objective responses
in patients with advanced ovarian cancer (FIGO stages III and IV). Response
rates greater than 30% have been reported for the following single agents:
melphalan, chlorambucil, thiotepa, cyclophosphamide, and cisplatin.(6-12)
Methotrexate and adriamycin appear to have activity of a lesser degree. (13,
14) Combination chemotherapy has been reported to have increased response
rates. The combination of hexamethylmelamine, cytoxan, methotrexate, and
5-fluorouracil (hexa-CAF) results in response rates of 42% to 75%. (15,16) The
use of cisplatin as part of a chemotherapy combination has had significant
additive effect. One investigator has reported a 67% response rate with
cytoxan, hexamethylmelamine, and adriamycin. The complete response rate was
44% and median survival has not been reached at 23.5 months. (17)
At the University of Texas M.D. Xxxxxxxx Cancer Center, the
combinationi of cisplatin plus melphalan has produced a remission rate of
approximately 55%.(19) Complete responders had an increased survival and a
small percentage of patients entered into an unmaintained-complete remission.
However, some of the patients never achieve complete remission or suffer
relapse. Furthermore, median survival among nonresponders and partial
responders even with effective combination regimens is only 7-16 months.
(15-17) Thus, there is a need to investigate chemotherapeutic agents with the
aim of increasing the duration and number of complete responses.
Carboplatin, a recently released analogue with different toxicities than
its parent drug, offers an equivalent alternative to the treatment of ovarian
cancer. It is also useful as a reinduction agent for prior platin resporders.
However, it does not resolve the problem of refractory patients or
-17-
significantly improve long term survival.(18-19) The problem of cisplatin or
carboplatin resistant patients is particularly acute.
Patients progressing on cisplatin therapy have median survivals of 6-8
months. Patients with macroscopic residual disease at restaging laparotomy
after cisplatin based therapy have median survivals of approximately 12 months
with no proven salvage therapy.(20)
In patients who develop recurrent and/or resistant disease after cisplatin
containing chemotherapy, the probability of obtaining significant therapeutic
results has traditionally been quite limited.(21) Cisplatin itself has been
used at high dosages (200 mg/m2 or 1 mg/kg/week) as a second- line treatment
in ovarian cancer.(22-25) However, this approach has presented two relevant
limitations: (a) the treatment toxicity, in particular peripheral
neuropathies, has been more severe than usually seen with cisplatin, due to
increased dosages and prolonged prior exposure to the compound; and (b) the
efficacy has been significant only in patients who had not previously
developed resistance to therapy containing cisplatin at standard doses.
A significant antitumor activity in previously treated ovarian cancer has
recently been reported with taxol. A natural product obtained from taxus
brevifolia (26), the compound has had a relatively limited clinical
development due to the scarcity of clinical supplies. After objective
responses have been observed in patients with ovarian cancer receiving taxol
during phase I clinical trials, three independent phase II studies have
confirmed the single agent activity of the compound.(2,3,4) Of particular
intefest, objective responses have been observed in patients who failed to
respond to (or had a rapid relapse after) cisplatin containing chemotherapy.
There has been no clearly established dose response relationship of Taxol.
(1,27) The main toxicities of taxol are myelosuppression, neuropathy and
allergic reactions. Previous trials have utilized doses of >=175 mg/m2.
Analysis of patients at our institution treated at fixed doses of 135 mg/m2
reveal only 1 partial response of 33 patients. Neutropenia requiring G-CSF is
still dose limiting. G-CSF lessens the duration and degree but does not
eliminate neutropenia (Product Monograph).
1.0 Drug Information
1.1 XXXXX, XXX 000000
1.1.1. Chemistry
Taxol is a diterpene plant product derived from Taxus brevifolia
(western yew). Chemical name: Xxx-xx-xx-x-xxx, 0, 00-xxxxx-0, 2, 4, 7,
10, 13 -hexahydroxy-l, 4, 10-diacetate-2-benzoate-13- (
-phenyl-hippurate). Molecular formula: C47H51NO14. Molecular weight:
853.9.
1.1.2 Molecular structure:
[diagram of molecular structure of Taxol]
-18-
1.1.3 Mechanism of Action:
Taxol has a unique mechanism of action in that rather than inhibiting
tubulin polymerization, it markedly enhances the reaction, and
microtubules formed in the presence of taxol are unusually stable.
Taxal blocks cell replication in HeLa cells, mainly during mitosis.
(13) Studies with purified microtubule protein have demonstrated that
taxol promotes the assembly of tubulin into calcium-stable
microtubules in vitro in the presence or absence of GTP or
microtubule-associated proteins.(13,15,18) Taxol binds directly to
polymerized tubulin (17,18) with saturation occurring at approximate
stoichiometry with tubulin dimer concentration. These activities of
taxol are in contrast to other known mitotic spindle poisons such as
colchicine, podophyllotoxin, and the vinca alkaloids which inhibit
microtubule formation.
Animal Tumor Data:
Taxol has demonstrated good antitumor activity against the murine i.p.
implanted L1210 and P388 leukemias and also CX-1 colon and LX-1 lung
xenografts. Taxol was not effective against the murine s.c. implanted
CDF1 mammary and colon 38 carcinomas and the i.v. implanted Xxxxx lung
carcinoma. The effect of treatment schedule on the antitumor activity
of taxol was evaluated in the i.p. implanted P388 leukemia system.
Neither toxicity nor activity was observed following oral daily
treatment, or the i.v. single or intermittent and daily
administrations. Intraperitoneal administration of several doses in
one day appeared to be more effective than a single drug injection per
day.(12)
1.1.4 Animal Toxicity:
Preclinical toxicity studies have been conducted with taxol by
intraperitoneal administration in CD2F1 mice (five daily i.p doses)
and by intravenous infusion in beagle dogs (single and daily i.v.
doses).
The main toxic effects of taxol were most evident in the tissues with
high cell turnover: e.g. lymphatic, hematopoietic, gastrointestinal,
and reproductive organs.
Drug-related lymphoid depletion was dose-related and reversible, with
the exception of mandibular lymph node inflammations (rat) and
tonsillitis (dogs), which were secondary to taxol-induced immune
suppression.
Hematopoietic toxicity was evident in all 3 species, although the cell
lines affected and severity varied among species. Dogs showed anemia,
reticulocytopenia, thrombocytopenia, and leukopenia in the lethal and
toxic dose high range. Effects at the toxic dose low and highest
non-toxic dose range were minimal and readily
-19-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
reversible. All drug-related lesions reported from bone marrow
evaluations were reversible.
Considerable toxicity to the gasstrointestinal system was seen
following taxol administration. Clinical sings included weight loss,
bloody diarrhea, anorexia, adipsia, emesis, and mucoid stools.
Histological findings in mice and rats included atypical hyperplasia
in the crypts of Lieberkuhn and duadenal lesions and inflammation, all
of which were reversible. In dogs, there was congestion of the ileum,
cecum, and colon, chronic inflammation of the duodenum and
inflammation and necrosis of the colon.
No histological evidence of drug or vehicle-related toxicity to the
central or peripheral nervous system was seen in any species. Clinical
signs suggestive of central effects were noted including lethargy,
tachypnea, ataxia, lacrimation, salivation, hypothermia, nystagmus,
and mydriasis.
Effects on other systems (e.g. cardiovascular, renal, and endocrine)
were relatively minor, with no histological confirmation of toxicities
suggested by clinical signs or clinical pathology.
1.1.5 Human toxicology:
Several phase I studies of taxol have been completed. Dose schedules
included daily x 5 by bolus i.v. injection where MTD was 30 mg/m2/day
and the dose limiting toxicity was myelosuppression, mainly
neutropenia.(19) In a study of one continuous infusion i.v. schedule
MTD was 200 mg/m2 and dose limiting toxicity was leukopenia.(20) A
third study was discontinued at a dose of 230 mg/m2 x dl by 3 hour
infusion due to occurrence of fatal anaphylactic reaction. Two other
patients experienced transient allergic reaction at 190 mg/m2 dose
i.v..(21) Anaphylactic reaction in 2 patients was also observed with
daily x 5 i.v. schedule every 4 weeks from which they recovered.(22)
These allergic reactions as well as occurrence of skin rashes are
attributed to the vehicle cremaphor EL. A total of 12% of patients
have experienced this side effect. None of these allergic reactions
occurred in patients receiving continuous infusion i.v. schedule with
the proposed premedication schedule.
Other toxicities included alopecia, nausea, vomiting, and stomatitis.
[ ***
]
-20-
The following four pages have been omitted pursuant to a request for
confidential treatment. A complete copy of this exhibit containing the omitted
portions has been separately filed with the Securities and Exchange
Commission.
-21-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
[ ***
]
F. VERTEBRATE ANIMALS: NONE
G. CONSOLTANTS/COLLABORATORS: NONE
H. CONSORTIUM/CONTRACTUAL ARRANGEMENTS: NONE
I. LITERATURE CITED:
22 Wiernick PH, et al: Phase I clinical and pharmacokinetic study of taxol.
Cancer Res 47:2486, 1987.
-22-
23 XxXxxxx XX, et al: A unique antineoplastic agent with significant activity
in advanced ovarian epithelial neoplasms. Xxx Intern Med 111:273, 1989.
