An Official Journal
`of the
`American Association
`for
`Cancer Research
`
`Clinical
`Cancer
`Research
`
`Unlv. of Minn.
`Bio-Medical
`Library
`
`.__ __ 11 7 ___!9~5=--------
`
`November 1995 • Volume 1 Number 11
`PP. 1235-1437 • ISSN 1078-0432
`
`LILLY EX. 1008 - 1/12
`
`

`
`Notice to Members of the American Association for Cancer Research
`
`Ofiicers for 1995-1996
`President: Joseph R. Bertino, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
`President Elect: Louise C. Strong, UT M. D. Anderson Cancer Center, Houston, TX 77030
`Treasurer: Bayard D. Clarkson, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
`Executive Director.‘ Margaret Foti, AACR, Public Ledger Bldg., Suite 816, Philadelphia, PA 19106-3483
`
`Annual Dues
`
`The annual dues of active members of the American Association for Cancer Research are $160, $40 of which may be applied to a
`subscription to Clinical Cancer Research. Corresponding members of the Association will be charged an appropriate fee to offset
`second-class postage costs. Payment of dues and changes of address of members of the Association should be sent promptly to
`Margaret Foti, Executive Director, American Association for Cancer Research, Public Ledger Bldg., Suite 816, 150 South
`Independence Mall West, Philadelphia, PA 19106-3483; Telephone: (215) 440-9300; FAX: (215) 440-9313.
`
`Submission of Manuscripts
`Clinical Cancer Research, a new journal of the American Asso-
`ciation for Cancer Research, publishes original articles describing
`clinical research on the cellular and molecular characterization,
`prevention, diagnosis, and therapy of human cancer. Its focus is on
`innovative clinical
`research and translational
`research which
`bridges the laboratory and the clinic. Clinical Cancer Research is
`especially interested in clinical trials evaluating new treatments for
`cancer; research on molecular abnormalities that predict incidence,
`response to therapy, and outcome; and laboratory studies of new
`drugs and biological agents that will
`lead to clinical
`trials in
`patients. All submissions undergo peer review. Papers should be
`sent to John Mendelsohn, M.D., Editor-in-Chief, Clinical Cancer
`Research, Department of Medicine, Memorial Sloan-Kettering
`Cancer Center, 1275 York Ave, New York, NY 10021; Tele-
`phone: (212) 639-5878; FAX: (212) 717-3629.
`
`Back Issues and Single Copy Sales of the Journal
`Copies of back stock of the journal may be ordered from
`Clinical Cancer Research, P.O. Box 3000, Denville, NJ 07834
`[Telephone: (800) 875-2997 or (201) 627-2427; FAX: (201)
`627-5872]. As long as supplies permit, single copies will be
`sold by this company at $12.50/copy. Orders outside the U.S.
`add $1.50/copy to offset postage costs.
`
`Advertisements
`
`Advertisement insertion orders and copy must be received one
`month prior to the date of issue in which the advertisement is
`to be published. The journal is mailed on approximately the 1st
`day of the month of issue. Inquiries about advertising should be
`directed to: M. J. Mrvica Associates, Inc., 155 South White
`Horse Pike, Berlin, NJ 08009 [Telephone: (609) 768-9360;
`FAX: (609) 753-()064].
`
`Copyright and Permissions
`Authors who wish to publish in Clinical Cancer Research must
`formally transfer copyright to the proprietor of the journal, the
`American Association for Cancer Research, Inc. It is under-
`stood by this transfer that the authors relinquish all exclusive
`rights of copyright ownership, including the rights of reproduc-
`tion, derivation, distribution, sale, and display.
`Authors who prepared their articles as part of their official
`duties
`employees of the U.S. Federal Government are not
`
`required to transfer copyright to the American Association for
`Cancer Research, Inc., since these articles are considered to be
`in the public domain.
`In the case of articles supported by
`federal grants or contracts, copyright transfer to the American
`Association for Cancer Research, Inc., is required. The federal
`government may retain a nonexclusive license to publish or
`republish such material.
