Exhibit 10.14


 


AGREEMENT


 


 


Between


 


 


NEW YORK UNIVERSITY


 


 


and


 


 


COLLATERAL THERAPEUTICS, INC.


 





















 


NYU/COLLATERAL THERAPEUTICS


Research & License Agreement


 


 


INDEX


 


	
  Section 1

  	
  Definitions

  	
  page 1

  
	
  Section 2

  	
  Effective Date

  	
  page 4

  
	
  Section 3

  	
  Performance of the NYU
  Research Project

  	
  page 4

  
	
  Section 4

  	
  Funding of the NYU
  Research Project

  	
  page 5

  
	
  Section 5

  	
  Title

  	
  page 5

  
	
  Section 6

  	
  Patents and Patent
  Applications

  	
  page 6

  
	
  Section 7

  	
  Grant of License

  	
  page 8

  
	
  Section 8

  	
  Payments for License

  	
  page 9

  
	
  Section 9

  	
  Method of Payment

  	
  page 12

  
	
  Section 10

  	
  Development and
  Commercialization

  	
  page 13

  
	
  Section 11

  	
  Confidential Information

  	
  page 15

  
	
  Section 12

  	
  Publication

  	
  page 15

  
	
  Section 13

  	
  Liability and
  Indemnification

  	
  page 16

  
	
  Section 14

  	
  Security for
  Indemnification

  	
  page 17

  
	
  Section 15

  	
  Expiry and Termination

  	
  page 18

  
	
  Section 16

  	
  Representations and
  Warranties by CORPORATION

  	
  page 19

  
	
  Section 17

  	
  Representations and
  Warranties by NYU

  	
  page 20

  
	
  Section 18

  	
  No Assignment

  	
  page 21

  
	
  Section 19

  	
  Use of Name

  	
  page 21

  
	
  Section 20

  	
  Miscellaneous

  	
  page 22

  
	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  
	
   

  	
  Appendix I       Pre Existing Inventions 

  	
   

  
	
   

  	
   

  	
   

  
	
   

  	
  Appendix II      Research Program

  	
   

  
	
   

  	
   

  	
   

  
	
   

  	
  Appendix III     Development Plan

  	
   

  



 





















 


RESEARCH AND  LICENSE  AGREEMENT


 


This
Agreement, effective as of March 24, 1997 (the “Effective Date”), is by and
between NEW YORK UNIVERSITY (hereinafter “NYU”), a corporation organized and
existing under the laws of the State of New York and having a place of business
at 70 Washington Square South, New York, New York 10012 and COLLATERAL
THERAPEUTICS, INC. (hereinafter “CORPORATION”), a corporation organized and
existing under the laws of the State of California, having its principal office
at 9360 Towne Centre Drive, San Diego, California 92121.


 


RECITALS


 


WHEREAS,
Dr. Claudio Basilico of NYU (hereinafter “the NYU Scientist”), together with
other co-inventors, has made certain inventions all as more particularly
described in an issued U.S. patent and U.S. patent applications and foreign
patent applications owned by NYU, in each case identified in annexed Appendix I
and forming an integral part hereof (hereinafter “the Pre-Existing Inventions”);


 


WHEREAS, NYU is willing to perform the NYU Research
Project (as hereinafter defined);


 


WHEREAS, CORPORATION is prepared to sponsor the NYU
Research Project;


 


WHEREAS, subject to the terms and conditions hereinafter set forth, NYU
is willing to grant to CORPORATION and CORPORATION is willing to accept from
NYU the License (as hereinafter defined);


 


NOW,
THEREFORE, in consideration of the mutual promises and agreements contained
herein, the parties hereto hereby agree as follows:


 


1.             Definitions.


 


(a)          “Calendar Year” shall mean any consecutive
period of twelve months commencing on the first day of January of any year.


 


(b)         “Corporation Entity” shall mean any company
or other legal entity which controls, or is controlled by, or is under common
control with, CORPORATION; control means the holding of fifty percent (50%) or more
of (i) the capital and/or (ii) the voting rights and/or (iii)
the right to elect or appoint directors.


 





















 


(c)          “Date of First Commercial Sale” shall have
the meaning set forth in Section 7(b) hereof.


 


(d)         “FGF-4” shall mean Fibroblast Growth Factor 4
the amino acid sequence of which is provided in Figure 1 in the article by P.
Delli Bovi, A.M. Curatola, F. G. Kern, A. Greco, M. Ittmann, and C. Basilico published
in Cell, Volume 50, pages 729-737, August 28, 1987.


 


(e)          “Field” shall mean gene therapy for coronary
artery disease, congestive heart failure, and peripheral vascular disease.


 


(f)          “GI Agreement” shall mean, collectively (i)
the Agreement, dated as of February 6, 1989, between NYU and Genetics
Institute, Inc. (“GI”), as amended by the Amendment, dated February 25, 1997,
and (ii) the Settlement
Agreement, dated August 22, 1996, between NYU and GI.


 


(g)         “License” shall mean the exclusive worldwide
license to practice the Research Technology (as hereinafter defined) for the
development, manufacture, use and
sale of the Licensed Products (as hereinafter defined) in the Field and the
exclusive worldwide right to sublicense such rights in accordance with Section
7(c).


 


(h)         “Licensed Products” shall mean products
comprising a nucleic acid sequence encoding FGF-4 or fragments or analogs
thereof, in each case which are covered by a claim of any unexpired patent
within the NYU Patents (as hereinafter defined) which has not been disclaimed
or held invalid by a court of competent jurisdiction from which no appeal can
be taken or of any active patent application within the NYU Patents, or which
utilize all or any portion of NYU Know-How.


 


(i)           “Net Sales” shall mean the total amount
invoiced in connection with sales of Licensed Products by CORPORATION, any
Corporation Entity or any sublicensee of CORPORATION, any Corporation Entity or
a sublicensee in accordance with Section 7(c)(iii), in each case to end users; provided
that Net Sales shall (i) not include any amounts invoiced in connection
with sales of Licensed Products for (A) transportation charges,
including insurance relating thereto, or (B) sales and excise taxes,
value-added taxes or customs duties paid by the


 


2





















 


person selling or distributing any Licensed
Product or any other governmental charges imposed upon the sale or distribution
of any Licensed Product; and (ii) be adjusted to reflect any deductions
to amounts invoiced to take account of (X) distributors’ fees, rebates,
allowances or sales commissions actually granted, allowed or incurred and
credits for returns or (Y) quantity or case discounts, cash discounts or
chargebacks actually granted, allowed or incurred in the ordinary course of
business in connection with the sale or distribution of any Licensed Product; provided,
further, that Net Sales shall not include amounts invoiced by
CORPORATION to any person or entity that is a Corporation Entity or a
sublicensee of CORPORATION or a Corporation Entity under the License.


 


(j)           “NYU Know-How” shall mean the Pre-Existing
Inventions, any proprietary information or proprietary materials including, but
not limited to, pharmaceutical, chemical, biological and biochemical products,
information and trade secrets, know-how, technical and non-technical data,
materials, methods and processes and any drawings, plans, diagrams,
specifications and/or other documents containing such information, discovered,
developed or acquired by, or on behalf of students or employees of NYU during the term and in the course of
the NYU Research Project.


 


(k)          “NYU Patents” shall mean all United States
and foreign patents and patent applications, and any divisions, continuations, in whole or in part,
reissues, re-examinations, renewals and extensions thereof, and pending
applications therefor:


 


(1)           which claim Pre-Existing Inventions and which
are identified on annexed Appendix I; or


 


(2)           which claim inventions that are made, in
whole or in part, by students or employees of NYU during the term and in the
course of the NYU Research Project.


 


(l)           “Research Period” shall mean the three-year
period commencing on the Effective Date hereof and any extension thereof as to
which NYU and CORPORATION shall mutually agree in writing.


 


(m)         “NYU Research Project” shall mean the
investigations at NYU during the Research Period into the Field under the
supervision of the NYU Scientist in


 


3





















 


accordance
with the research program, described in annexed Appendix II, which forms an
integral part hereof.


 


(n)         “Research Technology” shall mean all NYU
Patents and NYU Know-How.


 


(o)         “Total Net Sales” shall mean the aggregate
Net Sales of CORPORATION, any Corporation Entity and any sublicensee of
CORPORATION or any Corporation Entity to end users of Licensed Products.


 


2.             Effective Date.


 


This Agreement shall be effective as of the
date first written above and shall remain in full force and effect until it
expires or is terminated in accordance with Section 16 hereof.


 


3              Performance of the NYU Research Project.


 


(a)           In consideration of the sums to be paid to
NYU as set forth in Section 4 below, NYU undertakes to perform the NYU Research
Project under the supervision of the NYU Scientist during the Research Period,
as such Project may be amended in accordance with Section 20(f).  If, during the Research Period the NYU
Scientist shall cease to supervise the NYU Research Project, then NYU shall promptly so notify CORPORATION
and shall endeavor to find among the scientists of NYU a Scientist acceptable
to CORPORATION to continue the supervision of the NYU Research Project.  If NYU is unable to find such a Scientist
acceptable to CORPORATION within three months after such notice to CORPORATION,
CORPORATION shall have the option to terminate its funding of the NYU Research
Project.  CORPORATION shall promptly
advise NYU in writing if CORPORATION so elects. 
Such termination of funding pursuant to this Section 3(a) shall not
terminate this Agreement or the License granted herein.  Nothing herein contained shall be deemed to
impose an obligation on NYU to find a replacement for the NYU Scientist.


 


(b)           Nothing contained in this Agreement shall be construed as a warranty on
the part of NYU that any results or inventions will be achieved by the NYU
Research Project, or that the Research Technology and/or any other results or
inventions achieved by the NYU Research Project, if any, are or will be
commercially exploitable and furthermore, NYU makes


 


4





















 


no
warranties whatsoever as to the commercial or scientific value of the Research
Technology and/or as to any results which may be achieved in the NYU Research
Project.


 


(c)           NYU will have full authority and
responsibility for the NYU Research Project. All students and employees of NYU
who work on the NYU Research Project will do so as employees or students of
NYU, and not as employees of CORPORATION.


 


(d)           NYU shall provide to CORPORATION a report on
the NYU Research Project within ninety (90) days following the end of each
twelve-month period occurring during the Research Period.


 


4.             Funding of the NYU Research Project.


 


(a)           As compensation to NYU for work to be
performed on the NYU Research Project during the Research Period, subject to
any earlier termination of the Research Project pursuant to Section 3(a)
hereof, CORPORATION will pay NYU the total sum of $600,000, payable in six
equal consecutive installments of $100,000, on the Effective Date and at six
month intervals following such Date.


 


(b)           Nothing in this Agreement shall be
interpreted to prohibit NYU (or the NYU Scientist) from obtaining additional
financing or research grants for the NYU Research Project from government
agencies, which grants or financing may render all or part of the NYU Research
Project and the results thereof subject to the patent rights of the U.S.
Government and its agencies, as set forth in Title 35 U.S.C.§200 et  seq.


5.             Title.


 


(a)           Subject to the License granted to CORPORATION
hereunder, it is hereby agreed that all right, title and interest, in and to
the Research Technology, and in and to any drawings, plans, diagrams,
specifications, and other documents containing any of the Research Technology
shall vest solely in NYU.  At the request
of NYU, CORPORATION shall take all steps as may be necessary to give full
effect to said right, title and interest of NYU including, but not limited to,
the execution of any documents that may be required to record such right, title
and interest with the appropriate agency or government office.


 


5





















 


(b)           Subject to the License granted to CORPORATION
hereunder, for so long as the NYU Scientist is employed by NYU, any and all
inventions made by such NYU Scientist and relating to the Field shall be owned
solely by NYU.


 


6.             Patents and Patent Applications.


 


(a)           NYU will promptly disclose to CORPORATION in
writing any inventions which constitute potential NYU Patents developed in the
course of the NYU Research Project.


 


(b)           At the initiative of CORPORATION or NYU, the
parties shall consult with each other regarding the prosecution of all patent
applications within NYU Patents (excluding any Pre-Existing Invention).  Such patent applications shall be filed,
prosecuted and maintained by the law firm of Darby & Darby or by other
patent counsel jointly selected by NYU and CORPORATION.  Copies of all such patent applications and
patent office actions shall be forwarded to each of NYU and CORPORATION.


 


NYU
and CORPORATION shall each also have the right to have such patent applications
and patent office actions independently reviewed by other patent counsel
separately retained by NYU or CORPORATION, upon prior notice to and consent of
the other party, which consent shall not unreasonably be withheld.


 


(c)           All applications and proceedings with respect
to NYU Patents (other than those relating to any Pre-Existing Invention) shall
be filed, prosecuted and maintained by NYU at the expense of CORPORATION. Against
the submission of invoices, CORPORATION shall reimburse NYU for all costs and
fees incurred by NYU during the term of this Agreement, in connection with the
filing, maintenance, prosecution, protection and the like of such patents.