00 Xxxxxx XX, et al: Phase II study of taxol in patients with advanced
ovarian cancer. Proc Am Assoc Clin Oncol 8:158, 1989.
25 Xxxxxxx T, et al: Phase II trial of taxol as second-line therapy for
ovarian carcinoma: a Gynecologic Oncology Group Study. Proc Am Asso Clin
Oncol 9:156, 1990.
26 Xxxxxxxxxx XX, Xxxxxx S, Xxxxxxxxx M, Pastan I, Xxxxxx D, and Xxxxxxxx XX:
Positive election in vivo for hematopoietic cells expressing the multidrug
resistance gene following retroviral mediated gene transfer. Blood Vol
78(10):191, Suppl 1, 1991.
27 Xxxxx XX: Gynecologic malignancies. IN: Cancer Chemotherapy, (ed, XX
Xxxxxx), Elsevier, New York, Annual I, pp 340-451, 1979.
28 Xxxxx MS, et al: Melphalan chemotherapy in advanced ovarian cancer. Obstet
and Gynecol 51;352, 1978.
29 Xxxxx XX, et al: Chemotherapy in the treatment of cancer of the ovary. Am
J Obstet Gynecol 107:691, 1978.
30 Xxxxxx XX, et al: Cyclophosphamide in the treatment of ovarian cancer.
Clin Obstet Gynecol 11:382, 1968.
31 Xxxxx XX: Treatment of ovarian cancer. IN: Advances in Chemotherapy. (eds,
Umezawa N) Xxxxxxxxx Xxxxxxxxxx Xxxx Xxxxx, Xxxxxxxx, pp 493-503, 1978.
00 Xxxxx XX, et al: Cancer of the ovary: survival studies based upon
operative therapy, chemotherapy and radiotherapy. Am J Obstet Gynecol
108:878, 1970.
33 Xxxxxxxxx XX, et al: The role of chemotherapy in the treatment of
gynecologic malignancy. Am J Obstet Gynecol 93:1102, 1965.
34 Xxxxxxxxx XX: Thiotepa and methotrexate therapy of advanced ovarian
cancer. J Mt Sinai Hosp. 35:52-67, 1968.
35 Xxxxx G, et al: Adriamycin in ovarian cancer patients resistant to
cyclophosphamide. Eur J Cancer 14:1401, 1978.
00 Xxxxx X,X et al: Advanced ovarian adenocarcinoma: A prospective clinical
trial of melphalan (L-XXX) vs combination chemotherapy. NEJM 299:1261,
1978.
-23-
37 XxXxxx G, et al: Prospective study with Hexa-CAF combination in ovarian
carcinoma. Cancer Chemother and Pharmacol 5:157, 1981.
38 Xxxxx XX, et al: Cyclophosphamide, hexamethylmelamine, adriamycin, and
diamminedichloroplatinum - "XXXX" vs Melphalan for advanced ovarian cancer
- A randomized prospective trial of the eastern cooperative group. ASCO
(abst. #C-548), 1981.
39 Xxxxxxx D, et al: Carboplatin in the treatment of ovarian cancer. Sem
Oncol 16(5):19, 1989.
40 Xxxxxxxx J, et al: Carboplatin in refractory epithelial ovarian cancer.
Sem Oncol 16(2):1, 1989.
41 Berek J: Epithelial ovarian cancer. IN: Practical Gynecologic Oncology,
(ed, J Berek and N Hacker), Xxxxxxxx and Xxxxxxx, Baltimore, p. 352-55,
1989.
42 Xxxxxxx XX, et al: Developing new drugs for ovarian cancer: a challenging
task in a changing reality. J Cancer Res Clin Oncol 107:111, 1984.
43 Xxxxx XX, et al: High-dose cisplatin in hypertonic saline in refractory
ovarian cancer. J Clin Oncol 3:1246, 1985.
44 Xxxxx MS, et al: Effective weekly cis-dichloro diammine platinum (CDDP) as
third line chemotherapy in ovarian carcinoma. Proc Am Soc Clin Oncol
22:467, 1981.
45 Xxxxx MS, et al: Cis-diammine dichloroplatinum (II): Secondline induction
chemotherapy in advanced ovarian adenocarcinoma. J Surg Oncol 24:323,
1983.
46 Xxxxxxxxxx XX, et al: Re-treatment of patients with recurrent epithelial
ovarian cancer with cisplatin-based chemotherapy. Obstet Gynecol 73:798,
1989.
47 Xxxxxx XX, et al: Promotion of microtubule assembly in vitro by taxol.
Nature 22:665, 1979.
48 Donehover RC, et al: Phase I trial of taxol in patients withadvanced
cancer. Cancer Treat Rep 71:1171, 1987.
49 Xxxxx XX, et al: Hydroxyurea-induced inhibition of deoxyribonucleotide
synthesis: studies in intact cells. Cancer Res 27:526, 1967.
50 Krakoff IH: Clinical and pharmacological effects of hydroxyurea. IN:
Xxxxxxxxxx XX, Xxxxx XX (eds): Hanbuch der experimentellen Pharmakologie,
Vol 38/II. Springer, Berlin, pp 789- 792, 1975.
51 Xxxxxxx XX, et al: Evaluation of hydroxyurea (NSC-32065) by parenteral
infusion. Cancer Chemother Rep 49:27, 1965.
-24-
52 Krakoff IH, et al: Phase II studies of hydroxyurea (NSC 43065) in adults:
clinical evaluation. Cancer Chemother Rep 40:53, 1964.
53 Belt RJ, et al: Studies of hydroxyurea administered by continuous
infusion. Cancer 46:455, 1980.
54 Xxxxx D, et al: Phase I study of high-dose hydroxyurea in lung cancer.
Cancer Chemother Pharmacol 21:53, 1988.
55 Xxxxxxxx D, et al: Pharmacokinetics of parenteral hydroxyurea (HU) in rat
and man. Proc Am Asso Cancer Res 32:383, 1990.
56 Xxxxxxxxxxx MS, et al: Long term hydroxyurea intravenous infusion in
patients with advanced cancer: a phase I trial. Proc Am Asso Cancer Res
31:204, 1990.
57 Simon R, et al: Randomized Phase II Clinical Trials, Cancer Treat Rep
69:1375-1381, 1985.
58 Xxxxx XX, et al: Measuring quality of life today: Methodological aspects.
Oncology 4(5):29- 38, 1990.
59 Xxxxxxx WO, et al: Measuring the quality of life of cancer patients: A
concise QL-Index for use by physicians. J Chron Dis 34:585-597, 1981.
60 Simon R: Optimal two-stage designs for phase II clinical trials.
Controlled Clinical Trials. 10:1-10, 1989.
61 Xxxxx XX, Xxxxxxxxx XX, Xxxxxxx PH, et al: Hypersensitivity reactions from
Taxol. J Clin Oncol 8(7):1236-1268, 1990.
62 Xxxxx XX, Xxxxx XX, Xxxxx XX, et al: Phase I trial of high dose, 24 hour
continuous infusion hydroxyurea,. Proc Am Assoc Cancer Res 32:200, abst.
1193, 1991, and personal communication.
63 O'Xxxxx Xxxxxxx: Biometrics 35:549-556, 1979.
-25-
SPECIFIC AIMS 5-6:
PROJECT 1C
CHEMOPROTECTION OF HEMATOPOIETIC CELLS BY
GENETIC THERAPY IN BREAST CANCER
-26-
ABSTRACT
The project outlines a program with curative intent for cancers of the
breast for development of dose intensive chemotherapy followed by infusion of
involving cells which have been genetically modified so as to make them
resistant to chemotherapy.
Dose intensive chemotherapy, designed to take advantage of the steep
dose-response antitumor effects of many chemotherapeutic agents, is well
documented to increase the complete response rates, a prerequisite for cure of
breast cancer. The challenge for the future is to optimally incorporate these
principles in developing more effective, curative treatment strategies. The
strategy of modifying bone marrow stem cells with mdr-1 (Project 1A) or other
genes mediating resistance to chemotherapy, such as glutathione S transferase
(see Project 2) may allow prolonged, outpatient dose intensive chemotherapy to
improve elimination of the malignancy with little risk, toxicity or cost to
the patient.
-27-
A. SPECIFIC AIMS
1. Utilize high dose chemotherapy with autologous bone marrow
transplantation to establish hematopoiesis derived from genetically
engineered bone marrow cells.
2. Use this approach with safety modified retroviruses to transduce
viruses carrying mdr-1 o glutathione S transferase into hematopoietic
cells to aIlow intensive therapy of poor prognosis carcinoma of the
breast.
B-C. BACKGROUND AND PRELIMINARY DATA
Many common cancers such as leukemias, lymphoma, germ cell malignancies
and cancers of the breast and ovary are sensitive to chemotherapy, yet few
cases are cured with conventional dose treatment. These diseases exhibit a
dose dependent response, and dose intensity over time is an important aspect
of therapy (1,2). The dose of antineoplastic agents that can be administered
clinically is limited by toxicity to normal tissues and bone marrow
suppression is the dose limiting toxicity for most agents. Doses can be
substantially escalated to more effective levels if followed by
transplantation of normal hematopoietic cells, collected either from the bone
marrow or peripheral blood, rescuing the patient from severe and prolonged
myelosuppression (2). This method to produce dose intensity is limited,
however, to administradon of one or two courses of treatment which may not be
sufficient to eradicate the malignancy.