`The Journal will routinely allow authors (or others with the
`permission of the authors) to include select parts of a copy-
`righted article in reviews, books, or subsequent papers, upon
`written request to the AACR Director of Publications. Requests
`to reproduce an article in its entirety will be considered on an
`individual basis and permission may be granted contingent upon
`payment of an appropriate copyright fee. All reproduction requests
`must include a brief description of intended use and a stamped,
`self-addressed envelope. Third parties should obtain the approval
`of the authors before corresponding with the AACR.
`Those who wish to photocopy articles should Contact the
`AACR Publications Department [Telephones (215) 440-9300;
`FAX: (215) 440-9354].
`
`No responsibility is accepted by the Editors, by the Amer-
`ican Association for Cancer Research, Inc., or by Cadmus
`Journal Services for the opinions expressed by contributors
`or for the content of the advertisements.
`
`Clinical Cancer Research is abstracted and/or indexed in Can-
`cer Lit, Chemical Abstracts, and Current Contents (Clinical
`Medicine).
`
`Clinical Cancer Research (ISSN 1078-0432) is published monthly
`by the American Association for Cancer Research, Inc., Public
`Ledger Bldg., Suite 816, 150 South Independence Mall West,
`Philadelphia, PA 19106-3483 for $40 annually for members and
`$95 for individual nonmembers. Clinical Cancer Research is only
`available to institutions as a combined subscription with Cancer
`Research. The combined 1995 institutional subscription price of
`$495 includes $65 for a subscription to Clinical Cancer Research.
`Second-class postage is pending at Philadelphia, PA and addi-
`tional mailing offices. POSTMASTER: Send address changes to
`Clinical Cancer Research, PO. Box 3000, Denville, NJ 07834.
`Copyright 1995 by the American Association for Cancer Re-
`search, Inc. Printed on acid-free paper in the U.S.A.
`LILLY EX. 1008 - 2/12
`
`LILLY EX. 1008 - 2/12
`
`

`
`Clinical
`Cancer
`ReSeO|rCh
`
`it
`
`Volume 1, Number ii
`
`November 1995
`
`
`
`1235
`
`1245
`
`1253
`
`1259
`
`1267
`
`1275
`
`1285
`
`Minireview
`
`CONTENTS
`
`1295
`
`Preclinical Studies of Water-insoluble Camptothecin
`Congeners: Cytotoxicity, Development of Resistance,
`and Combination Treatments. Panayotis Pantazis.
`
`Advances in Brief
`
`1301
`
`Telomerase Activity in Preneoplastic and Neoplastic
`Gastric and Colorectal Lesions. Hidetoshi Tahara,
`Hiroki Kuniyasu, Hiroshi Yokozaki, Wataru Yasui,
`Jerry W. Shay, Toshinori Ide, and Eiichi Tahara.
`
`Human Glioma Cells Overexpress Receptors for In-
`terleukin 13 and Are Extremely Sensitive to a Novel
`Chimeric Protein Composed of Interleukin 13 and
`Pseudomonas Exotoxin. Waldemar Debinski, Nicholas 1.
`
`Obiri, Stephen K. Powers, Ira Pastan, and Raj K. Puri.
`
`Research Articles
`
`Phase Ia Trial of Murine Immunoglobulin A Anti-
`transferrin Receptor Antibody 42/6. Donald Brooks,
`Charles Taylor, Betty Dos Santos, Hannah Linden, Alan
`Houghton, Toby T. Hecht, Stephen Kornfeld,
`and
`Raymond Taetle.
`
`Rapidly Cycled Courses of High-Dose Alkylating
`Agents Supported by Filgrastim and Peripheral
`Blood Progenitor Cells in Patients with Metastatic
`Breast Cancer. Linda Vahdat, George Raptis, David
`Fcnnelly, Nicola Hamilton, Lillian Reich, Amy Tiersten,
`Meg Harrison, Clifford Hudis, Michael Moore, T-J. Yao,
`Larry Norton, and John Crown.
`
`Clinical Pharmacokinetic and Pharmacodynamic
`Studies with the Nonclassical Antifolate Thymidylate
`Synthase Inhibitor 3,4-Dihydro-2-amino-6-methyl-4-
`oxo-5-(4-pyridylthio)-quinazolone Dihydrochloride
`(AG337) Given by 24-Hour Continuous Intravenous
`Infusion.