 


(d)           NYU and CORPORATION shall assist, and cause
their respective employees and consultants to assist each other, in assembling
inventorship information and data for the filing and prosecution of patent
applications on inventions pertaining to the Research Technology.


 


6





















 


(e)           If at any time during the term of this
Agreement CORPORATION decides that it is undesirable, as to one or more
countries, to prosecute or maintain any patents or patent applications within
the NYU Patents (other than those relating to any Pre-Existing Invention), it
shall give prompt written notice thereof to NYU, and upon receipt of such
notice CORPORATION shall be released from its obligations to bear all of the
expenses to be incurred thereafter as to such countries in conjunction with
such patent(s) or patent application(s) and such patent(s) or application(s)
shall be deleted from the Research Technology and NYU shall be free to grant
rights in and to the Research Technology in such countries to third parties,
without further notice or obligation to CORPORATION, and the CORPORATION shall
have no rights whatsoever to exploit the Research Technology in such countries.


 


(f)            Under the GI Agreement, provisions exist to
determine the circumstances under which patent protection will be obtained by
NYU with respect to any Pre-Existing Invention. 
For patent applications with respect to Pre-Existing Inventions, copies
of such applications and office actions shall be forwarded to CORPORATION who
may consult with NYU with regard thereto. 
CORPORATION agrees, upon presentation of supporting documentation, to
reimburse NYU for one quarter (1/4) of the expenses incurred by NYU as of the
Effective Date in connection with obtaining such patent protection.  In the event that such separate provisions
result in a situation where patent protection in any country is not pursued by
NYU because of a lack of funding pursuant to such provisions, then NYU shall
notify CORPORATION thereof and CORPORATION shall have the option to pay NYU to
pursue such patent protection.


 


(g)           Nothing herein contained shall be deemed to be a warranty by NYU that


 


(i)            NYU can or will be able to obtain any patent or patents on any patent application
or applications in the NYU Patents or any portion thereof, or that any of the
NYU Patents will afford adequate or commercially worthwhile protection, or


 


7





















 


(ii)           that the manufacture, use, or sale of any element of the Research
Technology or any Licensed Product will not infringe any patent(s) of a third
party.


7.             Grant of License.


 


(a)           Subject to the terms and conditions
hereinafter set forth, and subject to any rights of the U.S. Government
pursuant to Title 35 of the United States Code §200 et  seq., NYU
hereby grants to CORPORATION and CORPORATION hereby accepts from NYU the License.


 


(b)           The License granted to CORPORATION in Section
7(a) hereto shall commence upon the Effective Date and shall remain in force on
a country-by-country basis, if not previously terminated under the terms of
this Agreement, for fifteen (15) years from the Date of First Commercial Sale
in such country or until the expiration date of the last patent within the NYU
Patents in any such country to expire, whichever shall be later.  CORPORATION shall inform NYU in writing of
the Date of First Commercial Sale with respect to each Licensed Product in each
country as soon as practicable after the making of each such first commercial
sale.


 


(c)           CORPORATION shall be entitled to grant
sublicenses under the License on terms and conditions in compliance and not
inconsistent with the terms and conditions of this Agreement (except that the
rate of royalty may be at higher rates than those set forth in this Agreement)
(i) to a Corporation Entity or (ii) to other third parties for
consideration and in an arms-length transaction. All sublicenses shall only be
granted by CORPORATION under a written agreement, a copy of which shall be provided
by CORPORATION to NYU as soon as practicable after the signing thereof.  Each sublicense granted by CORPORATION
hereunder shall be subject and subordinate to the terms and conditions of this License
Agreement and shall contain (inter-alia) the following provisions:


 


(1)           the sublicense shall expire automatically on the
termination of the License;


 


(2)           the sublicense shall not be assignable, in whole
or in part;


 


8





















 


(3)           the sublicensee shall not grant further sublicenses,
except that a sublicensee may grant a further sublicense solely for purposes of
effecting distribution of Licensed Products to end users on the same terms
required for sublicenses under this Section 7(c);


 


(4)           both during the term of the sublicense and thereafter
the sublicensee shall agree to a confidentiality obligation similar to that imposed
on CORPORATION in Section 11 below, and that the sublicensee shall impose on
its employees, both during the terms of their employment and thereafter, a
similar undertaking of confidentiality; and


 


(5)           the sublicense agreement shall include the text
of Sections 13 and 14 of this Agreement and shall state that NYU is an intended
third party beneficiary of such sublicense agreement for the purpose of
enforcing such indemnification and insurance provisions.


 


8.             Payments for License.


 


(a)           In consideration for the grant and during the term of the License with
respect to each Licensed Product, CORPORATION shall pay to NYU:


 


(1)           On the Effective Date, a non-refundable,
non-creditable license issue fee of one hundred thousand dollars ($100,000);


 


(2)           On the first anniversary of the Effective Date
and on each subsequent anniversary thereof, a non-refundable, non-creditable license
maintenance fee of twenty-five thousand dollars ($25,000); provided that
such fee shall cease to be payable on the first anniversary of the Effective
Date following the completion by CORPORATION of one full year of sales of
Licensed Products in accordance with the terms of this Agreement;


 


(3)           Upon the achievement of the following technical
milestones with respect to any Licensed Product, the payments as indicated
below:


 


9





















 


	
  Milestone

  	
   

  	
  Payments

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  Upon the filing of an initial
  Investigational New Drug Application (or foreign equivalent thereof) for each
  new Licensed Product

  	
   

  	
  $

  	
  250,000

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  Upon the filing of a Product License
  Application (or foreign equivalent thereof) for each Licensed Product

  	
   

  	
  $

  	
  500,000

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  Upon the approval of a Product License
  Application (or foreign equivalent thereof) for each Licensed Product

  	
   

  	
  $

  	
  1,000,000

  	
   

  



 


provided that only one payment shall be made at each
milestone for each separate Licensed Product notwithstanding the number of
applications that may be filed and approved in various countries for each such
separate Licensed Product.


 


(4)           With respect to sales of Licensed Products a royalty
of 3% of Total Net Sales during each calendar year.


 


(b)           For the purpose of computing the royalties due to NYU hereunder, the
year shall be divided into two parts ending on June 30 and December 31.  Not later than one hundred thirty (130) days
after each December and June in each Calendar Year during the term of the
License, CORPORATION shall submit to NYU a full and detailed report of
royalties or payments due NYU under the terms of this Agreement for the
preceding half year (hereinafter “the Half-Year Report”), setting forth the
Total Net Sales and Net Sales of each of CORPORATION, each Corporation Entity
and each sublicensee of CORPORATION, any Corporation Entity or sublicensee
permitted under Section 7(c)(iii) and/or lump sum payments and all other payments
or consideration from sublicensees upon which such royalties are computed and
including at least:


 


(i)            the quantity of Licensed Products used, sold,
transferred or otherwise disposed of on a country-by-country basis;


 


10





















 


(ii)           the
selling price of each Licensed Product;


 


(iii)          the
deductions permitted under subsection l(i) hereof to arrive at Net Sales; and


 


iv)           the royalty
computations and subject of payment.


 


If no royalties or other payments are due, a statement shall be sent to
NYU stating such fact. Payment of the full amount of any royalties or other
payments due to NYU for the preceding half year shall accompany each Half-year
Report on royalties and payments. 
CORPORATION shall keep for a period of at least six (6) years after the
date of entry, full, accurate and compete books and records consistent with
sound business and accounting practices and in such form and in such detail as
to enable the determination of the amounts due to NYU from CORPORATION pursuant
to the terms of this Agreement.


 


(c)           Within
ninety (90) days after the end of each Calendar Year, commencing on the Date of
First Commercial Sale CORPORATION shall furnish NYU with a report (hereinafter
the “Annual Report”), certified
by an independent certified public accountant, relating to the royalties and
other payments due to NYU pursuant to this Agreement in respect of the Calendar
Year covered by such Annual Report and containing the same details as those
specified in Section 8(b) above in respect of the Half-Year Report.


 


(d)           On
reasonable notice and during regular business hours, NYU or the authorized
representative of NYU shall each have the right to inspect the books of
accounts, records and other relevant documentation of CORPORATION or of
Corporation Entity and the sublicensees of CORPORATION, Corporation Entity and
any sublicensee insofar as they relate to the production, marketing and sale of
the Licensed Products, in order to ascertain or verify the amount of royalties
and other payments due to NYU hereunder, and the accuracy of the information
provided to NYU in the aforementioned reports. 
NYU shall also have the right, not more than once each calendar year, to
audit CORPORATION’s books and financial records for the purpose of verifying
full


 


11





















 


payment by CORPORATION of
its royalty obligations hereunder. Such audits shall be conducted during normal
business hours and shall not interfere with CORPORATION’s conduct of its
business. Each such audit shall be at NYU’s expense, unless a particular audit
reveals an underpayment of ten percent (10%) or more of the amount that should
have been paid to NYU for the period audited, in which case CORPORATION shall
bear the expense of such audit.  In the
event of any underpayment of royalties, CORPORATION shall promptly remit to NYU
all amounts due.


 


(e)           Beginning
in the year in which CORPORATION completes one full year of sales of Licensed
Products and continuing thereafter until this Agreement shall terminate or
expire, CORPORATION agrees that if the total royalties paid to NYU under
subsection 8 (a) (4) hereof do not amount to five hundred thousand dollars
($500,000) in each Calendar Year, CORPORATION will pay to NYU within one
hundred thirty (130) days after the end of each such Calendar Year, as
additional royalty, the difference between the amount of the total royalties
paid to NYU by CORPORATION in such Calendar Year and five hundred thousand
dollars ($500,000), failing which NYU shall have the right solely at its
election, upon written notice to CORPORATION, to either terminate this
Agreement for cause or to declare the License granted herein to CORPORATION to
be non-exclusive.


 


(f)            CORPORATION
shall, and shall cause each Corporation Entity and sublicensee of CORPORATION,
Corporation Entity or a sublicensee, to effect sales of Licensed Products to
third parties on commercially reasonable, arm’s length terms.


 


9.             Method
of Payment.


 


(a)           Royalties
and other payments due to NYU hereunder shall be paid to NYU in United States
dollars. Any such royalties on or other payments relating to transactions in a
foreign currency shall be converted into United States dollars based on the
closing buying rate of the Morgan Guaranty Trust Company of New York applicable
to transactions under exchange regulations for the particular currency on the
last business day of the accounting period for which such royalty or other payment
is due.


 


12





















 


(b)           CORPORATION
shall be responsible for payment to NYU of all royalties due on sale, transfer
or disposition of Licensed Products by Corporation Entity or by the
sublicensees of CORPORATION, Corporation Entity or a sublicensee.


 


10.           Development
and Commercialization.


 


(a)           It
shall be within the judgment of CORPORATION in what manner to proceed with the
development of Licensed Products for commercialization; provided that
CORPORATION shall use efforts, consistent with its sound and reasonable
business practices and technical judgment, to effect introduction of Licensed
Products into the commercial market. CORPORATION shall be deemed to satisfy the
due diligence requirements of this Section 10(a) by: (i) preparing and
filing an Investigational New Drug Application for a Licensed Product within
three (3) years following the Effective Date; (ii) preparing and filing
an application for marketing approval of a Licensed Product in the United
States, Canada, or a country within the European Union within six (6) years
following the Effective Date; and (iii) obtaining marketing approval of
a Licensed Product in the United State, Canada, or a country within the
European Union within eight (8) years following the Effective Date.  Corporation’s Development Plan is annexed
hereto as Appendix III.


 


(b)           Provided
that applicable laws, rules and regulations require that the performance of the
tests, trials, studies and other activities required by subsection (a) above
shall be carried out in accordance with FDA current Good Laboratory Practices,
current Good Manufacturing Practices and current Good Clinical Practices and in
a manner acceptable to the relevant health authorities, CORPORATION shall carry
out such tests, trials, studies and other activities in accordance with such
Practices and in a manner acceptable to the relevant health authorities.  Furthermore, the Licensed Products shall be
produced in accordance with FDA current Good Manufacturing Practice procedures
in a facility which has been licensed by the FDA to manufacture such Licensed
Products, provided that applicable laws, rules and regulations so require.


 


(c)           CORPORATION
undertakes to begin the regular commercial production, use, and sale of the Licensed


 


13





















 


Products in each country
in which approval has been received (as described in Section 10(a)) and to
continue diligently thereafter to commercialize the Licensed Products in each
such country in a manner consistent with sound and reasonable business
practices.


 


(d)           CORPORATION
shall provide NYU with written reports on all activities and actions undertaken
by CORPORATION to develop and commercialize the Licensed Products; such reports
shall be made within sixty (60) days after each six (6) months of the duration
of this Agreement, commencing six months after the Effective Date.