An innovative approach to improve dose intensity would be to induce drug
resistance in the normal bone marrow to reduce myelosuppression and allow
prolonged treatment with higher dose chemotherapy. Many drug resistance
mechanisms have been described in vitro, based mostly on the studies using
cultured tumor cells. Different mechanisms of resistance may develop,
depending upon prior exposure to antitumor agents, type of experimental cell
culture system and culture conditions. In clinical situations, the mechanisms
of drug resistance are more difficult to define. The mechanisms found in
cultured cells may not be operative in vivo. Recent success in molecular
cloning of multidrug-resistance (mdr) genes encoding a group of 170- to 190-kD
membrane protein termed P-glycoproteins or multidrug transporters has revealed
a mechanism for detoxifying a wide spectrum of natural products, including
plant alkaloids (vincristine vinblastine), antibiotics (doxorubicin and
actinomycin) and epipodophyllotoxins (teniposide and etoposide). It is
currently believed that P-gp function as efflux pumps by expelling these
cytotoxic substance, thereby reducing the intracellular drug concentrations to
sublethal levels (for reviews, see 49,50). In animal cells, overexpression of
P-gp via a variety of mechanisms including gene amplification and/or
transcriptional activation resulted in the development of the multidrug
resistance phenotype. In humans, there are two MDR genes, MDRl (51) and MDR2
(52). Transfection experiments using cDNA linked to expression vectors
demonstrated that only MDR1 can confer MDR phenotype in otherwise drug
sensitive cells; whereas MDR2 can not. These results suggest a diverse role of
MDR gene family. Using monoclonal antibody C219 which recognizes both MDR1 and
MDR2 gene products in immunohistochemical study, we previously analyzed the
expression of P-gp in sample from 48 patients with locally advanced breast
cancer P-gp was not expressed in eight tumor specimens obtained at the time of
diagnosis, prior to chemotherapy. The percentage of the tumor
-28-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
cell population expressing P-gp varied from <5% to >30%; expression was
observed significantly more often in tumors that showed less than partial
response to the preoperative chemotherapy (53).
Thus, the program in marrow chemoprotection is one which impacts on the
other two areas of suppression of the transformed phenotype and
immunoenhancement. What is the evidence that chemoprotection will work?
Pastan, Xxxxxxxxx and their colleagues have reported that transgenic mice
overexpressing the human MDR1 cDNA, which codes for the 107 kDa glycoprotein
which codes for a drug efflux pump in the plasma membrane of cells, exhibit
normal patterns of hematopoiesis, expression of levels of MDR glycoprotein
which are 3-fold higher than most MDR resistant fresh tumor cells or tumor
cell lines, and exhibit a 10-fold increase in the resistance of hematopoietic
cells to drugs like Taxol (1-4). Retroviral vectors have also been used to
infect mammalian cells and in populations selected by FACS for the modified
cells, the resistance to MDR drugs increased up to 26-fold (5). It is clear,
however, that in vivo or in vitro selection is necessary following retroviral
transduction to maintain the resistant phenotype in mammalian cells. Several
mouse and large animal model studies have also been recently reported (6-8),
which document the feasibility of introduction of resistance genes into human
marrow.
Clinical trials of chemoprotection have been proposed in carcinomas of the
breast and ovary. It is clear, from published studies, that the human MDR1 is
not naturally expressed in normal breast or ovarian tissue, and that all of
the fresh specimens of breast or ovarian cancer do not exhibit elevated levels
of MDR providing they were not previously exposed to chemotherapy (9-11). The
levels of MDR in ovarian and breast cancers following therapy in the advanced
disease setting are elevated by 2 to 3-fold. Thus, the introduction of MDR1
into the normal hematopoietic cells of patients with poor prognosis disease,
would make possible the testing of therapeutic questions that would not be
possible with drugs like Taxol. [ ***
] In our
institutional experience (12-13) and that of others, the toxicity of Taxol is
primarily myelosuppression. The clinical programs proposed for breast, ovary
and a third model, trophoblastic disease of pregnancy, are summarized in
detail in Project 1. The presence of marrow contamination with neoplastic
cells, which is a problem with breast cancer, is not a significant risk in
ovary or trophoblastic disease. The technical details of this program,
including marrow purging before retroviral transduction, will be dealt with in
Project 1.
Studies conducted at MD Xxxxxxxx and elsewhere in patients with leukemia,
lymphoma, metastatic breast cancer and ovarian cancer have documented that
high dose combination chemotherapy and autologous bone marrow transplantation
results in a higher complete response rates compared to standard dose
treatment and a fraction of patients surviving disease free beyond 5 years
(3-9). The most effective regimens have included combinations of agents
primarily limited by myelosuppression such as cyclophosphamide, melphalan,
carmustine, thiotepa, busulfan or
-29-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
carboplatin. [ ***
] The toxic effect of other drugs, such as Taxol and
doxorubicin, will be educed by introduction of MDR-1. We propose to use high
dose chemotherapy with autologous bone marrow transplantation to induce major
antitumor cytoreduction as well as a method to introduce autologous bone
marrow cells transduced with MDR-1 retrovirus [ ***
] to produce hematopoietic resistance to many chemotherapeutic
agents and allow continued higher dose chemotherapy.
Hematopoietic Growth Factors to Accelerate Hematopoietic Regeneration
The proliferation, differentiation and function of hematopoietic cells is
regulated by a complex family of glycoprotein hormones, collectively described
as hematopoietic growth factors. These factors regulate a hierarchy of stem
cells and progenitors (12,13). Granulocyte colony- stimulating factor (G-CSF)
and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been
extensively studied to accelerate hematopoietic recovery of hematopoiesis
after high dose chemotherapy alone or with autologous marrow transplantation.
Granulocyte recovery is significantly accelerated with each factor (14-18).
Erythrocyte and platelet recovery is unaffected by G-CSF and GM-CSF treatment.
Interleukin-6 appears promising to enhance thrombopoiesis and exhibits
synergism with many other hematopoietic growth factors on other lineages.
Interleukin-3 is a multipotential hematopoietic growth factor which has
recently been evaluated in clinical trials. IL-3 affects early and
intermediate myeloid progenitors (12). Treatment with this agent produces only
modest increases in circulating granulocytes and platelets (19,20). There is a
synergistic interaction, however, with IL-3 and GM-CSF leading to a marked
granulocytosis and importantly, a marked increase in the level of circulating
progenitors (21,22). Treatment with a synergistic combination of hematopoietic
growth factors after Taxol, anthrocyclines or other chemotherapy affected by
mdr-1 may enhance the selective growth of mdr-1 transfected hematopoietic
cells.
Minimal Residual Disease
In this project, a patient's bone marrow is collected, transfected with
mdr-1, cryopreserved, and later reinfused after high dose chemotherapy
treatment. A limitation in this approach is the possible involvement of the
bone marrow by the malignancy. Breast cancer, for example, frequently involves
the bone marrow. Standard diagnostic techniques are relatively insensitive to
identify bone marrow metastases. More sensitive assays have been proposed
including cell culture techniques, monoclonal antibody based
immunofluorescence or immunohistochemistry (23-29). The recent development of
molecular methods using polymerase chain reaction may allow more sensitive
detection of subclinical disease. This has been successfully employed for
detection of minimal residual disease in leukemias and lymphomas (30).
Xxxxxxxx et al have recently employed a PCR technique for mucin gene
expression for detection of occult breast cancer cells in the marrow (31).
-30-
In this project we will apply this approach to evaluating marrow involvement
both for prognosis and for assessment of bone marrow purging techniques.
Using monoclonal antibodies directed at cell surface antigens of breast
cancer cells and flow cytometry or immunoperoxidase techniques, many patients
with newly diagnosed clinically stage II breast cancer can be shown to have
occult bone marrow involvement; this finding was associated with a relatively
poor prognosis and a short disease free interval (31,32). Several
investigators have reported a high incidence of occult bone marrow involvement
by metastatic breast cancer in patients with histologically normal bone marrow
biopsies. Up to 28 % of patients with normal marrow biopsies by light
microscopy were positive by immuno-cytochemical methods. Other studies
reported that in 40-57% of similar patients, tumor cells can be grown in
tissue culture (27).
Purging and Stem Cell Selection
The clinical implications of occult bone marrow involvement on the risk of
breast cancer relapse following autologous marrow or peripheral blood cell
transplantation is unknown, but malignant cells capable of forming marrow
metastases may be able to reestablish the malignancy following reinfusion with
the autologous marrow. If drug resistance genes are to be transfected into
marrow, it is critical that malignant cells not be present.
Recent advances in technology may make it possible to eliminate malignant
cells from the autologous marrow/stem cell product. A number of methods have
been proposed, to eliminate or "purge" malignant cells, including treatment
with antitumor monoclonal antibodies, antibody-toxin conjugates, chemotherapy
or physical techniques (33-36). These techniques are capable of a 99 - 99.9%
reduction of target cells (two to three logs), but this degree of depletion is
likely insufficient to completely eliminate all malignant cells.
An alternative method is to positively select hematopoietic stem cells.