`Imran Rafi, Gordon A. Taylor, Joanne A.
`Calvete, Alan V. Boddy, Kathryn Balmanno, Nigel
`Bailey, Michael Lind, A. Hilary Calvert, Stephanie
`Webber, Robert C. Jackson, Amanda Johnston, Neil
`Clendeninn, and David R. Newell.
`
`Immune Responses in Patients with T-Cell Lym-
`phoma Treated with an Anti-Idiotype Antibody Mim-
`icking a Highly Restricted T-Cell Antigen. Kenneth A.
`Foon, Allan R. Oseroff, Louis Vaickus, Steven J.
`Greenberg, David Russell, Zale Bernstein, Stephanie
`Pincus, Heinz Kohler. Ben K. Seon, Elvan Tahaoglu,
`Teri
`Beers, Mala
`Chakraborty,
`and Malaya
`Bhatacharya-Chatterjee.
`
`1311
`
`1319
`
`1327
`
`1337
`
`1345
`
`1353
`
`p53 Nuclear Protein Expression Is an Independent
`Prognostic Marker in Clinically Localized Prostate
`Cancer Patients Undergoing Radical Prostatectomy.
`John J. Bauer,
`Isabell A. Sesterhenn, K. F. Mostofi,
`David G. McLeod, Shiv Srivastava, and Judd W. Moul.
`
`Expression of the Multidrug Resistance-associated
`Protein (MRP) Gene in Human Cancers. Kees Nooter,
`Anne Marie Westerman, Marcel J. Flens, Guido J. R.
`Zaman, Rik J. Scheper, Kyra E. van Wingerden, Herman
`Burger, Robert Oostrum,
`Ton Boersma,
`Pieter
`Sonneveld, Jan Willem Gratama, Tjebbe Kok, Alexander
`M. M. Eggermont, Fre T. Bosnian, and Gerrit Stoter.
`
`Biological Efficacy of a Chimeric Antibody to the
`Epidermal Growth Factor Receptor in a Human Tu-
`mor Xenograft Model. Neil I. Goldstein, Marie Prewett,
`Kazys Zuklys, Patricia Rockwell, and John Mendelsohn.
`
`Monocyte-mediated Lysis of Acute Myeloid Leuke-
`mia Cells in the Presence of the Bispecific Antibody
`251 X 22 (Anti-CD33 X Anti-CD64). Jian Chen,
`Jie-Hua Zhou, and Edward D. Ball.
`
`Alterations iii '1‘ Cell Receptor and Signal Transduc-
`tion Molecules in Melanoma Patients. Arnold H. Zea,
`Brendan D. Curti, Dan L. Longo, W. Gregory Alvord,
`Susan L. Strobl, Hiromoto Mizoguchi, Stephen P.
`Creekmore, John J. O’Shea, Gerry C. Powers, Walter].
`Urba, and Augusto C. Ochoa.
`
`In Vitro Synergism between 5-Fluorouracil and Nat-
`ural B Interferon in Human Colon Carcinoma Cells.
`Alessandra Guglielmi, Carlo Aschele, Ave Mori, Chiara
`Baldo, Patrizia Russo, Domizia Debernardis, Monica
`Valenti, Silvia Bruno, Monica Taverna, Riccardo Rosso,
`and Alberto Sobrero.
`
`Effects of 9-Aminocamptothecin on Newly Synthe-
`sized DNA in Patient Bone Marrow Samples. Francois
`Geoffroy, William Dahut, Chris H. Takimoto, and
`Jean L. Grem.
`
`Involvement of Human Interleukin 6 in Experimental
`Cachexia Induced by a Human Uterine Cervical Car-
`cinoma Xcnograft. Sumie Tamura, Kaori Fujimoto
`Ouchi, Kazushige Mori, Mika Endo, Takehisa Matsumoto,
`Hiroyuki Eda, Yutaka Tanaka, Hideo Ishitsuka, Hisashi
`Tokita, and Ken Yamaguchi.