 


(e)           If
CORPORATION shall not satisfy the requirements set forth in Section 10(a)
(unless such delay or failure is necessitated by FDA or other regulatory agencies or unless NYU and
CORPORATION have mutually agreed to amend the Development Plan because of
unforeseen circumstances) NYU shall notify CORPORATION in writing of
CORPORATION’S failure and shall allow CORPORATION sixty (60) days to cure such
failure.  Upon receiving such notice,
CORPORATION may elect to extend such diligence period and all subsequent
diligence periods relating to such Licensed Product for one twelve (12) month
period upon written certification to NYU that CORPORATION is continuing product
development work with respect to a Licensed Product and payment to NYU of a fee
equal to $100,000. After the expiration of any such twelve-month period,
CORPORATION may elect to further extend its diligence obligations under Section
10(a) with respect to such Licensed Product for successive one-year periods
upon (i) written notice to NYU, (ii) certification by CORPORATION that it is
continuing to diligently develop such Licensed Product and, together with its
sublicensee(s), will spend no less than three million dollars ($3,000,000) in
each Calendar Year on development of such Product and (iii) payment to NYU
prior to the beginning of such year of an amount equal to $500,000,
representing minimum annual royalties with respect to such Product.  CORPORATION’s failure to cure a delay in the
diligence requirements to NYU’s reasonable satisfaction or elect and satisfy
the requirements of one of the options set forth above within such 60-day
period shall be a material breach of this Agreement.


 


14





















 


11.           Confidential
Information.


 


(a)           Except
as otherwise provided in Section 11(b) and 11(c) below CORPORATION shall
maintain any and all of the Research Technology in confidence and shall not
release or disclose any tangible or intangible component thereof to any third
party without first receiving the prior written consent of NYU to said release
or disclosure; provided that CORPORATION may, without NYU’s consent,
disclose Research Technology to sublicensees pursuant to Section 7, CORPORATION
Entities and consultants engaged by CORPORATION, in each case pursuant to a
confidentiality agreement requiring such party to maintain any and all of the
Research Technology in confidence and not release or disclose any tangible or
intangible component thereof to any third party without first receiving the
prior written consent of NYU to said release or disclosure.


 


(b)           The
obligations of confidentiality set forth in Section 11(a) shall not apply to
any component of the Research Technology which was part of the public domain
prior to the Effective Date of this Agreement or which becomes a part of the
public domain not due to some unauthorized act by or omission of CORPORATION
after the effective date of this Agreement or which is disclosed to CORPORATION
by a third party who has the right to make such disclosure.


 


(c)           The
provisions of Section 11(a) notwithstanding, CORPORATION may disclose the
Research Technology to third parties who need to know the same in order to
secure regulatory approval for the sale of Licensed Products.


 


12.           Publication.


 


(a)           Prior
to submission for publication of a manuscript describing the results of any
aspect of the NYU Research Project, NYU shall
send CORPORATION a copy of the manuscript to be submitted by overnight mail or
facsimile transmission, and shall allow CORPORATION thirty (30) days from the
date of such mailing to determine whether the manuscript contains such subject
matter for which patent protection should be sought prior to publication of
such manuscript, for the purpose of protecting an invention made by the NYU
Scientist during the course and within the term of the NYU Research Project.


 


15





















 


Should CORPORATION believe
the subject matter of the manuscript contains a patentable invention, then,
prior to the expiration of such 30-day period from the mailing date of such
manuscript to CORPORATION by NYU, CORPORATION shall give written notification
to NYU of:


 


(i)            its determination
that such manuscript contains patentable subject matter for which patent
protection should be sought; and


 


(ii)           the
countries in which such patent protection should be sought.


 


(b)           After
the expiration of such 30-day period from the date of mailing such manuscript
to CORPORATION, unless NYU has received the written notice specified above from
CORPORATION, NYU shall be free to submit such manuscript for publication to
publish the disclosed research results in any manner consistent with academic,
standards.


 


(c)           Upon
receipt of such written notice from CORPORATION, NYU will thereafter delay
submission of the manuscript for an additional period of up to sixty (60) days
to permit the preparation and filing in accordance with Section 6 hereof of a
U.S. patent application by NYU on, the subject matter to be disclosed in such
manuscript.  After expiration of such
60-day period, or the filing of a patent application on each such invention,
whichever shall occur first, NYU shall be free to submit the manuscript and to
publish the disclosed results.


 


13.           Liability
and Indemnification.


 


(a)           CORPORATION
shall indemnify, defend and hold harmless NYU and its trustees, officers,
medical and professional staff, employees, students and agents and their
respective successors, heirs and assigns (the “Indemnitees”) , against any
liability, damage, loss or expense (including reasonable attorneys’ fees and
expenses of litigation) incurred by or imposed upon the Indemnitees or any one
of them in connection with any claims, suits, actions, demands or judgments (i)
arising out of the design, production, manufacture, sale, use in commerce or in
human clinical trials, lease, or promotion by CORPORATION, a Corporation Entity
or an agent of CORPORATION, or by a sublicensee of CORPORATION, a


 


16





















 


Corporation Entity or a sublicensee,
of any Licensed Product, process or service relating to, or developed pursuant
to, this Agreement or (ii) arising out of any other activities to be carried
out pursuant to this Agreement.


 


(b)           With
respect to an Indemnitee, CORPORATION’S indemnification under subsection (a)(i)
of this Section 13 shall apply to any liability, damage, loss or expense
whether or not it is attributable to the negligent activities of such
Indemnitee, CORPORATION’s indemnification obligation under subsection (a)(ii)
of this Section 13 shall not apply to any liability, damage, loss or expense to
the extent that it is attributable to the negligent activities of any such
Indemnitee.


 


(c)           CORPORATION
agrees, at its own expense, to provide attorneys reasonably acceptable to NYU
to defend against any actions brought or filed against any Indemnitee with
respect to the subject of indemnity to which such Indemnitee is entitled
hereunder, whether or not such actions are rightfully brought.


 


14.           Security
for Idemnification.


 


(a)           At
such time as any Licensed Product, process or service relating to, or developed
pursuant to, this Agreement is being commercially distributed or sold (other
than for the purpose of obtaining regulatory approvals) by CORPORATION or by a
sublicensee, Corporation Entity or agent of CORPORATION, CORPORATION shall at
its sole cost and expense procure and maintain, or cause, a sublicensee,
Corporation Entity or agent of CORPORATION to procure and maintain, policies of
comprehensive general liability insurance in amounts not less than
$5,000,000.00 per incident and $10,000,000.00 annual aggregate and naming the
Indemnitees as additional insureds.  Such
comprehensive general liability insurance shall provide (i) product liability
coverage and (ii) broad form contractual liability coverage for CORPORATION’s
indemnification under Section 13 of this Agreement.  If CORPORATION elects to self-insure all or
part of the limits described above (including deductibles or retentions which
are in excess of $250,000 annual aggregate) such self-insurance program must be
acceptable to NYU.


 


17





















 


The minimum amounts of
insurance coverage required under this Section 14 shall not be construed to
create a limit of CORPORATION’s liability with respect to its indemnification
under Section 13 of this Agreement.


 


(b)           CORPORATION
shall provide NYU with written evidence of such insurance upon request of NYU.  CORPORATION shall provide NYU with written
noticed at least sixty (60) days prior to the cancellation, non-renewal or
material change in such insurance; if CORPORATION does not obtain replacement
insurance providing comparable coverage within such sixty (60) day period, NYU
shall have the right to terminate this Agreement effective at the end of such
sixty (60) day period without notice or any additional waiting periods.


 


(c)           CORPORATION
shall maintain such comprehensive general liability insurance beyond the expiration
or termination of this Agreement during (i) the period that any product,
process or service, relating to, or developed pursuant to, this Agreement is
being commercially distributed or sold (other than for the purpose of obtaining
regulatory approvals) by CORPORATION or by a sublicensee, Corporation Entity or
agent of CORPORATION and (ii) a reasonable period after the period referred to
in (c)(i) above which in no event shall be less than fifteen (15) years.


 


15.           Expiry
and Termination.


 


(a)           Unless
earlier terminated pursuant to this Section 15 or Section 8(e), hereof, this
Agreement shall expire upon the expiration of the period of the License in all
countries as set forth in Section 7(b) above.


 


(b)           At
any time prior to expiration of this Agreement, either party may terminate this
Agreement forthwith for cause, as “cause” is described below, by giving written
notice to the other party.  Cause for
termination by one party of this Agreement shall be deemed to exist if the
other party materially breaches or defaults in the performance or observance of
any of the provisions of this Agreement and such breach or default is not cured
within sixty (60) days or, in the case of failure to pay


 


18





















 


any amounts due
hereunder, thirty (30) days (unless otherwise specified herein) after the
giving of notice by the other party specifying such breach or default, or if
either NYU or CORPORATION discontinues its
business or becomes insolvent or bankrupt.


 


(c)           In
the event that CORPORATION determines, at any time following the end of the
Research Period, to cease all development or commercialization of all Licensed
Products covered by this Agreement, CORPORATION may terminate this Agreement by
notifying NYU in writing thereof no less than one hundred twenty (120) days
prior to the date of termination.


 


(d)           Any
amount payable hereunder by one of the parties to the other, which has not been
paid by the date on which such payment is due, shall bear interest from such
date until the date on which such payment is made, at the rate of two percent
(2%) per annum in excess of the prime rate prevailing at the Citibank, N.A., in
New York, during the period of arrears and such amount and the interest thereon
may be set off against any amount due, whether in terms of this Agreement or
otherwise, to the party in default by any non-defaulting party.


 


(e)           Upon
termination of this Agreement for any reason and prior to expiration as set
forth in Section 15(a) hereof, all rights in and to the Research Technology
shall revert to NYU, and CORPORATION shall not be entitled to make any further
use whatsoever of the Research Technology.


 


(f)            Termination
of this Agreement shall not relieve either party of any obligation to the other
party incurred prior to such termination.


 


(g)           Sections
5, 11, 13, 14, 15 and 19 hereof shall survive and remain in full force and
effect after any termination, cancellation or expiration of this Agreement.


 


16.           Representations
and Warranties by CORPORATION.


 


CORPORATION hereby represents and warrants to NYU as follows:


 


(1)           CORPORATION
is a corporation duly organized, validly existing and in good standing under
the laws of the State of California. 
CORPORATION has been granted all requisite power and authority to carry


 


19





















 


on its business and to
own and operate its properties and assets. 
The execution, delivery and performance of this Agreement have been duly
authorized by the Board of Directors of CORPORATION;


 


(2)           There
is no pending or, to CORPORATION’s knowledge, threatened litigation involving
CORPORATION which would have any effect on this Agreement or on CORPORATION’s
ability to perform its obligations hereunder;


 


(3)           There
is no indenture, contract, or agreement to which CORPORATION is a party or by
which CORPORATION is bound which prohibits or would prohibit the execution and
delivery by CORPORATION of this Agreement or the performance or observance by
CORPORATION of any term or condition of this Agreement; and


 


(4)           CORPORATION
has received and reviewed copies of the GI Agreement (with the exception of
those sections of the February 6, 1989 Agreement following section 4.1) and
understands and accepts the terms thereof that it has received and reviewed.


 


17.           Representations
and Warranties by NYU.


 


NYU hereby represents and warrants to CORPORATION as follows:


 


(1)           NYU
is a corporation duly organized, validly existing and in good standing under
the laws of the State of New York.  NYU
has been granted all requisite power and authority to carry on its business and
to own and operate its properties and assets. The execution, delivery and
performance of this Agreement have been duly authorized by the Board of
Trustees of NYU.


 


(2)           There
is no pending or, to NYU’s knowledge, threatened litigation involving NYU which
would have any effect on this Agreement or on NYU’s ability to perform its
obligations hereunder; and


 


(3)           There
is no indenture, contract, or agreement to which NYU is a party or by which NYU
is bound which prohibits or would prohibit the execution and delivery by NYU of
this Agreement or the performance or observance by NYU of any term or condition
of this Agreement.


 


20





















 


(4)           As
of the Effective Date, NYU is not aware of any prior art that would invalidate
any patent or patent claim, or that would prevent from issuing any patent
application covered by the NYU Patents.


 


18.           No
Assignment.


 


Neither CORPORATION nor
NYU shall have the right to assign, delegate or transfer at any time to any
party, in whole or in part, any or all of the rights, duties and interest
herein granted without first obtaining the written consent of the other to such
assignment, which consent shall not be unreasonably withheld; provided that
(i) CORPORATION may, without the prior consent of NYU, assign all of its rights
and obligations under this Agreement to a third party in connection with a
merger or corporate restructuring of CORPORATION or a sale of all or
substantially all of its assets, following written notice thereof and execution
by the third party with NYU of an agreement to be bound by the terms of this
Agreement and (ii) NYU may assign its interest in this Agreement in whole or in
part without the consent of CORPORATION if such assignee (A) is a parent,
subsidiary, affiliate or related entity to NYU or (B) is an entity that
acquires substantially all of the ownership interests or assets of NYU or New
York University Medical Center (or any successor to the foregoing) or (C) is an
entity formed by NYU or New York
University Medical Center (or any successor to the foregoing) and other
institutions, one of the purposes of which is to perform the activities for
which NYU is obligated pursuant to this Agreement.