CD34 positive cells represent < 1% of the bone marrow but encompass
progenitors capable of reconstituting hematopoiesis (37). These cells can be
selectively separated from the remaining marrow by reactivity with a
biotinylated anti-CD34 monoclonal antibody and adherence to a avidin column,
immunomagnetic separation or by panning (37,38). Each of these methods yields
approximately 1% of the starting cell number, but collecting > 60% of CD34
positive cells. We will be collaborating with CellPro, Inc. to implement this
CD34 selection (see letter). One problem is nonspecific contamination by
CD34-negative cells, which constitute approximately 35-50% of the final
product. This procedure results in approximately a 2 log reduction of
malignant cells. Combination of positive selection of CD34 positive cells with
negative depletion of malignant cells would be expected to have an additive
effect. In addition, the reduction in cell number associated with the positive
selection of stem cells may allow a more effective depletion step because of
the lower number of cells. In this project, we will assess the degree of bone
marrow involvement in patients with early and advanced breast an ovarian
cancer, evaluate methods for marrow purging using immunomagnetic separation,
and assess the effect of positive selection of stem cells and purging on the
elimination of malignant cells from the bone marrow.
-31-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
D. WORK PROPOSED
The clinical implementation of the work will not take place until all
of the purging risks have been resolved and the regulatory approvals outlined
in Table I have been obtained. We propose to utilize high dose chemotherapy
and autologous bone marrow transplantation as a means to both produce major
anti-tumor cytoreduction and reestablish hematopoiesis with mdr-l transfected
bone marrow to allow continued escalated dose treatment with mdr-l susceptible
drugs. [ ***
]
Patients with selected malignancies including breast cancer, will have
bone marrow collected while in a minimal disease state (bone marrow in
clinical remission). The marrow will be processed by positive selection of
CD34 positive cells followed by a negative depletion of malignant cells using
immunomagnetic separation. The residual cells will be assessed by flow
cytometry, FISH and molecular studies for residual malignant cells.
The stem cell enriched purged bone marrow will then be transduced with....
mdr-l safety modified retroviruses, as described in Project 1A. The frequency
of transduction will be determined.
For preparing probes for MDRI/MDR2, we (Kuo et al) have subcloned a 174
base pair Bst Xl/AccI fragment from human MDR1 cDNA (nucleotide coordination
1594 to 1768 from the translation start site into the EcoRI and AccI site of
pGEM (Promega) vector). Antisense RNA probe will be prepared using XX0 XXX
polymerase. Because a 65-nt sequence in the MDR2 cDNA was identical to a
sequence in this fragment, this probe can simultaneously detect both MDRI and
MDR2 transcripts, yielding protected fragments of 174- and 65-nt,
respectively.
[ ***
]
Clinical trials of dose intensive chemotherapy for breast cancer.
-32-
Approximately 200 patients per year receive dose intensive chemotherapy
with autologous bone marrow transplantation at the MD Xxxxxxxx Cancer Center,
including 80 for patients with breast and ovarian cancers.
Quantitative PCR assay for Breast Cancer Cells
The tremendous sensitivity of the PCR method permit a detection of mRNA
levels probably at the levels of a few copies in 100 - 1000 cells. Concerns
about its quantitation have been addressed by the recent developments with
this technique. Xxxxxx et al (67) and Xxxxxx et al (68) reported that PCR can
be both sensitive (qualitative) and quantitative in the detection of MDR mRNA
in various tumor samples and in MDR cell lines in which different levels of
mRNA are present. Accurate quantitative were demonstrated in an exponential
range determined by fixed number of cycles on serial dilutions of thee RNA
reverse transcription (cDNA) products or performing the reaction with a
varying number of cycles on a fixed quantity of cDNA. Normalization of the
results can be achieved by independent amplification of a control gene, e.g.,
b2-microglobulin. Quantitative PCR method is particularly useful for detecting
expression of MDR gene in breast cancer because of its low level of
expression. We feel that the method should be applicable for detection of
thymidylate synthetase and GST-pi mRNA.
Complementary DNA will be synthesized from RNA by Xxxxxxx murine leukemia
virus reverse transcriptase (Bethesda Research Laboratories). PCR will be
carried out with cDNA derived from 50 ng of RNA according to the method
described by Xxxxxx et al. (67) using AmpliTaq reaction kit purchased from
Xxxxxx-Xxxxx/Cetus. Paired amplimers used for amplification of MDR1. GST-pi
and thymidylate synthetase will be synthesized separately using the flanking
sequences to the 5' side and 3' (complementary sequence) of the restriction
sites used in the preparations of antisense RNA. Each pair of amplimers will
be tested individually. PCR reactions will be carried out using [-32P] dCTP in
the triplicated reaction mixtures. PCR products will be separated on 12%
polyacrylamide gels and stained with ethidium bromide. Bands corresponding to
each specific PCR product will be excised from the gels and the amount of
incorporated radioactivity will be determined by scintillation counting.
Quantitative PCR will be established using positive controls with known levels
of MDR mRNA and negative controls in which Escherichia coli tRNA will be used.
Once Successful quantitative PCR for each gene product is established, we will
include all three paired amplimers in the same reaction mixture to establish a
simultaneous detection for all the three gene transcripts. The PCR products
for the three mRNA have distinct sizes and can be separated by the same gel
system and quantified.
Assessment of minimal residual disease in the bone marrow
We propose to develop methodology to detect minimal metastatic involvement
in the bone marrow using both immunofluoreseence directed against mucin,
cytokeratins or other breast cancer cell surface proteins not shared by normal
bone marrow cells as well as molecular detection of mucin gene transcripts.
-33-
A number of monoclonal antibodies to breast cancer cells and
oligonucleotide probes to the mucin gene are available. The sensitivity and
specificity of these techniques for detection of minimal residual disease will
be assessed. This data will be used to evaluate the incidence and clinical
importance of subclinical bone marrow involvement in patients with localized
disease before and after surgery and adjuvant chemotherapy as well as patients
with metastatic breast cancer, including patients receiving bone marrow
transplantation. The presence of marrow involvement by these techniques will
be assessed as a prognostic factor for response and duradon of response. In
preliminary studies, approximately 30% of newly diagnosed patients with stage
I and II breast cancer have marrow involvement detected by immunoperoxidase
with anti-cytokeratin antibodies. In this project we will also develop
methodology using anti breast cancer monoelonal antibodies and immunomagnetic
separation to deplete breast cancer cells from normal bone marrow and to
quantitatively assess the efficacy of this approach for ex vivo elimination of
breast cancer cells from autologous bone marrow.
We also propose to evaluate minimal residual disease in bone marrow using
oligonucleotide probes to the mucin gene and the polymerase chain reaction.
Breast mucin is a highly glycosylated large molecular weight protein present
in breast epithelium and virtually all breast eaneers (69), but absent from
the normal bone marrow. The cDNA sequence for the core protein of breast mucin
has recently been determined and has revealed it to be composed of an extended
series of 20 amino acid tandem repeats (40 to 80) flanked by 3' and 5' unique
sequences. Xxxxx Xxxxxxxx, Ph.D., a collaborator on this project (see letter),
synthesized primers for the PCR reaction which would that would amplify a
region of the unique sequence contained by introns. The down stream primer
overlapped an mRNA splice region so that no amplification of contaminating DNA
would occur. Using a reverse transcriptasePCR technique and oligonucleotide
primers for the unique sequence just 3' from the tandem repeat of the breast
mucin mRNA, as little as 50 pg of RNA from MCF-7 or other breast cancer eell
lines can be detected equivalent to approximately 3 cells. This technique was
positive for 5 of 5 breast cancer cell lines, but not in two lymphoblastoid
lines or in 7 normal bone marrows (31). Work is in progress to define the
limit of detection for breast cancer cells in normal marrow, but we anticipate
it will be approximately one in 100,000 cells. In order to use this approach
to quantify the level of bone marrow involvement by tumor cells, and the
effectiveness of bone marrow purging techniques, a competitive PCR procedure
will be employed. This involves cloning the sequence to be amplified into a
plasmid from a genomic clone containing an intron, or alternatively, a
restriction site can be introduced into the sequence to distinguish the
competing DNA from the specific sequence. In order to use this approach to
quantify the level of bone marrow involvement by tumor cells, and the
effectiveness of bone marrow purging, techniques, a competitive PCR technique
will be employed. This involves cloning the sequence to be amplified into a
plasmid from a genomic clone containing an intron, or alternatively, a
restriction site can be introduced into the sequence to distinguish the
competing DNA from the specific sequence. This competing DNA sequence is
amplified by the same primers, but yields a different sized fragment(s). The
competitor is added in different amounts to the PCR reaction mixture. The
samples are analyzed by gel electrophoresis and the relative amounts of the
amplified specific and competing sequence are determined. The concentration of
the competitor which results in both bands being equal is considered to be the
concentration of the specific mRNA.
-34-
This technique will be studied for its sensitivity and specificity to
identify breast cancer cells within normal marrow and peripheral blood stem
cell collections. Normal marrows will be spiked with cells from breast cancer
cells lines and fresh tumor cells to assess the limits of detection. It is
likely that there will be some variability between tumors related to the level
of expression of mucin. This approach promises to be considerably more
sensitive than nonmolecular methods. We will directly compare the sensitivity
of this PCR based approach and its quantitative assessment with
immunohistochemistry and immunofluorescent methods on the same specimens.
Subsequently fresh bone marrow aspiration and biopsy specimens from patients
with breast cancer will be analyzed and results correlated with routine
histology, immunocytochemistry (8) and clonogenic assay (27).
Bone marrow samples from patients with stage one and two disease at
diagnosis as well as metastatic disease eligible for bone marrow
transplantation will be assessed for occult marrow involvement by flow
cytometry using a panel of anti breast cancer reactive antibodies and the for
mucin gene expression by this PCR technique. These assays will be used to
evaluate the presence of contaminating malignant cells in the bone marrow
harvests and peripheral blood progenitor collections used for transplantation,
and to assess the efficacy of the stem cell selection and purging procedures
to be studied to eliminate breast cancer from the hematopoietic cell
infusions. We will validate this detection by sorting the rare breast tumor
cells detected by flow cytometry and confirm their genotype by PCR.
Development of bone marrow purging and stem cell selection to eliminate breast
cancer cells from normal hematopoietic cells for autologous transplantation
As an approach to transplant marrow or peripheral blood progenitors free
of contaminating malignant cells we will initially isolate CD34-positive cells
using the avidin-biotin column separation procedure described by Berenson et
al (38) whose company is participating with us (see letter). In brief, bone
marrow or peripheral blood buffy coat cells are incubated with biotinylated
anti CD34 monoclonal antibody and passed through an avidin column (Cell Pro,
Inc., Seattle WA) (see end for letter of collaboration). The adherent CD34
positive cells are released by agitation and collected. This results in a
yield of approximately 1% of the starting cell number including > 60% of the
CD34-positive cells. The efficacy of the system to deplete malignant cells
from normal marrow will be studied by spiking normal marrow samples with
various numbers of breast cancer cells and assessed for residual cancer cell
involvement of the separated CD34 positive cells. Marrow and peripheral blood
collections intended for transplantation will be processed in using this
technique; assuming > 106 CD34-positive cells/kg are collected, these cells
will be used as a source of hematopoietic progenitors for autologous
transplantation. The pre and post processed collections will be studied as
described above for contamination by malignant cells.
It is likely that the stem cell selection procedure will reduce, but not
totally eliminate malignant cells from the nonnal hematopoietic cells. As a
subsequent step, we study several methods for further depletion of breast
cancer cells. We will initially evaluate the efficacy of purging using
anti-mucin and other anti-breast cancer monoclonal antibodies with
immunomagnetic separation to selectively deplete breast cancer cells from
normal bone marrow and assess the efficacy of this approach for ex vivo
elimination of breast cancer cells from autologous bone marrow. In
-35-
brief, normal marrow or the CD34-positive cell fraction will be spiked with
varying numbers of breast cancer cells or clonogenic breast cancer cell lines
and treated with the immunotoxin in concentrations ranging from 10-6 to 10-2
M, as descubed in project 4. Recovery of normal hematopoietic colonies CFU-GM
vs. breast cancer cells and colonies will be assessed.
Immunomagnetic separation will be studied as an alternative approach for
depleting malignant cells. A number of monoclonal antibodies reactive with
breast cancer cells will be screened for reactivity against a large number of
primary tumors and tumor metastatic to the marrow using immunohistology and
flow cytometry. Candidate antibodies will be uniformly reactive with the
breast cancer cells within a given patient and reactive with breast cancer
cells from > 90% of samples. The antibody Bre3 (52) a murine monoclonal IgG1
antibody reactive with a mucin-like glycoprotein complex, a characteristic
cell surface antigen present on breast cancer cells will be initially studied.
Four other anti mucin antibodies are also available. These antibodies will be
tested for ex vivo depletion of clonogenic breast cancer cells and cell lines
using immunomagnetic separation (70). This immunomagnetic separation procedure
is presently used in our program for purging lymphoid malignancies and is
capable of a 2 to 4 log reduction of tumor cells depending on the model
system. Monoclonal antibodies reactive with breast carcinoma cells which do
not bind to normal bone marrow will be utilized. Percoll separated bone marrow
cells (1 x 108/ml) are incubated with monoclonal antibody in various doses on
ice for 30 minutes and washed. The cells are resuspended with Dynal
immunomagnetic beads conjugated with goat anti-mouse antibody (30 Beads/cell).
The suspension is gently rocked at 4(degree)C. for 30 minutes and passed
through the magnetic separation column. The targeted tumor cells bound by the
anti-breast cancer antibody are bound to the beads and are separated in the
magnetic field. The residual breast cancer cells will be assessed by
immunofluorescence, clonogenic assay and the PCR technique.
Recovery of normal hematopoietic progenitors (CFU-GM) will also be
assessed. As a model, clonogenic breast cancer cell lines or fresh breast
cancer cells (ideally from marrow or a malignant effusion) will be spiked in
various doses into irradiated human marrow with the resulting depletion
assessed by clonogenic assay of the cells pre and post separation. Alternative
antibodies, alone and in combination will also be studied and conditions will
be adjusted to optimize the depletion results.
The techniques which are most successful in eliminating the malignant
cells without toxicity to normal hematopoietic elements in vitro will
subsequently be integrated into the MD Xxxxxxxx clinical program for
autologous bone marrow and peripheral blood cell transplantation.
Development of Therapeutic Model for Chemoprotection in Breast Cancer:
This program will not be initiated until the retrovirus producer cell
lines undergo the analysis outlined in Tables I and the regulatory review
outlined in Table II and Figure 1.
First, marrow will be taken from advanced disease patients and purged so
as to remove all breast cancer cells by the methods summarized above. PCR for
mucin mRNA will be used to determine if the marrow is devoid of breast cancer
cells. If that assay is negative, the cells will be
-36-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
transduced and infused following intensive preparative therapy
(cyclophosphamide, BCNU and thiotepa. We will have an unmodified marrow as
backup containing 2x108 nucleated cells/kg).
Following hematopoietic recovery of an autologous transplant, patients are
usually very sensitive to receive chemotherapy for maintenance although they
exhibit impressive responses with the intensive therapy. The transduction of
the autologous cells before transplant is designed to make the patient's
marrow more tolerant of maintenance therapy. The presence of MDR-I modified
cells may permit one to deliver intensive doses of drugs like Taxol, or the
combination of Doxorubicin and Velban, all MDR drugs. The delivery of cyclical
therapy over many months may permit one to eradicate the minimal residual
disease which is left after transplant. If this program is successful, we may
attempt to use MDR-1 genetically-modified cells or infusion in the adjuvant
setting in poor prognosis patients (greater than 10 nodes positive at
diagnosis) to support the delivery of multiple cycles of intensive adjuvant
cyclical chemotherapy. The delivery of intensive consolidation
(cyclophosphamide, BCNU and Thiotepa) following initial regional therapy (e.g.
surgery or radiation) for poor prognosis patients followed by the extension of
maintenance therapy (Doxorubicin, Velban, or Taxol) over time in the minimal
residual disease setting may also alter the relapse frequency of poor
prognosis patients after initial therapy. The patients will be followed after
infusion of modified cells to evaluate the persistence of the modified cells,
using the PCR and FACS sorting, and functional assays for MDR outlined in Core
C and above in this project following the outline of assays summarized in
Table III. A major risk is introduction of the MDR virus in breast cancer
cells which are beneath the level of detection of the PCR. However, this level
will be less than 1/1000,000. It is possible that most patients have much
higher levels than this before the purging in advanced disease patients. We
will be able to desect this if it occurs but it is unlikely that this will be
clinically significant as most relapses occur in extramedullary sites.
[ ***
]
-37-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
[ ***
]
F. VERTEBRATE ANIMALS: NONE
G. CONSULTANTS/COLLABORATORS: NONE
H. CONSORTIUM/CONTRACTUAL ARRANGEMENTS: NONE
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4-hydroperoxycyclophosphamide, monoclonal antibodies, and magnetic
microspheres. Cancer. 1991 ;68: 1272-1278.
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-44-
The following seven pages have been omitted pursuant to a request for
confidential treatment. A complete copy of this exhibit containing the omitted
portions has been separately filed with the Securities and Exchange
Commission.
-45-
Table II
EVALUATION OF RETROVIRAL SUPERNATANTS
------------------------------------------------------------------------------
TYPE OF ASSAY YEAR YEAR YEAR YEAR
------------------------------------------------------------------------------
1 2 3 4
NORMAL LEVEL OF EXPRESSION OF LOW OR ABSENT IN X X
TRANSGENOME MATURE MYELOID
CELLS
MINIMAL LEVEL OF EXPRESSION SUFFICIENT TO X X
REQUIRED INCREASE RESISTANCE
10-FOLD
MAXIMAL LEVEL OF EXPRESSION TO UNKNOWN X X
BE OBTAINED
CONSEQUENTS OF OVEREXPRESSION UNKNOWN X X
AND INAPPROPRIATE SPECIFICITY
ABSENCE OF HELPER VIRUS S+V LOCUS ASSAY; X X
NIH3T3 AMPLIFICATION
ASSAY + ELUTION
MICROSCOPY
ABSENCE OF INFECTIOUS AGENTS CULTURE X X
(BACTERIA, MYCOPLASMA, VIRUS)
TOXIC EFFECT OF VIRUS MOUSE ASSAY X X
(EXTRANEOUS TOXIN TEST)
ENDOTOXIN ASSAY
ANALYSIS OF STRUCTURE OF SOUTHERN PCR X X
RETROVIRAL TRANSGENOME (IS
RETROVIRUS INTACT IN PRODUCER
CELL LINE?; IS THERE A SINGLE
COPY?)