 


19.           Use
of Name.


 


Without the prior written
consent of the other party, neither CORPORATION nor NYU shall use the name of
the other party or any adaptation thereof or of any staff member, employee or
student of the other party:


 


(i)            in any
product labeling, advertising, promotional or sales literature;


 


(ii)           in
connection with any public or private offering or in conjunction with any
application for regulatory approval, unless disclosure is otherwise required by
law, in which case either party may make factual statements concerning the
Agreement or file copies of the Agreement after providing the other party with
an opportunity


 


21





















 


to comment and reasonable
time within which to do so on such statement in draft.


 


Except as provided
herein, neither NYU nor CORPORATION will issue public announcements about this
Agreement or the status or existence of the NYU Research Project without prior
written approval of the other party.


 


20.           Miscellaneous.


 


(a)           In
carrying out this Agreement the parties shall comply with all local, state and
federal laws and regulations including but not limited to, the provisions of
Title 35 United States Code §200 et  seq. and 15 CFR §368 et
seq.


 


(b)           If
any provision of this Agreement is determined to be invalid or void, the
remaining provisions shall remain in effect.


 


(c)           This
Agreement shall be deemed to have been made in the State of New York and shall
be governed and interpreted in all respects under the laws of the State of New
York.


 


(d)           Any
dispute arising under this Agreement shall be resolved in an action in the
courts of New York State or the federal courts located in New York State, and
the parties hereby consent to personal jurisdiction of such courts in any
action.


 


(e)           All
payments or notices required or permitted to be given under this Agreement
shall be given in writing and shall be effective when either personally
delivered or deposited, postage prepaid, in the United States registered or
certified mail, addressed as follows:


 


To NYU:                New
York University Medical Center

550 First Avenue

New York, NY       10016


 


Attention:              Isaac
T. Kohlberg

Vice President for

    Industrial Liaison


 


and


 


22





















 


Office of Legal Counsel

New York University

Bobst Library

70 Washington Square South

New York, NY  10012


 


Attention:              Kathy
L. Schulz

Associate General Counsel


 


To CORPORATION:


 


Collateral Therapeutics,
Inc.

9360 Towne Centre Drive

San Diego, California 92121


 


Attention:              Jack
W. Reich, PhD

President and Chief

Executive Officer


 


or such other address or
addresses as either party may hereafter specify by written notice to the
other.  Such notices and communications
shall be deemed effective on the date of delivery or fourteen (14) days after
having been sent by registered or certified mail, whichever is earlier.


 


(f)            This
Agreement (and the annexed Appendices) constitute the entire Agreement between
the parties and no variation, modification or waiver of any of the terms or
conditions hereof shall be deemed valid unless made in writing and signed by
both parties hereto.  This Agreement
supersedes any and all prior agreements or understandings, whether oral or
written, between CORPORATION and NYU.


 


(g)           No
waiver by either party of any non-performance or violation by the other party
of any of the covenants, obligations or agreements of such other party
hereunder shall be deemed to be a waiver of any subsequent violation or
non-performance of the same or any other covenant, agreement or obligation, nor
shall forbearance by any party be deemed to be a waiver by such party of its
rights or remedies with respect to such violation or non-

performance.


 


(h)           The
descriptive headings contained in this Agreement are included for convenience
and reference only and shall not be held to expand, modify or aid


 


23





















 


in the interpretation, construction or meaning of this Agreement.


 


(i)            It
is not the intent of the parties to create a partnership or joint venture or to
assume partnership responsibility or liability. 
The obligations of the parties shall be limited to those set out herein
and such obligations shall be several and not joint.


 


IN WITNESS WHEREOF, the parties hereto have executed this Agreement
effective as of the date and year first above written.


 


	
   

  	
  NEW YORK UNIVERSITY

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
  By:

  	
  /s/ Isaac T. Kohlberg

  	
   

  
	
   

  	
   

  	
  Isaac T. Kohlberg

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
  Title:

  	
  Vice President for Industrial Liaison

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
  Date:

  	
  3/24/97

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
  Collateral Therapeutics, Inc.

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
  By:

  	
  /s/ Christopher J. Reinhard

  	
   

  
	
   

  	
   

  	
  Christopher J. Reinhard

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
  Title

  	
  Chief Operating Officer

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
   

  	
  Date:

  	
  3-21-97

  	
   

  



 


24





















 


Appendix I


 


Pre-existing NYU Patent and Patent Applications:


 


US patent 5,459,250 entitled “Truncated Mammalian
Growth Factor DNA Sequence” and US patent applications Serial No. 08/056,482
filed May 3, 1993, Serial No. 08/478,485 filed June 7, 1995, Serial No. 08/478,486 filed June 7, 1995, Rule 60
continuing patent application filed December 31, 1996 and US divisional patent application
filed February 13, 1997.


 


PCT filing /US90/06702 filed November 15, 1990

Serial No. 91900453 Europe filed June 12, 1992

Serial No, 2,068,871  Canada filed May
15, 1992

Serial No, 501065/1991 Japan filed August 19, 1992

Serial No, 68942/91 Australia filed November 15, 1990


 





















 


APPENDIX I


 


PRE-EXISTING
NYU PATENTS AND PATENT APPLICATIONS


 


	
  U.S. APPLICATIONS

  	
   

  	
  Serial No.

  	
   

  	
  Filing Date

  	
   

  	
  Status

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Mammalian
  Growth Factor

  	
   

  	
  07/062,925

  	
   

  	
  6/16/87

  	
   

  	
  ABANDONED

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Mammalian
  Growth Factor 

  	
   

  	
  07/177,506

  	
   

  	
  4/4/88

  	
   

  	
  ABANDONED

  
	
  (CIP of 07/062,925)

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Mammalian
  Growth Factor 

  	
   

  	
  07/806,771

  	
   

  	
  12/6/91

  	
   

  	
  ABANDONED

  
	
  (Cont. of 07/177,506)

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Mammalian
  Growth Factor 

  	
   

  	
  07/901,705

  	
   

  	
  6/22/92

  	
   

  	
  ABANDONED

  
	
  (CIP
  of 07/806,771)

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Mammalian
  Growth Factor 

  	
   

  	
  08/056,482

  	
   

  	
  5/3/93

  	
   

  	
  Allowed - Issue 

  Fee
  Paid 1/1/97

  
	
  (Cont.
  of 07/806, 771)

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Truncated
  Mammalian Growth 

  Factor
  DNA Sequence 

  (Cont. of 07/901,705)

  	
   

  	
  08/187,780

  	
   

  	
  1/25/94

  	
   

  	
  Issued -

  U.S.
  Patent No. 

  5,459,250

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Mammalian
  Growth Factor 

  (Div.
  of 08/187,780)

  	
   

  	
  08/478,485

  	
   

  	
  6/7/95

  	
   

  	
  Pending

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Mammalian
  Growth Factor 

  (Div.
  of 08/187,780)

  	
   

  	
  08/478,486

  	
   

  	
  6/7/95

  	
   

  	
  Pending

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Mammalian
  Growth Factor 

  (Cont. of 08/056,482)

  	
   

  	
  Not yet assigned

  	
   

  	
  12/31/96

  	
   

  	
  Pending

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Mammalian
  Growth Factor
 (Div. of 08/056,482) 

  	
   

  	
  Not yet assigned

  	
   

  	
  2/13/97

  	
   

  	
  Pending 

  



 





















 


FOREIGN
APPLICATIONS


 


The following patents
and patent applications are based upon International Application No. PCT/US90/06702, filed November 15, 1990 and
all are entitled “NON GLYCOSYLATED FGF-4 AND COMPOSITIONS CONTAINING THE SAME”.


 


	
  Country

  	
   

  	
  Application No.

  	
   

  	
  Filing Date

  	
   

  	
  Status

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Australia

  	
   

  	
  68942/91

  	
   

  	
  11/15/90

  	
   

  	
  Issued 2/24/94

  Patent No. 642,947

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Canada

  	
   

  	
  2,068,871

  	
   

  	
  11/15/90

  	
   

  	
  Pending

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  EPC

  	
   

  	
  91900453

  	
   

  	
  11/15/90

  	
   

  	
  Pending

  
	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  Japan

  	
   

  	
  501065/1991

  	
   

  	
  11/15/90

  	
   

  	
  Pending

  



 





















 


APPENDIX II


 


RESEARCH PROGRAM


 


THIRD GENERATION
FGF-4 MOLECULES


 


BACKGROUND


 


The FGFs family includes at least nine growth factors which show a
variable degree of homology within a conserved
“core” region. Although originally identified
for their growth promoting activity in fibroblasts, FGFs affect the proliferation or differentiation of a variety of
cells of mesenchymal and -neuroectodermal
origin.  Furthermore, many FGFs have been
shown to be angiogenic in vivo and in
vitro. The action of FGFs is mediated by their interaction with the high
affinity FGF receptors that also constitute a gene family of Receptor Tyrosine Kinases (RTK). (Basilico and Moscatelli,
1992)


 


The
receptors for the FGF family of growth factors are encoded by four distinct but homologous genes called FGFR-1/flg,
FGFR-2/bek, FGFR-3 and FGFR-4 These membrane-spanning
receptors have tyrosine kinase domains in their intracellular regions while their extracellular portion consists
of three immunoglobulin (Ig)-like loops.
Alternative splice variants of receptors 1, 2 and 3 have been isolated which
have alternative amino acids in the second half of Ig-loop III which are
encoded in three separate exons (IIIa,
IIIb and IIIc). This region is important in ligand-binding and variants of each receptor type have different
affinities for the various FGFs, In general,
the receptors have overlapping ligand-binding specificities with each receptor being able to bind more than one ligand
(Ornitz et al, 1996). The most restricted specificity is seen in the
case of the KGF receptor - a IIIb-splice variant of FGFR-2, which binds only FGF-7 (KGF), while the IIIc form can bind
FGF-1, FGF-2, FGF-4, FGF-5 and FGF-9 but not FGF-7. Binding of FGF to FGFR is
modulated by the


 





















 


interaction of the ligand with heparin or Heparin sulfate
proteoglycans (HSPG) (Yayon
et al, 1991). While the major effect of heparin binding on the FGF-FGFR interaction is not fully clarified, it is generally
agreed that a sustained activation of FGFR signaling cannot be achieved in the
absence of heparin or HSPG. Heparin binding does not appear to alter the
conformation of FGFs, but is known to stabilize them and affect binding affinity. This could result from ligand oligomerization,
which in turn influences sustained
receptor dimerization (Roghani et al, 1994 Spivak-Kroizman et al, 1994). As mentioned above, binding of FGFs to
their receptors exhibits only partial specificity. Thus the tissue distribution
of FGFR expression, the presence and concentration of a specific ligand, and
the relative affinity of the ligand -receptor interaction will all determine
whether a specific FGF can activate
a specific FGFR in a tissue or organ.


 


FGFs (and thus FGF signaling) have been implicated
in a variety of physiological and pathological processes. As mentioned
above, many FGFs are angiogenic,
because they can interact with specific receptors (mainly FGFR-1) which are present on the surface of vascular endothelial
cells. In addition to induce proliferation
of fibroblasts and in some cases of epithelial cells, they are potent neurotrophic factors, as they increase the
survival of neurons in culture, and may influence the proliferation and differentiation of neuronal progenitor
cells (Temple and Qian, 1995). FGFs
can also induce or inhibit differentiation in a variety of cell types (e.g. of myoblasts into myotubes).
Furthermore, several FGFs have been identified as oncogenes, because their
ectopic expression can create an autocrine growth loop in cells expressing the appropriate receptors. They can also
function as tumor progression agents
by inducing tumor vascularization (Basilico and Moscatelli, 1992).


 





















 


In spite of an
impressive number of publications, the roles that FGFs play vivo in tissue
homeostasis, regeneration and development are only beginning to be
understood.  Several lines of evidence
indicate that FGF signaling plays a major role in development. FGF signaling
has been shown to be important for mesoderm induction in Xenopus (Amaya et al,
1991; Amaya et al, 1993). Developmental abnormalities are associated with
knock-out of the FGF-4, FGF-8 and FGF-3 genes as well as of FGFR-1 and FGFR-2
(Mansour et al, 1993; Deng et al, 1994; Yamaguchi et al, 1994; Feldman et al, 1995) and targeting of
dominant-negative receptor molecules to various organs in transgenic mice also
results in a variety of defects in development (Werner et al, 1993; Peters et
al, 1994; Mima et al, 1995; Campochiaro et al, 1996). Furthermore, a number of
autosomally dominant genetic syndromes leading to bone malformations, including
achondroplasia, Crouzon syndrome, thanatophoric dysplasia etc., have been
linked to mutations in FGFR-1, 2 and 3 (Muenke and Schell, 1995). These
mutations, which occur in different regions of the receptor molecules, appear
to share the property of being “activating”/mutations, ie. to produce a
receptor which signals in a ligand-independent manner (Webster and Donoghue,
1996; Galvin et al 1996; Naski et al, 1996; Li et al, 1997). However, the
precise mechanism by which uncontrolled FGF signaling leads to skeletal and
skull malformations has not yet been elucidated.


 


The situation in the adult is far less clear. While
many FGFs have been shown to be angiogenic in a variety of experimental
settings, and exert other important effects on tissue homeostatis (e.g. promote
neuronal survival) (Basilico and Moscatelli, 1992; Baird, 1994), studies on
mice in which specific FGF genes which are normally expressed in the adult have
been inactivated by homologous recombination has produced somewhat surprising
results. Knock-out of FGF-5 only produces a long-hair phenotype (Hebert et al,
1994), and no defect in wound healing


 


3





















 


has been associated with the knock-out of FGF-7 (Guo
et al, 1996).  Knock-out of one of the
FGF prototypes, FGF-2 (basic FGF) produces no overt phenotype, although subtle
defects have begun to emerge (Ortega S. and Basilico, C., unpublished results).
A likely explanation for these observations is that the FGF family of growth
factors exhibits a high degree of redundancy, such that one FGF can easily
fulfill the function of another (e.g. FGF-1 for FGF-2).


 


The answer to some of the questions raised
above will undoubtedly derive from the many experiments of gene knock-out,
transgenic expression etc: that are being carried out in our as well as in
other laboratories, and will certainly suggest rational and perhaps unexpected
indications for FGFs in clinical interventions. For present and future usages
of FGFs in a clinical setting it will be however desirable to create new FGF
molecules with broader spectrum of action and higher potency or stability. We plan
to create such molecules, starting from FGF-4, which is the main subject of
this research agreement FGF-4 is normally only expressed during development,
where it plays an important role in post-implantation development and limb bud
formation. It is a secreted protein which has already been shown to be a potent
mitogen for a variety of cells and to be angiogenic in vivo.


 


SPECIFIC AIMS OF THE RESEARCH PROGRAM


 


As mentioned above, FGFs exhibit only partial
specificity for binding to their receptors. For example, while at present FGF-1
appears to be the ligand capable of binding and activating with very high
affinity all known FGF receptor isoforms (Ornitz et al, 1996), FGF-1 is quite
unstable and may not be the best universal activator. Similarly, FGF-4 binds
with high affinity to FGFR-2 and with somewhat lower affinity to FGFR-1, but
does not bind with high affinity FGFR-3 and-4, FGF-2


 


4





















 


binds equally well FGFR-1 and-2, but is a poor ligand
for all other receptors. It would be desirable therefore for experiments of
gene therapy or for any clinical use to develop FGF ligands which either bind
only one of the receptors, or exhibit broad specificity together with high
potency.  Furthermore, in clinical
application of molecules, such as FGF-4, immunogenicity could pose a problem.
Thus, if possible, it would be also desirable to generate a new FGF ligand with
low immunogenicity.


 


We already know from previous studies that
subtle alterations in the ligand structure can result in higher affinity
receptor binding. A case in point is the K140 truncated FGF-4 molecule
(Bellosta et al, 1993), which binds FGFR-1 and FGFR-2 (but not FGFR-3 and -4)
with higher affinity than FGF-4, and is consequently about 5 times more potent
as a mitogen in cells expressing these receptors. Preliminary studies also
indicate that K140 is more stable than FGF-4. We propose therefore to study the
possibility of creating, starting from the FGF-4 molecule, and maintaining its
secreting properties, new FGF ligands with increased binding efficiency to
specific receptors, with an emphasis on receptors expressed in cardiac myocytes
or heart endothelial cells. In addition we will try to create molecules which
are more stable and possibly less immunogenic than the ones presently
available. This investigation will require a preliminary study of exactly what
type of receptors are expressed in the heart tissues. Although the available
evidence suggests that it is mainly FGFR-1, this will have to be confirmed.
Furthermore, the design of new FGF-4 molecules would obviously benefit from the
knowledge of the tridimensional structure of FGF-4. We plan to collaborate with
a group of x-ray crystallographers to obtain this information. The crystal
structures of FGF-1 and FGF-2 are known, and some knowledge can also be derived
from those.


 


5





















 


The strategy that we plan to follow will
consist of creating either N-terminally shorter FGF-4 molecules, similar to the
K140 molecule previously described, chimeric molecules between FGF-4 and other
FGFs, or specific mutations in conserved and non-conserved domains of FGF-4 (or
K140).  The receptor binding domain(s) of
FGF molecules have not yet been precisely identified, possibly because they are
conformational domains. It is however known that specific regions of these
molecules contribute to receptor binding specificity (Seddon et al, 1995) and
this knowledge will help in designing new FGF molecules. According to the most
recently published data (Ornitz et al, 1996) FGF-4 is a highly potent activator
of FGFR-1, FGFR-2 (the IIIc isoforms) and of FGFR-4. These data however are at
some variance with our binding data, which indicate a binding affinity
decreasing from FGFR-2c > FGFR-lc > FGFR4.
While these discrepancies may be explained by a variety of factors, it is clear
that FGF-4 binds to and activates poorly the FGFR-2b form. Since this receptor
is preferentially expressed in cells of ectodermal origin (e.g. keratinocytes)
it would be desirable to obtain an FGF-4 molecule capable of activating FGFR-2b
as well as the other receptors. This will be the first objective of this
program.  Mutant FGF-molecules will be
expressed in mammalian cells as secreted proteins, or in bacteria as mature
proteins, and tested in vitro for receptor activation, mitogenicity, stability,
etc. Cell lines expressing specific FGF receptors as well as a variety of
endothelial cell lines are all available in our laboratory.


 


REFERENCES


 


Amaya, E., Musci, T.J. and Kirschner, M.W. (1991) Cell
66:257-270


 


Amaya E., Stein, P.A., Musci, T.J. and Kirschner, M.W.
(1993) Development 118:477-487


 


6





















 


Baird, A. (1994) Curr. Opin. Neurobiol. 4:78-86 


 


Basilico, C. and Moscatelli, D. (1992) Adv. Cancer
Res, 59:115-165 


 


Bellosta, P., Talarico, D., Rogers, D. and Basilico, C.
(1993) J of Cell Biol 121:705-713


 


Campochiaro, P.A., Chang, M., Ohsato, M., Vinores,
S.A., Nie, Z., Hjelmeland, L., Mansukhani, A., Basilico, C. and Zack, D.J.
(1996) J. Neurosci. 16:1679-1688


 


Deng, C.X., Wynshaw-Boris, A., Shen, M.M., Daugherty,
C., Ornitz, D.M. and Leder, P. (1994) Genes & Dev. 8:3045-3057


 


Feldman, B., Poureymirous, W., Papaioannou, V.E.,
DeChiara, T.M. and Goldfarb, M.(1995) Science 267:246-248


 


Galvin, B.D., Hart, K.C., Meyer, A.N., Webster, M.K.
and Donoghue, D.J. (1996) Proc. Natl. Acad. Sci. 93:7894-7899


 


Li, Y., Mangasarian, K., Mansukhani, A. and Basilico,
C. (1997) Oncogene, in press


 


Mima, T., Ueno, H., Fischman, D.A., Williams, L.T. and
Mikawa, T. (1995) Proc. Natl. Acad. Sci. 92:467-471


 


Muenke, M. and Schell V. (1995) TIGS 11:308-313


 


Naski, M.C., Wang, Q., Xu, J. and Ornitz, D.M. (1996)
Nature Genetics 13:233-237


 


7





















 


Ornitz, D.M., Xu, J., Colvin, J.S., McEwen, D.G.,
MacArthur, C.A., Coulier, F., Gao, G. and Goldfarb, M. (1996) J. Biol. Chem.
271:15292-15297


 


Peters, K., Werner, S., Liao, X., Wert, S., Whitsett,
J. and Williams, L. (1994) EMBO J. 13:3296-3301


 


Roghani, M., Mansukhani, A., Dell’Era, P., Bellosta,
P., Basilico, C., Rifkin, D.B. and Moscatelli, D. (1994) J. Biol. Chem.
269:3976-3984


Seddon, A.P., Aviezer, D., Li, L.Y., Bohlen, P. and
Yayon, A. (1995) Biochemistry 34:731-736


 


Spivak-Kroizman, T., Lemmon, M.A., Dikic, I., Ladbury,
J.E., Pinchasi, D, Huang, J., Jaye, M., Crumley,
G., Schlessinger, J., and Lax L. (1994) Cell 79:1015-1024


 


Temple, S. and Qian, X. (1995) Neuron
15:249-252


 


Webster, M.K. and Donoghue, D.J. (1996) EMBOJ.
15:520-527


 


Werner, S., Weinberg, W., Liao, X., Peters, K.G.,
Blessing, M., Yuspa, S.H., Weiner, R.L. and Williams, L.T. (1993) EMBO J.
12:2635-2643


 


Yamaguchi, T.P., Kendraprasad, H., Henkemeyer, M and
Rossant, J. (1994) Genes & Dev. 8:3032-3044


 


8





















 


Appendix
III. Collateral Therapeutics
Development Plan


 


Anglogenesis:
Myocardial Ischemia Draft Timeline


 


	
  Activity

  	
   

  	
  Duration

  	
   

  	
  Start

  	
   

  	
  Finish

  	
   

  	
  1997

  	
   

  	
  1998

  	
   

  	
  1999

  	
   

  	
  2000

  	
   

  	
  2001

  	
   

  	
  2002

  	
   

  	
  2003

  	
   

  	
  2004

  	
   

  
	
  Anglogensis: Myocardial Ischemia

  	
   

  	
  344w

  	
   

  	
  3/1/97

  	
   

  	
  10/3/03

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
   

  	
   

  
	
  FGF-4 gene Identified

  	
   

  	
  0w

  	
   

  	
  3/1/97

  	
   

  	
  3/1/97

  	
   

  	
  **

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  File IND 

  	
   

  	
  0w

  	
   

  	
  5/29/98

  	
   

  	
  5/29/98

  	
   

  	
   

  	
   

  	
  **

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  File PLA with FDA

  	
   

  	
  0w

  	
   

  	
  9/27/02

  	
   

  	
  9/27/02

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
  **

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  PLA approval

  	
   

  	
  0w

  	
   

  	
  10/3/03

  	
   

  	
  10/3/03

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
  **

  	
   

  	
   

  	
   

  



 


Project: Anglogenesis


Date: 3/3/97


 










	
  *

  	
  Task

  
	
  **

  	
  Milestone

  
	
  ***

  	
  Critical

  
	
  ****

  	
  Progress

  
	
  @

  	
  Summary

  



 





















 


Anglogenesis:
Peripheral Vascular Disease Draft Timeline


 


	
  ID

  	
   

  	
  Activity

  	
   

  	
  Duration

  	
   

  	
  Start

  	
   

  	
  Finish

  	
   

  	
  1997

  	
   

  	
  1998

  	
   

  	
  1999

  	
   

  	
  2000

  	
   

  	
  2001

  	
   

  	
  2002

  	
   

  	
  2003

  	
   

  	
  2004

  	
   

  
	
  1

  	
   

  	
  Anglogensis: Peripheral Vascular Diseas

  	
   

  	
  312w

  	
   

  	
  1/1/97

  	
   

  	
  12/24/03

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  2

  	
   

  	
  Pre-clinicial research

  	
   

  	
  98w

  	
   

  	
  1/1/97

  	
   

  	
  11/3/93

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  3

  	
   

  	
  Recombinant adenovirus/In vitro testi

  	
   

  	
  39w

  	
   

  	
  1/1/97

  	
   

  	
  9/30/97

  	
   

  	
  *

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  4

  	
   

  	
  In vivo studies(plg) 

  	
   

  	
  62w

  	
   

  	
  2/26/97

  	
   

  	
  6/5/98

  	
   

  	
  *

  	
   

  	
  *

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  5

  	
   

  	
  Toxcology studies

  	
   

  	
  28w

  	
   

  	
  5/6/98

  	
   

  	
  11/3/98

  	
   

  	
   

  	
   

  	
  *

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  6

  	
   

  	
  Pharmacodynamics study

  	
   

  	
  39.2w

  	
   

  	
  6/27/97

  	
   

  	
  5/27/98

  	
   

  	
  *

  	
   

  	
  *

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  7

  	
   

  	
  Formulation & stability studies

  	
   

  	
  39w

  	
   

  	
  10/1/97

  	
   

  	
  6/30/98

  	
   

  	
  *

  	
   

  	
  *

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  8

  	
   

  	
  File IND

  	
   

  	
  0w

  	
   

  	
  11/3/98

  	
   

  	
  11/3/98

  	
   

  	
   

  	
   

  	
  **

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  9

  	
   

  	
  Clinical

  	
   

  	
  221w

  	
   

  	
  10/1/97

  	
   

  	
  12/25/01

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
  @

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  10

  	
   

  	
  Pilot product manufacturing 

  	
   

  	
  70w

  	
   