PROPERTIES OF VIRUS:
TRANSDUCTION FREQUENCY; 10-60% CHEMO- X X
BIOLOGICAL EFFECT OF THE VECTOR RESISTANCE
TRANSDUCTION
EVIDENCE FOR DESIRED PHENOTYPIC CHEMORESISTANCE X X X
CHANGE AFTER TRANSDUCTION
ANIMAL MODEL DATA FOR EFFICACY REDUCTION OF X X X
MYELOSUPPRESSION
------------------------------------------------------------------------------
-46-
The following four pages have been omitted pursuant to a request for
confidential treatment. A complete copy of this exhibit containing the omitted
portions has been separately filed with the Securities and Exchange
Commission.
-47-
Figure 3
Analysis of Chemotherapy Indcued
Myalosuppression in BALB/c Mice
-48-
Figure 4
ANALYSIS OF NEO AND
BCR-ABL BY PCR
-49-
Figure 5
PCR OF COLONIES
-50-
The following six pages have been omitted pursuant to a request for
confidential treatment. A complete copy of this exhibit containing the omitted
portions has been separately filed with the Securities and Exchange
Commission.
-51-
EXHIBIT II
LICENSE AGREEMENT
This License Agreement (hereinafter referred to as the "License
Agreement"), effective as of the [DAY] of [MONTH], 1992 is by and between the
BOARD OF REGENTS ("BOARD") OF THE UNIVERSITY OF TEXAS SYSTEM ("SYSTEM"), an
agency of the State of Texas whose address is 000 Xxxx 0xx Xxxxxx, Xxxxxx,
Xxxxx 00000, THE UNIVERSITY OF TEXAS M. D. XXXXXXXX CANCER CENTER
("UNIVERSITY"), a component institution of SYSTEM, and INGENEX, INC., a
corporation duly organized and existing under the laws of the State of
California and having a principal place of business at 000 Xxxxxx Xxxxx Xxxx,
Xxxxx #000, Xxx Xxxxxxxxx, Xxxxxxxxxx 00000 ("LICENSEE").
WHEREAS, BOARD is the owner of U.S. Patent Application
Serial No. _________________________, filed _____________________________
hereinafter referred to as "the initial application", which is based on
inventions and confidential information relating to the Research Program, and
particularly relating to the invention of a genetically engineered retroviral
pharmaceutical reagent incorporating LICENSEE'S MDR-1 gene (or LICENSEE's RB
Tumor Suppressor gene), and whereas LICENSEE now desires to obtain a license,
under the Initial Application, upon the terms and conditions hereinafter set
forth; and
WHEREAS, LICENSEE has represented to UNIVERSITY, to induce UNIVERSITY
to enter into this License Agreement, that it shall commit itself to a
thorough, vigorous and diligent program of exploiting said inventions and
confidential information, including any patent rights which may be obtained
therein, so that public utilization shall result therefrom; and
WHEREAS, UNIVERSITY and LICENSEE have entered into a Research Agreement
and to which this License Agreement is attached contemporaneous herewith,
attached hereto as Exhibit II (the "Research Agreement"), granting LICENSEE an
option to license inventions, know-how, and patent rights arising out of the
Research Program described in the Research Agreement pursuant to the terms and
conditions set forth in this License Agreement.
NOW, THEREFORE, it is agreed as follows:
ARTICLE 1 - DEFINITIONS
For the purpose of this License Agreement, the following words and
phrases shall have the following meanings:
1.1 "LICENSEE" shall mean INGENEX, INC., a corporation duly organized
and existing under the laws of the State of California and having a principal
place of business at 000 Xxxxxx Xxxxx Xxxx, Xxxxx #000, Xxx Xxxxxxxxx,
Xxxxxxxxxx 00000.
1.2 "AFFILIATE" shall mean any company or entity, the voting control
of which is at least fifty percent (50%), directly or indirectly, owned or
controlled by LICENSEE.
1.3 "Patent Rights" shall mean:
1.3.1 Any United States and/or foreign, patent applications and/or
patents, arising out of the Research Program as set forth in the Research
Agreement, which may, at Licensee's option, be added to Exhibit III of this
Agreement;
1.3.2 Any later-filed United States and/or foreign patent
applications based on the patent applications and/or patents listed in Exhibit
III, or corresponding thereto, including any continuations,
continuations-in-part, divisional, reissues, reexaminations, or extensions
thereof; and
1.3.3 Any United States and/or foreign patents issuing from any of
the foregoing.
1.4 "Licensed Product(s)" shall mean:
1.4.1 Any product which is covered in whole or in part by a valid
and unexpired claim contained in the Patent Rights in the country in which the
product is made, used, leased or sold;
1.4.2 Any product which is manufactured by using a process which
is covered in whole or in part by a valid and unexpired claim contained in the
Patent Rights in the country in which the process is used;
1.4.3 Any product which is used according to a method which is
covered in whole or in part by a valid and unexpired claimed contained in the
Patent Rights in the country in which the method is used.
1.5 "Licensed Process(es)" shall mean any process or method, which is
covered, in whole, or in part, by a valid and unexpired claim contained in the
Patent Rights in the country in which the process or method is used.
1.6 "First Year Milestone" shall mean the scientific achievement or
commercial development event described in Appendix III, attached hereto.
1.7 "Net Sales" shall mean LICENSEE's or an AFFILIATE's xxxxxxxx for
Licensed Products and Licensed Processes less the sum of the following:
(a) discounts allowed in amounts customary in the trade;
(b) sales, tariff duties and/or use taxes directly imposed and with
reference to particular sales;
(c) outbound transportation prepaid or allowed;
(d) amounts allowed or credited or returns; and
(e) bad debt deductions actually written off during the period.
-2-
No deductions shall be made for commissions paid to individuals
whether they be independent sales agencies or regularly employed by LICENSEE
or an AFFILIATE and on their payroll. LICENSED PRODUCTS and LICENSED PROCESSES
shall be considered "sold" when billed out or invoiced.
ARTICLE 2 - GRANT
2.1 BOARD hereby grants to LICENSEE a worldwide license to practice
under the Patent Rights, and to make, have made, use, lease and/or sell the
Licensed Products and to practice the Licensed Processes, to the full end of
the term for which the Patent Rights are granted, unless sooner terminated as
hereinafter provided, said license to include the right to sublicense (with
the prior written approval of BOARD and UNIVERSITY, which approval shall not
be unreasonably withheld) and to be exclusive to LICENSEE.
2.2 LICENSEE agrees that any sublicenses granted by it shall provide
for the same obligations as those obligations imposed only by this License
Agreement.
2.3 LICENSEE agrees to forward to UNIVERSITY annually a copy of such
reports received from any sublicensee as may be pertinent to an accounting of
royalties.
2.4 LICENSEE understands that the Patent Rights may have been
developed, in part, under a funding agreement with the Government of the
United States of America and, if so, that the Government may have certain
rights relative thereto. This License Agreement is explicitly made subject to
the Government's rights under any such agreement and any applicable law or
regulation. To the extent that there is a conflict between any such agreement,
applicable law or regulation and this License Agreement, the terms of such
Government agreement, applicable law or regulation shall prevail.
ARTICLE 3 - DUE DILIGENCE
3.1 LICENSEE shall use its reasonable best efforts to bring Licensed
Products or Licensed Processes to market through a thorough, vigorous and
diligent program for exploitation of the Patent Rights and continue active,
diligent marketing efforts for Licensed Products or Licensed Processes
throughout the life of this License Agreement, consistent with the business
plan described in Paragraph 3.2.3, below.
3.2.1 LICENSEE shall fund the research at the UNIVERSITY in
accordance with the funding schedule contained in the Research Agreement;
3.2.2 Within six (6) months of the achievement of the First Year
Milestone, LICENSEE shall develop a business plan setting forth LICENSEE's
detailed commercial development plans and proposed product applications
showing the scheduled development goals, amount of money, number and kind of
personnel and time budgeted and planned for each phase of
-3-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
the clinical trials and regulatory approval processes of the Licensed Products
and/or Licensed Processes.
3.3 LICENSEE'S failure to perform the due diligence obligations
described in Paragraphs 3.2.1 or 3.2.2 shall constitute a material breach or
default for purposes of the termination provisions of Paragraph 7.3.
3.4 In the event LICENSEE fails to meet the scheduled development
goals set forth in the business plan prepared in accordance with Paragraph
3.2.2, UNIVERSITY and LICENSEE shall engage in good faith negotiations to
revise the initial business plan and establish substitute development goals.
In the event LICENSEE fails to meet the substitute scheduled development goals
as set forth in the revised business plan as a result of events or non-events
within the control of LICENSEE, such failure shall constitute a material
breach or default for purposes of the termination provisions of Paragraph 7.3.
ARTICLE 4 - ROYALTIES
4.1 For the rights, privileges and license granted hereunder, LICENSEE
shall pay to UNIVERSITY, as set forth below, to the end of the term of the
Patent Rights or until this License Agreement shall be terminated as
hereinafter provided:
4.1.1 In each calendar year, a royalty on Net Sales of the
Licensed Products or Licensed Processes leased or sold, by LICENSEE, or any
AFFILIATE, payable according to the following schedule:
(A) For Licensed Products or Licensed Processes described by Section
1.3.1, above, and which incorporate and/or utilize technology
disclosed in patents and patent applications listed in Exhibit III [
*** ] of Net Sales.
(B) For Licensed Products or Licensed Processes described by Section
1.3.2, above, which do not incorporate and/or utilize technology
disclosed in patents and patent applications listed in Exhibit III.
(a) [ *** ] of Net Sales for annual Net Sales less than or equal ---
to [ *** ]; and ---
(b) [ *** ] of Net Sales for annual Net Sales greater than [ *** ],
but less than or equal to [ *** ];
-4-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
(c) [ *** ] of Net Sales for annual Net Sales greater than [ *** ---
--- ] but less than or equal to [*** --- ];
(d) [ *** ] of Net Sales for annual Net Sales greater than [*** ], but
less than or equal to[ *** ];
(e) [ *** ] of Net Sales for annual Net Sales greater than [*** ---
--- ].
4.1.2 In each calendar year, a royalty on Net Sales of the leased
or sold, by LICENSEE, or any AFFILIATE, payable according to the following
schedule:
4.1.3 LICENSEE may reduce the amount of the royalties due to
UNIVERSITY pursuant to 4.1.1, above, by the amount required to be paid to any
third party to avoid infringement of any of such third party's patent rights
required to practice the rights granted under this License Agreement, but in
no event shall UNIVERSITY receive less than one-half (1/2) the applicable
royalty provide in 4.1.1, above.
4.1.4 In each calendar year, a royalty on the consideration
received by LICENSEE from sublicensees, pursuant to the following schedule:
a. for sublicenses within the scope of Article 4.1.1 (A), hereinabove,
CANCER CENTER shall receive one and one-half percent (1.5%) of of all
royalties, fees or other consideration received by LICENSEE; for
sublicenses within the scope of 4.1.1 (B), hereinabove:
b. where LICENSEE receives a royalty less than or equal to ten percent
(10%) and/or fees or other consideration valued less than or equal to
five million dollars ($5,000,000), MDA shall receive twenty percent
(20%) of the total consideration;
c. where LICENSEE receives a royalty greater than ten percent (10%) and
less than or equal to fifteen percent (15%) and/or fees or other
consideration valued greater than five million dollars ($5,000,000)
and less less than ten millio dollars ($10,000,000), MDA shall receive
twenty-five percent (25%) of the total consideration;
d. where LICENSEE receives a royalty greater than [ *** ] and/or fees or
other consideration valued greater than [ *** ], MDA shall receive [
*** ] of the total consideration.
-5-
The information marked by "[***]" has been omitted pursuant to a request for
confidential treatment. The omitted portion has been separately filed with the
Securities and Exchange Commission.
4.1.5 To maintain the exclusivity of LICENSEE'S license to the
Patent Rights, LICENSEE shall pay minimum annual royalties in accordance with
the following schedule:
(a) In January of the fifth (5th) calendar year after
execution of the license, a minimum of [ *** ]
(b) In January of the sixth (6th) calendar year after
execution of the license, a minimum of [ *** ]
(c) In January of the seventh (7th) calendar year after the
execution of the license, a minimum of [ *** ]
(d) In January of the eighth (8th) calendar year after the
execution of the license, a minimum of [ *** ]
(e) Each calendar year thereafter, a minimum of [ *** ]
4.2 No multiple royalties shall be payable because the use, lease or
sale of any Licensed Product or Licensed Process is, or shall be, covered by
more than one valid and unexpired claim contained in the Patent Rights.
4.3 Royalty payments shall be paid in United States dollars to THE
UNIVERSITY OF TEXAS M.D. XXXXXXXX CANCER CENTER, 0000 Xxxxxxxx Xxxxxxxxx,
Xxxxxxx, Xxxxx 00000 or at such other place as UNIVERSITY may reasonably
designate, consistent with the laws and regulations controlling in any foreign
country. Any withholding taxes which LICENSEE or any sublicensee shall be
required by law to withhold on remittance of the royalty payments shall be
deducted from royalty paid to UNIVERSITY. LICENSEE shall furnish UNIVERSITY
the original copies of all official receipts for such taxes. If any currency
conversion shall be required in connection with the payment of royalties
hereunder, such conversion shall be made by using the exchange rate prevailing
a Citibank, N.A. in New York, on the last business day of the calendar
quarterly reporting period to which such royalty payments relate.
ARTICLE 5 - REPORTS AND RECORDS
5.1 LICENSEE shall keep full, true and accurate books of account
containing all particulars that may be necessary for the purpose of showing
the amount payable to UNIVERSITY by way of royalty as aforesaid. Said books of
account shall be kept at LICENSEE's principal place of business. Said books
and the supporting data shall be open up to three (3) times per year, upon
reasonable notice to LICENSEE and no more than twice per calendar year, for
five (5) years
-6-
following the end of the calendar year to which they pertain, for inspection
by the UNIVERSITY Internal Audit Division and/or by an independent certified
public accountant employed by UNIVERSITY, to which LICENSEE has no reasonable
objection, for the purpose of verifying LICENSEE's royalty statement or
compliance in other respects with this License Agreement.
5.2 LICENSEE, within thirty (30) days after the end of each quarter of
each calendar year, shall deliver to UNIVERSITY true and accurate reports,
giving such particulars of the business conducted by LICENSEE during the
preceding quarter under this License Agreement as shall be pertinent to a
royalty accounting hereunder. These shall include at least the following:
(a) All Licensed Products and Licensed Processes used, leased
or sold, by or for LICENSEE, its AFFILIATES and
sublicensees.
(b) Total amounts invoiced for Licensed Products and Licensed
Processes used, leased or sold, by or for LICENSEE, its
AFFILIATES or its sublicensees.
(c) Deductions applicable in computed "Net Sales" as defined
in Paragraph 1.7.
(d) Total royalties due based on Net Sales by or for LICENSEE,
its AFFILIATES or its sublicensees.
(e) Names and addresses of all sublicensees and AFFILIATES of
LICENSEE.
(f) On an annual basis, LICENSEE's Annual Report.
5.3 With each such report submitted, LICENSEE shall pay to UNIVERSITY
the royalties due and payable under this License Agreement. If no royalties
shall be due, LICENSEE SHALL so report.
ARTICLE 6 - PATENT PROSECUTION
In accordance with Paragraph 3 (c) of the Research Agreement, the
LICENSEE, at its own expense and utilizing patent counsel of its choice (and
agreed to by BOARD and UNIVERSITY), shall have the sole right and
responsibility for the filing, prosecution, and maintenance of any patent
applications and patents contained in the Patent Rights. All such patents and
patent applications shall be assigned to BOARD. LICENSEE, or its patent
counsel, shall provide UNIVERSITY with copies of all correspondence and
documents filed with, or received from, the United States Patent and Trademark
Office or any foreign patent office or patent agent. In addition, LICENSEE
agrees that any and all official or "ribbon" copies of issued patents shall be
forwarded to, and retained by, UNIVERSITY.
ARTICLE 7 - TERMINATION
-7-
7.1 If LICENSEE shall become bankrupt or insolvent, or shall file a
petition in bankruptcy, or if the business of LICENSEE shall be placed in the
hands of a receiver, assignee or trustee for the benefit of creditors, whether
by the voluntary act of LICENSEE or otherwise, this License Agreement shall
automatically terminate.
7.2 Should LICENSEE fail in its payment to UNIVERSITY of royalties due
in accordance with the terms of this License Agreement, UNIVERSITY shall have
the right to serve notice upon LICENSEE, by certified mail to the address
designated in Article 14 hereof, of its intention to terminate this License
Agreement within thirty (30) days after receipt of said notice of termination
unless LICENSEE shall pay to UNIVERSITY, within (30) day period, all such
royalties due and payable. Upon the expiration of the thirty (30) day period,
if LICENSEE shall not have paid all such royalties due and payable, the
rights, privileges and license granted hereunder shall thereupon immediately
terminate.
7.3 Upon any material breach or default of this License Agreement by
LICENSEE, other than those occurrences set out in Paragraphs 7.1 and 7.2
hereinabove, which shall always take precedence in that order over any
material breach or default referred to in this Paragraph 7.3, UNIVERSITY shall
have the right to terminate this License Agreement and the rights, privileges
and license granted hereunder by ninety (90) days' notice to LICENSEE by
certified mail to the address designated in Article 14 hereof. Such
termination shall become effective unless LICENSEE shall have cured any such
breach or default prior to the expiration of the ninety (90) day period from
receipt of the notice of termination.
7.4 LICENSEE shall have the right to terminate this License Agreement
at any time on nine (9) months' notice by certified mail to UNIVERSITY.