  	
  10/1/97

  	
   

  	
  2/2/99

  	
   

  	
  *

  	
   

  	
  *

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  11

  	
   

  	
  Clinicial studies 

  	
   

  	
  143w

  	
   

  	
  12/16/98

  	
   

  	
  9/11/01

  	
   

  	
   

  	
   

  	
   

  	
   

  	
  *

  	
   

  	
  *

  	
   

  	
  *

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  12

  	
   

  	
  File
  market approval with FDA

  	
   

  	
  0W

  	
   

  	
  12/25/01

  	
   

  	
  12/25/01

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
  **

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  13

  	
   

  	
  Marketing

  	
   

  	
  52W

  	
   

  	
  12/26/01

  	
   

  	
  12/24/02

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
  @

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  14

  	
   

  	
  Large
  scale production

  	
   

  	
  52W

  	
   

  	
  12/26/01

  	
   

  	
  12/24/02

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
  *

  	
   

  	
   

  	
   

  	
   

  	
   

  
	
  15

  	
   

  	
  Product
  marketing/sales

  	
   

  	
  0W

  	
   

  	
  12/24/02

  	
   

  	
  12/24/02

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
   

  	
  **

  	
   

  	
   

  	
   

  	
   

  	
   

  



 


Project: Peripheral Vascular Dise


Date: 3/13/97


 










	
  *

  	
  Task

  
	
  **

  	
  Milestone

  
	
  ****

  	
  Progress

  
	
  @

  	
  Summary

  



 





















 


FIRST AMENDMENT TO AGREEMENT


 


This First Amendment to Agreement (hereafter “Amendment”)
is effective on April 28, 1998 by and between COLLATERAL THERAPEUTICS INC., a
corporation organized and existing under the laws of California, having a place
of business at 9360 Town Centre Drive, San Diego, California 92121 (hereafter “CORPORATION”);
and NEW YORK UNIVERSITY, a corporation organized and existing under the laws of
the State of New York, having a place of business at 70 Washington Square
South, New York, New York 10012 (hereafter “NYU”),


 


WITNESSETH:


 


WHEREAS, CORPORATION and NYU entered into a
certain agreement made and effective as of March 24, 1997 (the “Agreement”),
pursuant to which, inter alia. CORPORATION undertook to sponsor certain
research at NYU and NYU granted to CORPORATION a license to certain Research
Technology (as such term is defined in the Agreement); and


 


WHEREAS, CORPORATION and NYU desire to expand
the scope of the research under the terms and conditions of the Agreement as
specified herein; and


 


WHEREAS, NYU desires to perform such research;
and


 


WHEREAS, CORPORATION desires to provide
additional research funds to NYU for the performance of such research by NYU;


NOW, THEREFORE, in consideration of the
premises and the covenants, conditions and promises set forth below, the
parties hereto hereby agree as follows:


 


1.             Except as expressly provided for herein, all
terms and conditions of the Agreement shall remain in full force and effect.


 


2.             Terms which are defined in the Agreement shall
have the same meanings when used in this Amendment, unless a different
definition is given herein.


 





















 


3.             Section 1(m) of the
Agreement shall be, and hereby is, amended in its entirety so that, as amended,
said Section 1(m) shall read as follows:


 


1(m)        “NYU Research Project” shall mean the investigations
at NYU during the Research Period into the Field under the supervision of the
NYU Scientist in accordance with the research program, described in annexed
Appendix II and in annexed Exhibit A to the Amendment, each of which forms an
integral part hereof.


 


4.             Section 4(a) of the
Agreement shall be, and hereby is, amended in its entirety so that, as amended,
said Section 4(A) shall read as follows:


 


4(a)         As compensation to NYU for work to be performed on the
NYU Research Project during the Research Period, subject to any earlier
termination of the Research Project pursuant to Section 3(a) hereof,
CORPORATION will pay NYU the total sum of $675,000, payable in six successive
semiannual installments, commencing on the Effective Date; the first three
installments shall be in the amount of $100,000 each; the fourth installment
shall be in the amount of $175,000; the fifth and sixth installments shall be
in the amount of $100,000 each.


 


IN WITNESS WHEREOF, the parties hereto have executed
this Amendment as follows:


 


	
  NEW YORK UNIVERSITY

  	
  COLLATERAL THERAPEUTICS,
  INC,

  
	
   

  	
   

  
	
  By:

  	
  /s/ [ILLEGIBLE]

  	
   

  	
  By:

  	
  /s/ [ILLEGIBLE]

  
	
   

  	
   

  
	
  Title:

  	
  Vice
  President for Industrial Liaison

  	
  Title:

  	
  COO & CFO

  
	
   

  	
   

  
	
  Date

  	
  5.1.98

  	
  Date:

  	
  April 29, 1998

  
									



 





















 


EXHIBIT A


 


Specific Aims


 


Our plan is to construct a mammalian expression vector
expressing a truncated form of FGF-4 (K140, Bellosta, et al, J Cell. Biol.
121:705-713, 1993). This truncated form of FGF-4 was shown to result from the
spontaneous cleavage of unglycosylated FGF-4 molecules at position 66 of the
FGF-4 precursor protein, and was shown to have higher biological activity and
receptor binding affinity than the wild-type, mature FGF-4. Thus such a
molecule should have desirable properties in experiments aimed at inducing
collateral circulation in the myocardium of patients with coronary stenosis.


 


Research Plan


 


We have plasmids expressing the unglycosylated mutant
of FGF-4, whose protein product is cleaved in tissue culture by a cellular
protease to produce K140. However, since this protease is unknown, there is no
guarantee that it may be expressed in all tissues and operate efficiently in
vivo.  Therefore we will construct a
plasmid which should only produce directly the K140 protein. The strategy to
produce this plasmid will be as follows: We will create two deletions (see
diagram) linking the FGF-4 signal peptide to the N-terminus of K140, that
should produce three possible peptides differing only in the number of Alanines
(1, 2 or 3) at their N-terminus.
The FGF-4 signal peptide naturally causes cleavage in two positions in the
FGF-4 precursor protein, downstream of A30 or A31 and presumably this type of
cleavage will be preserved in the internally deleted molecule, which should
produce mature forms of 140 amino acids (K140, 2A at the N-terminus) 141 (3A at
the N-terminus), or 139 (1A at the N-terminus). 
We do not know whether the presence of an additional Alanine or the lack
of one will alter the properties of K140, although that seems unlikely, and
that is the reason why it may be useful to have two constructs.  The construction of these plasmids although
apparently simple is going to be laborious because of the high GC content of
the 5’ of the FGF-4 cDNA. The method which will be used is the following:


pGemKS3A (encoding
the wild type FGF-4) will be linearized with Pvul and subjected to PCR. Primers
used will be GGG GGC GCC GCC GCG GCC GTC CAG AGC GGC GCC GGC (spanning the
deletion from nt 342 to 441) and SP6. The PCR product will be cloned in the TA
vector (Invitrogen) and used as a template for two rounds of PCRs with oligos
extending the 5’ end till bp 258 of the FGF-4 cDNA (including a Eco47III site
at 281 bp). These two rounds of PCRs will be performed by preheating the
samples at 94° C for 10 minutes and PCR performed as before except that the
annealing temperature will be 65° C and include 10% DMSO. For each round the
PCR product will be gel purified. The product will be cloned into TA vector and
sequenced to verify the deletion junction. The presence of the Eco47III site
will be verified by restriction digestion. This site will be used to insert the
Eco47III fragment from pGEMKS3B containing the 281 bp 5’ end fragment of the
FGF-4 cDNA. All PCR generated mutations downstream of the deletion will be
eliminated by cloning the NgoM1 fragment containing the deletion into the
PGEMKS3B vector cut with NgOM1 in order to reconstitute the deletion-containing


 





















 


full length coding sequence. Finally, the EcoRI
fragment containing the full length cDNA encoding the internally deleted FGF-4
will be isolated, and subcloned into p91023B mammalian expression vector.


 


Once
the deleted molecules are constructed, they will be inserted into a mammalian
expression vector and tested for expression, production and secretion of the
correct protein, and biological activity of the protein produced. This will be
tested in a mitogenic assay comparing it to known amounts of recombinant FGF-4,
and by testing the ability of the plasmids encoding the deleted molecule to
transform NIH3T3 cells. We will also determine the stability of the protein
produced upon thermal inactivation. If these assays prove satisfactory, the
mutated cDNA will be available for insertion into Adenovirus vectors and
testing in the pig model of
coronary stenosis.


 


2





















 


	
  1

  	
   

  	
  MSGPGTAAVALLPAVLLALLAPWAGRGGAAAPTAPNGTLEAELERRWESL

  	
   

  	
  50

  
	
   

  	
   

  	
   

  	
   

  	
   

  
	
  51

  	
   

  	
  VALSLARLPVAAQPKE K140AAVQSGAGDYLLGIKRLRRLYCNVGIGFHLQALP

  	
   

  	
  100

  
	
   

  	
   

  	
   

  	
   

  	
   

  
	
  101

  	
   

  	
  DGRIGGAHADTRDSLLELSPVERGVVSIFGVASRFFVAMSSKGKLYGSPF

  	
   

  	
  150

  
	
   

  	
   

  	
   

  	
   

  	
   

  
	
  151

  	
   

  	
  FTDECTFKEILLPNNYNAYESYKYPGMFUALSKNGKTKKGNRVSPTMKVT

  	
   

  	
  200

  
	
   

  	
   

  	
   

  	
   

  	
   

  
	
  201

  	
   

  	
  HFLPRL

  	
   

  	
  206

  



 


1)      Amino acid sequence of the human K-FGF precursor
protein. The hydrophobic signal peptide is underlined. The arrows indicate the
sites of cleavage of the mature, secreted form of K-FGF. Asterisks indicate the
glycosylation signal. The bracket indicates the sites of cleavage which
generates the 15-kD K140 protein.


 


MSGPGTAAVALLPAVLLALLAPWAGRGGAAAAAVQSGAGD


 


MSGPGTAAVALLPAVLLALLAPWAGRGGAAAAVQSGAGD


 


2)      Sequence encoded in the deleted molecules. The arrows
indicate the expected sites of cleavage downstream of the signal peptide.


 


3





















 


SECOND AMENDMENT TO AGREEMENT


 


This Second Amendment to Agreement (hereafter “Second
Amendment”) is effective on March 24, 2000 by and between COLLATERAL
THERAPEUTICS INC., a corporation organized and existing under the laws of
Delaware, having a place of business at 11622 El Camino Real, San Diego,
California 92130 (hereafter “CORPORATION”); and NEW YORK UNIVERSITY, a
corporation organized and existing under the laws of the State of New York,
having a place of business at 70 Washington Square South, New York, New York
10012 (hereafter “NYU”).


 


WITNESSETH:


 


WHEREAS, CORPORATION and NYU entered into a
certain agreement made and effective as of March 24, 1997 and First Amendment
to Agreement effective as of April 28, 1998 (together the “Agreement”),
pursuant to which, inter alia, CORPORATION undertook to sponsor certain
research at NYU and NYU granted to CORPORATION a license to certain Research
Technology (as such term is defined in the Agreement); and


 


WHEREAS, CORPORATION and NYU desire to extend the term
of the research period and expand the scope of the research under the terms and
conditions of the Agreement as specified herein; and


 


WHEREAS, NYU desires to perform such research;
and


 


WHEREAS, CORPORATION desires to provide
additional research funds to NYU for the performance of such research by NYU;


 


NOW, THEREFORE, in consideration of the
premises and the covenants, conditions and promises set forth below, the
parties hereto hereby agree as follows:


 


1.             Except as expressly
provided for herein, all terms and conditions of the Agreement shall remain in
full force and effect.


 


2.             Terms which are
defined in the Agreement shall have the same meanings when used in this Second
Amendment, unless a different definition is given herein.


 


3.             Section 1(1) of the
Agreement shall be, and hereby is, amended in its entirety so that, as amended,
said Section 1(1) shall read as follows:


 


1(1)        “Research Period” shall mean the six-year
period commencing on the Effective Date hereof and any extension thereof as to
which NYU and CORPORATION shall mutually agree in writing.


 


4.             Section 1(m) of the
Agreement shall be, and hereby is, amended in its entirety so that, as amended,
said Section 1(m) shall read as follows:


 


1(m)        “NYU Research Project” shall mean the
investigations at NYU during the Research Period into the Field under the
supervision of the NYU Scientist in accordance with the research program,
described in annexed Appendix II, in annexed Exhibit A to the First Amendment,
and in annexed Exhibit A to the Second Amendment each of which forms an
integral part hereof.


 





















 


5.           Section 4(a) of the
Agreement shall be, and hereby is, amended in its entirety so that, as amended,
said Section 4(A) shall read as follows:


 


4(a)        As compensation to
NYU for work performed on the NYU Research Project during the Research Period,
subject to any earlier termination of the Research Project pursuant to Section
3(a) hereof, CORPORATION will pay NYU (i) the sum of $675,000, payable in six
successive semiannual installments, commencing on the Effective Date; the first
three installments shall be in the amount of $100,000 each; the fourth
installment shall be in the amount of $175,000; the fifth and sixth
installments shall be in the amount of $100,000 each; NYU acknowledges that as
of September 28, 1999, CORPORATION has paid this amount in full; and (ii) the
sum of $396,000 payable in six, equal, successive semiannual installments, the
first of which shall be due within fifteen (15) days after the signature of this
Second Amendment by the last party to sign (the “Execution Date”).


 


IN WITNESS WHEREOF, the parties hereto have executed
this Amendment as follows:


 


	
  NEW
  YORK UNIVERSITY

  	
  COLLATERAL THERAPEUTICS, INC.

  
	
   

  	
   

  
	
  By:

  	
  /s/ [ILLEGIBLE]

  	
   

  	
  By:

  	
  /s/ [ILLEGIBLE]

  
	
   

  	
  Vice Dean for Industrial Liaison

  	
   

  	
   

  	
  President and Chief Operating Officer

  
	
   

  	
  and Research Administration

  	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  Date:

  	
  7/24/00

  	
   

  	
  Date:

  	
  07-05-00

  	
   

  
									



 





















 


EXHIBIT A


to the Second
Amendment


 


Structural and biological analysis of the
interaction of FGF4 with FGF receptors


 


INTRODUCTION


 


The family of fibroblast growth factors (FGF)
is one of the largest family of growth factors with a multiplicity of
biological functions which impact on a variety of biological processes. FGFs
can affect the proliferation and differentiation of a variety of cells of
mesenchymal and neuro-ectodermal origin, and it has become evident that,
clearly the most important role of FGF signaling is during development (1,2).


 


FGF signaling has been shown to play a major
role in a number of developmental processes, including embryonic mesoderm
induction and post-implantation blastocyst development. FGF signaling is also
essential for the development of limb and lungs and unregulated FGF signaling
leads to a variety of human bone morphogenic disorders, including dwarfism and
craniosynostosis syndromes. FGFs are also potent angiogenic factors; the
sprouting of new blood vessels from a pre-existing one is an essential
physiological process in development, but also plays a major role in human
diseases, such as diabetic retinopathy, arteriosclerosis, and cancer (2).


 


The FGF family includes at present twenty-two
members including four (FGF11-  14, FHF) which may not be bonafide FGFs. Ten
(FGF1-10) are best characterized (1,2). FGFs action is mediated by their interaction
with high affinity receptors. FGF receptors (FGFR) also constitute a family of four
related, membrane spanning tyrosine kinases, with a conserved structure, including three
Ig-like loops in the extracellular domain, and a split cytoplasmic tyrosine kinase domain
(1-3). The FGFR genes produce m-RNAs which can undergo alternative splicing to
produce different FGFR isoforms. 
Splicing alternatives which result in modification of the sequence of the third
Ig loop are biologically important, as they can alter the binding specificity of the
receptor. Binding


 


1





















 


of FGFs to their receptors shows only partial and
overlapping specificity, such that one receptor can bind multiple ligands with
variable affinity (3,4).


 


This complex system of ligand-receptor
specificity is also characterized by the fact that FGFs need to interact with
the so called low-affinity receptors, which consist of cell surface Heparan
sulfate proteoglycans (HSPG) and can be replaced by soluble heparin, in order
to activate FGFRs (5). Receptor activation requires interaction of FGFs with
HSPG or heparin. These molecules simultaneously interact with the receptor
forming a trimeric receptor-heparin-ligand structure (6,7). Heparin oligosaccharides
also exhibit considerable specificity in ligand/receptor interaction (8). Thus
the interaction of FGFs with their receptors is regulated at a variety of
levels, including growth factor expression, specificity of receptor binding and
interaction with HSPG.


 


FGFs range in molecular weight from 18 to 29
kDa and show 13-71% aminoacid identity. All members share a conserved,
centrally located, “core” region, of about 100 aminoacids, common to all FGFs
(1,2). Outside of this “core” structure, their sequences diverge considerably.


 


The FGFR family includes four identified genes
and numerous subtypes of alternative spliced isoforms, particularly within the
FGFR1 and FGFR2 genes (3,9). FGFRs have an extracellular domain which binds the
ligand, a transmembrane segment and a cytoplasmic tyrosine kinase domain, which
can be activated by phosphorylation. The extracellular portion comprises three
Ig-like domains, Dl, D2, and D3, with an acidic stretch of approximately 30
residues, between Dl and D2. Isoforms generated by alternative splicing include
receptors that lack Ig-like domain D1 or both D1 and the acidic box, as well as
variants having two alternative sequences, called IIIb and IIIc for the
C-terminal half of the third Ig-like D3 domain (3,9). As alluded to above,
these latter splice forms alter ligand specificity, in agreement with the
finding that D3 is an


 


2





















 


important site for ligand binding (see below).
Furthermore, the IIIc forms of FGFR1 and FGFR2 are expressed exclusively in
mesenchymally derived cells, while the IIIb forms are expressed in cells of
epithelial origin. Members of the FGFs family bind FGFRs with varying
affinities. FGF1 binds with high affinity to the four receptors and to all the
known isoforms. FGF4 binds FGFR1 and FGFR2 with high affinity, and FGFR3 and
FGFR4 with a lower affinity. FGF3, FGF7 and FGF10 only interact with the IIIb
splice variant form of FGFR2, (also known as the KGFR)(4).


 


Experiments of gene knock-out in mice have
revealed considerable redundancy in the FGF gene family, perhaps not surprising
since multiple FGFs can activate the same receptor (2). This could be one of
the reasons why so far an angiogenic phenotype has not been demonstrated in any
single FGF knock-out mice. Nevertheless, a considerable body of evidence
indicates that most of the FGFs are potent angiogenic agents in a variety of
experimental animal models. The most important FGFs for angiogenesis are
probably those which can interact with FGFR1 and FGFR2 (IIIc), which are the
major FGFRs expressed by endothelial cells, FGF4, on which we have concentrated
most of our studies, is angiogenic in mice, rabbit and pig hearts.


 


Recently the crystal structures of FGF2 bound to the
extracellular domain of FGFR1, and that of FGF1 bound to FGFR2 have been
determined (6,7). The crystal structure of the extracellular ligand binding
domain of FGFRl (D2 and D3) in complex with FGF2 at 2.8 A resolution shows two
FGF2 molecules (residues 16-144), two D2-3 molecules (residues 149-359) in 1:1
complex, and four sulfate ions (6). The structure of the FGF1/FGFR2 complex is
very similar (7). The dimeric structure is stabilized by direct
receptor-receptor interaction (D2-D2) and interaction between FGF2 and D2 of
the other receptor in the dimer. The two FGF2 molecules are on opposite sides
of the dimer and are not in contact. A positively charged canyon is formed
between the two


 


3





















 


D2 domains of FGFR, extending onto the heparin binding
sites of the ligand. This canyon is likely to make-up the heparin binding site
of the receptor. A ternary structure is formed, probably occurring first by
binding of FGF to FGFR in a 1:1 complex, which then associate into signaling
dimers in the presence of heparin (6). Thus the specificity of FGF interaction
with FGFR depends on a variety of factors: 1) Binding of the ligand to the D2
and D3 domains and intervening sequences. 2) Binding of the ligand as well as
of the receptor to heparin or specific heparin sulfate proteoglycans. 3)
Interaction between the D2 domains of dimerizing receptor molecules.
Modification of any of these parameters would alter the ability of each ligand
to interact with specific FGER, suggesting that a structural and mutational
analysis of the precise contact points between ligand and receptor, and/or
ligand and heparin could be fruitful for the creation of novel FGF molecules.


 


SPECIFIC AIMS


 


1.       To obtain the crystal structure of FGF4 bound to its
highest affinity receptor, FGFR2.


 


2)      To identify the FGF4 residues that contact FGFR and
mediate its activation.


 


3)      To utilize the knowledge generated by the studies
described above to create FGF4 mutants with novel or broader affinity to FGFR
and possibly FGFR antagonists.


 


PRELIMINARY REULTS


 


In order to obtain a detailed insight into the
three-dimensional structure of FGF4, we determined its crystal structure at
2.8A resolution (Figure 1). For these studies we used a cDNA encoding only a
truncated FGF4 molecule missing the first 43 N-terminal


 


4





















 


aminoacids of the mature protein. The protein
expressed and purified from bacteria has full biological activity, in line with
the observation that a truncated FGF4 molecule lacking the 35 N-terminal residues
(32-67; residues 1-31 encode for the signal peptide) retains potent biological
activity and displays higher receptor binding affinity compared to that of full
length FGF4 (10). Based on the crystal structure, we predicted that residues
F129, FF135/136, FF15Q/151, Y172, that are exposed to the surface of the
molecule, could be relevant for FGF4 receptor binding. However, when FGF4
molecules in which these residues had been mutated were tested for biological
activity, some of these predictions turned out not to be correct, since
mutation of these residues did not affect the biological activity of FGF4 (see
Table 1). Because of the similarity between FGF2 and FGF4, we thus superimposed
the structure of FGF4 on that of FGF2 in complex with FGFR1 (Figures 2 and 3).
By analyzing the data of the complex, we identified residues Y87, Y166, E159,
L203, R205 as possible primary interacting sites between FGF4 and D2 and D3 of
FGFR1. Some of these predictions are in agreement with previous studies on the
importance of these FGF residues in receptor binding. Replacement of Y24 and
Y103 by alanine in FGF2, which correspond to residues 87 and166 in FGF4,
resulted in a dramatic decrease in FGFR1 binding. Substitution of E96 with
alanine in FGF2, corresponding to E196 in FGF4, also results in more than 100
fold reduction in receptor binding (11).


 


We
have created a series of FGF4 mutants by site directed mutagenesis (Table 1).
We have expressed and produced the mutants in a mammalian system using COS
cells and subsequently analyzed the biological activity of these mutants in a
DNA synthesis assay, using NIH3T3 cells (Figure 4). The results of these
experiments are summarized in Table 1. Unfortunately, many of the mutants
constructed appear not to be efficiently produced in COS cells. Thus we plan to
express these mutants in bacteria, purify these


 


5





















 


factors and determine their ability to bind and
activate FGF receptors in comparison to that of wild type FGF4 (see below). It
should also be mentioned that the use of bacterially produced, recombinant FGF4
molecules will allow us much more precise determination of their receptor
affinity, interaction with heparin, etc. than is possible using supernatants
from transfected COS cells.


 


AIM 1. Crystal structure of FGF4 bound to FGFR2.


 


As discussed above, a number of predictions on
the FGF4 domains which interact and play a role in the activation of FGFR were
made on the basis of the crystal structure of this growth factor in isolation,
but these predictions are not always correct, as clearly conformational changes
occur during receptor-ligand interaction or interaction of the ligand/receptor
with heparin. The superimposition of FGF4 on the known structure of FGF2 bound
to FGFR1 allows more accurate predictions, but still suffers from the problem
of relying on the conservation of FGF structure and is unlikely to highlight
unique features of FGF4 or of its interaction with FGFR (Figures 2 and 3). We
plan therefore to obtain the crystal structure of FGF4 bound to FGFR2, its
highest affinity receptor. We will use the FGF4 construct described above
(missing the 43 N-terminal aminoacids) because it has full biological activity
and easier to express and purify to high concentration. Similarly we will use
FGFR2 molecules containing only D2 and D3 as previously used for the structure
of the FGF2/FGFR1 complex. Proteins will be expressed in E. Coli and we will
follow the strategy described in ref. (6).


 


Another interesting aspect of the interaction between
FGF and FGFR is the role and sites of interaction of heparin with the receptor
and the ligand (Figure 3). The two structures recently described were obtained
in the absence of heparin. Thus the heparin sites of interaction were inferred
from the position of sulfate ions on the structure. It


 


6





















 


should be very interesting to verify whether these
hypotheses are correct, and whether they also apply to FGF4, and therefore we
will attempt to obtain the structure of FGF4/FGFR2 complex in the presence or
absence of specific heparin oligosaccharides (classical heparin preparations
are too heterogeneous for crystallographic studies). This will be important for
future studies and possible modification of the heparin binding sites on the
ligand or the receptor.


 


AIM 2. Identification of residues
that are relevant for the binding of FGF4 with its high affinity receptors.


 


Since the mutants, FF150/151GG, FF135/136AA,
Y166A and N167A are not produced and/or secreted in COS cells (see Table 1), we
have decided to change our strategy and to produce all mutants in
bacteria.  As described under Preliminary
Results we superimposed the structure of FGF4 with that of FGF2 in complex with
D2 and D3 of FGFR1 (Figure 2). Based on the model that was generated, we have
identified residues Y87, Y166, E159, L203, R205 of the FGF4 as likely to be
directly involved in high affinity receptor binding. By molecular dynamics
calculations however, we have determined that the residues F129, F136 and F151
could also be relevant for the stability of the binding of FGF4 with the
receptor.