7.5 Upon termination of this License Agreement for any reason, nothing
herein shall be construed to release either party from any obligation that
matured prior to the effective date of such termination. LICENSEE and/or any
sublicensee thereof may, however, after the effective date of such
termination, sell all Licensed Products, and complete Licensed Products in the
process of manufacture at the time of such termination, and sell the same,
provided that LICENSEE shall pay to UNIVERSITY the royalties therein as
required by Article 5 of this License Agreement and shall submit the reports
required by Article 5 hereof on the sales of Licensed Products.
ARTICLE 8 - INFRINGEMENT AND OTHER ACTIONS
8.1 LICENSEE and UNIVERSITY shall promptly provide written notice, to
the other party, of any alleged infringement by a third party of the Patent
Rights and provide such other party with any available evidence of such
infringement.
8.2 During the term of this License Agreement, LICENSEE shall have the
right, but not the obligation, to prosecute and/or defend, at its own expense
and utilizing counsel of its choice, any infringement of, and/or challenge to,
the Patent Rights. In furtherance of such right, UNIVERSITY hereby agrees that
LICENSEE may join UNIVERSITY hereby agrees that LICENSEE may join
-8-
UNIVERSITY as a party in any such suit, without expense to UNIVERSITY. No
settlement, consent judgment or other voluntary final disposition of any such
suit may be entered into without the consent of UNIVERSITY, which consent
shall not unreasonably be withheld. LICENSEE shall indemnify UNIVERSITY
against any order for costs that may be made against UNIVERSITY in any such
suit.
8.3 Any recovery of damages by LICENSEE, in any such suit, shall be
applied first in satisfaction of any unreimbursed expenses and legal fees of
LICENSEE RELATING to the suit. The balance remaining from any such recovery
shall be treated as royalties received by LICENSEE from sublicensees and
shared by UNIVERSITY and LICENSEE in accordance with Paragraph 4.1.2 hereof.
8.4 If within six (6) months after receiving notice of any alleged
infringement, LICENSEE shall have been unsuccessful in persuading the alleged
infringer to desist, or shall not have brought and shall not be diligently
prosecuting an infringement action, or if LICENSEE shall notify UNIVERSITY,
any time prior thereto, of its intention not to bring suit against the alleged
infringer, then, and in those events only, UNIVERSITY shall have the right,
but not the obligation, to prosecute, at its own expense and utilizing counsel
of its choice, any infringement of the Patent Rights, and UNIVERSITY may, for
such purposes, join the LICENSEE as a party plaintiff. The total cost of any
such infringement action commenced solely by UNIVERSITY shall be borne by
UNIVERSITY and UNIVERSITY shall keep any recovery or damages for past
infringement derived therefrom.
8.5 In any suit to enforce and/or defend the Patent Rights pursuant to
this License Agreement, the party not in control of such suit shall, at the
request and expense of the controlling party, cooperate in all respects and,
to the extent possible, have its employees testify when requested and make
available relevant records, papers, information, samples, specimens, and the
like.
ARTICLE 9 - PRODUCT LIABILITY
9.1 BOARD, by this License Agreement, makes no representation as to
the patentability and/or breadth of the inventions contained in the Patent
Rights. BOARD, by this License Agreement, makes no representation as to
patents now held or which will be held by others, or by BOARD, in the field of
the Licensed Products for a particular purpose.
9.2 LICENSEE agrees to defend, indemnify and hold BOARD and UNIVERSITY
harmless from and against all liability, demands, damages, expense or losses
for death, personal injury, illness or property damage arising (a) out of use
by LICENSEE or its transferees of inventions licensed or information furnished
under this License Agreement, or (b) out of any use, sale or other disposition
by LICENSEE or its transferees of products made by use of such inventions or
information. As used in this clause, UNIVERSITY includes its Regents,
Officers, Agents, Employees and Students, and "LICENSEE" includes its
Affiliates, Contractors and Sub-Contractors.
ARTICLE 10 - ASSIGNMENT
-9-
LICENSEE may assign or otherwise transfer this License Agreement and
the license granted hereunder and the rights acquired by it hereunder so long
as such assignment or transfer shall be accompanied by a sale or other
transfer of LICENSEE's entire business or of that part of LICENSEE's business
to which the license granted hereunder relates. LICENSEE shall give BOARD and
UNIVERSITY thirty (30) days prior written notice within which to reasonably
object to such assignment or transfer. If within thirty (30) days after the
giving of such notice, no written objection is received by LICENSEE, BOARD and
UNIVERSITY shall be deemed to have approved such assignment or transfer;
provided, however, BOARD shall not be deemed to have approved such assignment
and transfer unless such assignee or transferee shall have agreed in writing
to be bound by the terms and conditions of this License Agreement. Upon such
assignment or transfer and agreement by such assignee or transferee. If
LICENSEE shall sell or otherwise transfer its entire business or that part of
its business to which the license granted hereby relates and the transferee
shall not have agreed in writing to be bound by the terms and conditions of
this License Agreement, or new terms and conditions shall not have been
reasonably agreed upon within sixty (60) days of such sale or transfer, BOARD
shall have the right to terminate this License Agreement.
ARTICLE 11 - NON-USE OF NAMES
LICENSEE shall not use the name of UNIVERSITY or any adaptation
thereof in any advertising, promotional or sales literature without prior
written consent obtained from UNIVERSITY, in each case, except that LICENSEE
may state that it is licensed by UNIVERSITY, under one or more of the patents
and/or applications comprising the Patent Rights.
ARTICLE 12 - PAYMENTS, NOTICE AND OTHER COMMUNICATIONS
Any payment, notice or other communication pursuant to this License
Agreement shall be sufficiently made or given on the date of mailing is sent
to such party by certified first class mail, postage prepaid, addressed to it
at its address below or as it shall designate by written notice given to the
other party:
In the case of BOARD:
BOARD OF REGENTS
The University of Texas System
000 Xxxx Xxxxxxx Xxxxxx
Xxxxxx, Xxxxx 00000
ATTENTION: System Intellectual
Property Office
with copy to: The University of Texas
M.D. Xxxxxxxx Cancer Center
Office of Technology Development
0000 Xxxxxxxx Xxxx, Xxxxx 0000
Xxxxxxx, Xxxxx 00000
Attention: Xxxxxxx X. Xxxx
Director
-10-
In the case of LICENSEE:
Ingenex, Inc.
000 Xxxxxx Xxxxx Xxxx, Xxxxx #000
Xxx Xxxxxxxxx, Xxxxxxxxxx 00000
Attention: Xxxxx Xxxxxx, M.D.,
President
ARTICLE 13 - MISCELLANEOUS PROVISIONS
13.1 This License Agreement shall be construed, governed, interpreted
and applied in accordance with the laws of the State of Texas, U.S.A., and
adjudication may take place in courts of either State, except that questions
affecting the validity, enforceability, or infringement of any patent
contained in the Patent Rights shall be determined by the law of the country
in which the patent was granted.
13.2 The parties hereto acknowledge that this License Agreement sets
forth the entire agreement and understanding of the parties hereto as to the
subject matter hereof, and shall not be subject to any change of modification
except by the execution of a written instrument subscribed to by the parties
hereto.
13.3 The provisions of this License Agreement are severable, and in
the event that any provision of this License Agreement shall be determined to
be invalid or unenforceable under any controlling body of law, such invalidity
or unenforceability shall not in any way affect the validity or enforceability
of the remaining provisions hereof.
13.4 LICENSEE agrees to xxxx the Licensed Products sold in the United
States with all applicable United States patent numbers. All Licensed Products
shipped to, or sold in, other countries shall be marked in such a manner as to
conform with the patent laws and practice of the country of manufacture or
sale.
13.5 The failure of either party to assert a right hereunder or to
insist upon compliance with any term or condition of this License Agreement
shall not constitute a waiver of that right or excuse a similar subsequent
failure to perform any such term or condition by the other party.
-11-
IN WITNESS WHEREOF, the parties hereto have executed this License
Agreement, in duplicate by proper persons thereunto duly authorized.
THE UNIVERSITY OF TEXAS BOARD OF REGENTS OF THE
M.D. XXXXXXXX CANCER CENTER UNIVERSITY OF TEXAS SYSTEM
By By
--------------------------- --------------------------
Xxxxx X. Xxxxxxxx Xxx Xxxxxxx
Executive Vice President General Counsel
Administration and Finance
APPROVED AS TO CONTENT: APPROVED AS TO FORM
By By
--------------------------- --------------------------
Xxxxxxx X. Xxxx Xxxxxx X. Xxxxx, Xx.
Director, Technology Manager, Intellectual Property
Development
INGENEX, INC.
By
-------------------------
Xxxxx Xxxxxx, M.D.
President
-12-
EXHIBIT III
1. United States Patent Application #652311 dated February 6, 1991
entitled "DNA That Confers Multidrug Resistance"
2. United States Patent Application #07/622,836 filed September 24, 1990
entitled "Compositions and Methods for Clones Containing DNA Sequences
Associated with Multidrug Resistance in Human Cells"
3. Pending United States Patent Application entitled "Methods and
Modified Gene Products for Practicing Tumor Suppressor Gene Therapy",
Xxxxxx and Xxxxxxx attorney docket number 7409-025, Xxxxxx and
Xxxxxxx, 1155 Avenue of the Americas, N.Y. N.Y.
-13-