 


Switch aminoacid mutagenesis has and will be carried
out following the Quick Change TM site directed mutagenesis kit (Strategene)
using the pET15b-FGF4 vector as a template. This plasmid was used for the
production of FGF4 used for the crystal structure. In the pET15b vector the
FGF4 cDNA was cloned under the LacZ T7 inducible promoter (Novagene). The
presence of the mutation in the cDNA will be analyzed by sequencing and the
production and purification of the mutant protein will be done according to the
protocol described in Plotnikov et al (6), The biological activity


 


7





















 


of
the purified proteins will be tested in a DNA synthesis assay using NIH3T3
cells and compared to that of wild-type FGF4. Studies on the affinity of the
FGF4 mutants with its high receptors will be performed by Scatchard analysis on
a CHO cell line expressing FGFR2. Briefly, the purified protein will be
iodinated using the 125-1 labeled Bolton-Hunter reagent. Scatchard assays will
be performed as described in (10). Relatives dissociation constants (Kd) of the
mutants will be calculated and compared with that of wt-FGF4. These results
will be confirmed also by competition experiments for binding to FGFR2, using
iodinated FGF4 or FGF2 as the ligand (10). As previously described, FGF2 binds
FGFR2 with the same high affinity as FGF4 (4). In order to study if binding of
the FGF4 mutants with its high affinity receptor results in weaker receptor
activation, we will also analyze the ability of the mutants to phosphorylate
FGFRs. These experiments will be performed by conventional receptor
phosphorylation assays using CHO cells expressing FGFR2. Briefly, CHO-FGFR2
cells will be serum starved for 24 hours and treated for 10 minutes with 100 ng
of either wt FGF4 or the mutants. Cells will be lysed in 1% Triton buffer and
subjected to immunoprecipitation using anti-FGFR2 antiserum. Immunoprecipitates
will be resolved in SDS-PAGE and proteins blotted on a nitrocellulose membrane.
Immunoblotting will be performed using anti phosphoytyrosine and anti FGFR2
antisera.


 


While
these experiments are in progress, we should be able to obtain the crystal
structure of FGF4 bound to FGFR2, As discussed above, this is likely to provide
more precise information on the FGF4/FGFR2 interaction, that should complement,
extend or correct some of the predictions made on the basis of superimposing
FGF4 on the FGF2/FGFR1 complex. In particular, the results of the experiments
in the presence of heparin should be very useful. Thus, similarly to what is
described above, we will


 


8





















 


generate new mutants of FGF4 and test their ability to
interact with the receptor, and with heparin.


 


Taken together, these experiments should
provide a comprehensive description of the mechanisms determining the
interaction of FGF4 with its receptor and suggest strategies to design mutated
forms of FGF4 with increased, decreased, or broader binding of FGFRs.


 


AIM 3. Creation of FGF4 mutants with novel or broader binding
specificity to FGFR.


 


Structure-based site directed mutagenesis in FGFs identified two
putative receptor binding sites: a primary site that contributes to most of the
high affinity interaction of FGFs with FGFRs, and a secondary site that
exhibits low receptor binding affinity but is required for receptor activation.
It was suggested that the secondary binding site may confer to FGFs the ability
to bind the FGFRs with different affinities (12). FGF7 is ideal for this type
of study since it only recognizes the FGFR2bIII isoform, or KGFR (13). Since
FGF4 does not bind KGFR with appreciable affinity (4), it is possible to study
if substitution of particular residues of the FGF7 molecule into the FGF4
increases binding of the new chimeric FGF4/FGF7 for KGFR. Based on the model
that was generated with the crystal structure of FGF7 (14), we believe that the
b4/b5 loop from aminoacid 103 to 107 (RTVAV) of FGF7 is probably relevant
for its biological activity and for its receptor binding affinity to KGFR.
Based on subsequent studies of this and other models, we also predict that the
substitution of A106 of FGF7 with valine would increase the probability of binding
of the new chimera to KGFR. We will substitute the residues SRVER from
aminoacid 119 to 123 of FGF4 with the aminoacid RTVVV from aminoacid 103 to 107
of FGF7.


 


9





















 


Switch aminoacids mutagenesis will be carried out by
site directed mutagenesis as described above, using the pET15b-FGF4 vector as a
template. The protein will be expressed in bacteria and purified as described
(6). The chimeric FGF4/FGF7 will be tested for its ability to bind and activate
KGFR. We will initially express the KGFR in CHO-DG44 cells which do not express
any of the member of the FGFR family (1). The plasmid encoding for the KGFR
cDNA will be transfected into the CHO cells together with an excess of a
plasmid encoding for the resistance for Neomycin. Positive clones will be
selected and expression of the receptor will be tested in western blot analysis
using an antibody that recognizes the KGFR. Affinity of the chimera FGF4/FGF7
for KGFR will be calculated and compared with that of wt-FGF7. Schatchard
analysis and competition experiments will be performed using CHO expressing
KGFR or FGFR2, for which FGF4 has high affinity (4). We will also analyze the
ability of the chimeric FGF4/FGF7 ligand to activate the KGFR receptor in
phosphorylation experiments. Briefly, CHO cells expressing either the KGFR or
the FGFR2 will be serum starved for 24 hours and increasing concentration of
FGF4/FGF7 will be added to the cells for 10 minutes. KGFR will be
immunoprecipitated from total cell lysates and its level of phosphorylation
will be analyzed by immunoblot experiments using antiphosphotyrosine antiserum.
As a positive control we will use wt-FGF4 and wt-FGF7 for FGFR2 and KGFR
phosphorylation.


 


While these studies will provide useful information on
the possibility of designing new FGF4 molecules, it is not clear whether the
chimeric FGF4/FGF7 molecule will have practical uses. We will therefore construct new mutants of FGF4 based on
the structure of FGF4 complexed with FGFR2, compared to that of the FGFR2/FGFR1
complex. It is very difficult at this moment to indicate which mutations we
will perform; as the choice will dependent on identifying the critical sites of
FGF4


 


10





















 


interaction with its receptor and heparin. The
goal would be that of creating FGF4 molecules with tighter “fit” for the
receptors, or perhaps capable of a more stable association. In the long run,
although it is again difficult
to outline a precise strategy at the moment, we would like to create FGFR
antagonists, i.e. ligands which will bind FGFR with high affinity, but will not
activate signal transduction. These ligands would therefore compete with FGF4
(or other FGFs) and inhibit receptor activation. Such ligands could in principle
be truncated FGF polypeptides which only interact with the D2 or D3 domain of
the receptor or molecules which are incapable of binding the heparin “bridge”
spanning the heparin binding canyon of the receptor.


 


REFERENCES


 


1)      Basilico C., and Moscatelli, D. 1992. The FGF family
of growth factors and oncogenes. Adv. Cancer Res. 59:115-165.


 


2)      Goldfarb, M. 1996. Functions of fibroblast growth
factors in vertebrate development. Cytokine & Growth Rev. 7:311-325


 


3)      Johnson, D.E., and Williams, L.T. 1993. Structural and
functional diversity in the FGF receptor multigene family. Adv. Cancer Res.
60:1-41.


 


4)      Ornitz, D.M., Xu, J., Colvin, J.S., McEwen, D.G.,
MacArthur, C.A., Coulier, F., Gao, G., and Goldfarb, M. 1996. Receptor
specificity of the fibroblast growth factor family. J. Biol. Chem.
271:15292-15297.


 


5)      Yayon, A., Klagsbrun, M., Esko, J.D., Leder, P., and Ornitz, D.M. 1991. Cell
surface, heparin-like molecules are required for binding of basic fibroblast
growth factor to its high affinity receptor. Cell 64:841-848.


 


6)      Plotnikov, A.N., Schlessinger, J., Hubbard, S.R., and Mohammadi,
M. 1999. Structural basis for FGF receptor dimerization and activation. Cell
98:641-650.


 


7)      Stauber, D.J., DiGabriele, A.D., and Hendrickson, W.A.
2000. Structural interactions of fibroblast growth factor receptor with its
ligands. Proc. Natl. Acad. Sci. USA 97:49-54.


 


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8)      Ornitz, D.M. 20000. FGFs, heparan sulfate and FGFRs:
complex interactions essential for development. Bioessays 22:108-112.


 


9)      McKeehan, W.L., Wang, F., and Kan, M. 1998. The heparan
sulfate-fibroblast growth factor family: diversity of structure and function.
Prog. Nucleic Acid Res. Mol. Biol. 59:135-176.


 


10)    Bellosta, P., Talarico, D., Rogers, D., and Basilico, C. 1993.  Cleavage of K-FGF produces a truncated
molecule with increased biological activity and receptor binding affinity. J
Cell Biol. 121:705-713.


 


11)    Zhu, H., Ramnarayan, K. Anchin, J., Miao, W.Y., Sereno, A., Millman, L.,
Zheng, J., Balaji, V.N., and Wolff, M.E. 1995. Glu-96 of basic fibroblast
growth factor is essential for high affinity receptor binding. Identification
by structure-based site-directed mutagenesis. J. Biol. Chem. 270:21869-21874.


 


12)    Sher, I., Weizman, A., Lubinsky-Mink, S., Lang, T., Adir, N.,
Schomburg, D., and Ron, D. 1999. Mutations uncouple human fibroblast growth
factor (FGF)-7 biological activity and receptor binding and support broad
specificity in the secondary receptor binding site of FGFs. J. Biol. Chem.
274:35016-35022.


 


13)    Werner, S. 1998.
Keratinocyte growth factor: a unique playaer in epithelial repair processes.
Cytokine Growth Factor Rev. 9:153-165.


 


14)        Osslund, T.D., Syed, R., Singer, E., Hsu, E.W.,
Nybo, R., Chen, B.L., Harvey, T., Arakawa, T., Narhi, LO., Chirino, A., and
Morris, C.F. 1998. Correlation between the 1.6 A crystal structure and
mutational analysis of keratinocyte growth factor. Protein Sci. 7:1681-1690.


 


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figures


 


Figure 1.       Ribbon diagram of the FGF4 structure. The amino and
carboxyl termini are indicated by NT and CT. The  b strands of FGF4 are
labeled from 1 to 12, starting from the N-terminus.


 


Figure 2.        Superimposition of the FGF4 molecule in place of FGF2
binding to the D2 (green) and D3 (blue) domains of FGFRl. As can be seen by
comparison with ref (6), although the “fit” is quite good, three regions of
FGF4 (marked in red) clash with the receptor structure, highlighting the
limitations of this “superimposition” approach.


 


Figure 3.       A. Molecular surface representation of the putative
FGF4-FGFR1 complex, viewed from the top. FGF4 is in orange, the D2 domains of
FGFRl are in green.


B.     Surface
change distribution of the complex, also viewed from the top. Blue and red
represent positive or negative electrostatic potential, respectively. The
positively charged canyon, i.e. the putative heparin binding site, runs between
the two D2 domains in the dimer extending to the adjoining ligands. Sulfate
ions are marked.


 


Figure 4.       Mitogenic activity in quiescent NIH3T3 cells of FGF4
mutants. Cells were serum starved in 0.5% serum for 48 hrs. Supernatant from
COS cells containing the secreted proteins was added to the medium at the
relative concentration of 1:100, 1:250 and 1:1000 as indicated. Cells were labeled
after 16 hrs. with 1 μCi of 3H-Thymidine
for 6 hrs. and radioactivity incorporated into DNA was counted after TCA
precipitation.


 


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Table 1.
BIOLOGICAL ACTIVITY OF FGF4 MUTATIONS


 




	
  FGF4 WT

  	
  +++

  
	
   

  	
   

  
	
  Y87A

  	
  -

  
	
   

  	
   

  
	
  F129A (the F129 residue is
  unique to FGF4)

  	
  -

  
	
   

  	
   

  
	
  F135A

  	
  +/-

  
	
   

  	
   

  
	
  F136A

  	
  NT

  
	
   

  	
   

  
	
  FF135/136AA

  	
  not
  expressed/secreted

  
	
   

  	
   

  
	
  F150A

  	
  +++

  
	
   

  	
   

  
	
  F151A

  	
  NT

  
	
   

  	
   

  
	
  FF150/151GG

  	
  not expressed/secreted

  
	
   

  	
   

  
	
  E159A

  	
  NT

  
	
   

  	
   

  
	
  Y166A

  	
  not expressed/secreted

  
	
   

  	
   

  
	
  N167A

  	
  not expressed/secreted

  
	
   

  	
   

  
	
  Y172A

  	
  +++

  
	
   

  	
   

  
	
  L203A

  	
  NT

  
	
   

  	
   

  
	
  R205A (unique to FGF4)

  	
  NT

  






 


Mitogenic activity of the FGF4 mutants was calculated
by Thymidine incorporation assays as described in Figure 4 +++ high activity;
+/- low activity (less than 20,000 cpm at 1:100 conc.); - no activity (no
increase in cpm over background); NT = not tested.


 


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