`LILLY EX. 1008 - 3/“I2
`
`LILLY EX. 1008 - 3/12
`
`

`
`1359 Retroviral Transfer of :1 Bacterial Alkyltransferase
`Gene into Murine Bone Marrow Protects against
`Chloroethylnitrosourea Cytotoxicity. Linda C. Harris,
`Upendra K. Marathi, Carol C. Edwards, Peter
`J.
`Houghton, Deo Kurnar Srivastava, Elio F. Vanin,
`Brian P. Sorrentino, and Thomas P. Brent.
`
`1407 Pretreatment p53 Protein Expression Correlates with
`Decreased Survival in Patients with End-Stage Head
`and Neck Cancer. Edward R. Sauter, John A. Ridge,
`Samuel Litwin, and Corey J. Langer.
`
`Preclinical Toxicity of Liposome-incorporated Anna-
`mycin: Selective Bone Marrow Toxicity with Lack of
`Cardiotoxicity. Yiyu Zou, Waldemar Priebe, L. Clifton
`Stephens, and Roman Perez-Soler.
`
`Predictive and Prognostic Markers in a Series of
`Patients with Head and Neck Squamous Cell Invasive
`Carcinoma Treated with Concurrent Chemoradia-
`tion Therapy. Giampietro Gasparini, Pierantonio
`Bevilacqua, Emanuela Bonoldi, Alessandro Testolin,
`Andrea Galassi, Paolo Verderio, Patrizia Boracchi, Rosa
`Bianca Guglielmi, and Francesco Pezzella.
`
`Loss of Heterozygosity at 7q31 Is a Frequent and
`Early Event in Prostate Cancer. Alain Latil, Olivier
`Cussenot, Georges Fournier, Jean—Christophe Baron, and
`Rosette Lidereau.
`
`Expression of Topoisomerase II, Bcl-2, and p53 in
`Three Human Brain Tumor Cell Lines and Their
`
`Possible Relationship to Intrinsic Resistance to Eto-
`poside. Cynthia E. Herzog, Leonard A. Zwelling,
`Amanda McWatters, and Eugenie S. Kleinerman.
`
`1437
`
`Effects of Modulators of Protein Kinases on Taxol-
`induced Apoptosis of Human Leukemic Cells Possess-
`ing Disparate Levels of p26BCL-2 Protein. Vidya
`Ponnathpur, Ana Maria lbrado, John C. Reed, Swapan
`Ray, Yue Huang, Sally Self, Gloria Bullock, Amir
`Nawabi, and Kapil Bhalla.
`
`Enhanced erbB-3 Expression in Human Pancreatic
`Cancer Correlates with Tumor Progression. Helmut
`Friess, Yoichiro Yarnanaka, Michael S. Kobrin, David
`A. Do, Markus W. Bfichler, and Murray Kore.
`
`Elevated Levels of 2’,5’-linked 0ligoadenylate-depen-
`dent Ribonuclease L Occur as an Early Event in
`Colorectal Tumorigenesis. Liming Wang, Aimin Zhou,
`Sandip Vasavada, Beihua Dong, Huiqin Nie, James M.
`Church, Bryan R. G. Williams, Sipra Banerjee, and
`Robert H. Silverman.
`
`Insulin Receptor Substrate 1
`Overexpression of
`(IRS-1)
`in the Human Breast Cancer Cell Line
`MCF-7 Induces Loss of Estrogen Requirements for
`Growth and Transformation. Ewa Surmacz
`and
`Jean—Luc Burgaud.
`
`AACR Bulletin Board
`Annual Meeting
`Gertrude Elion Cancer Research Award
`New Research Fellowships Available
`Travel Grants from the Comprehensive Minority Biomedical
`Program of the National Cancer Institute
`AACR Special Conferences in Cancer Research
`
`i
`
`Instructions for Authors
`
`
`
`Pages ls—33s
`
`Call for Abstracts for 1996
`AACR Annual Meeting
`
`Includes: Abstract Submission Forms
`Advance Registration and
`Housing Forms
`
`
`
`LILLY EX. 1008 - 4/“I2
`
`LILLY EX. 1008 - 4/12
`
`

`
`Vo l. I, 13 11- 13 18, November 1995
`
`Cli nical Cancer Research 1311
`
`Biological Efficacy of a Chimeric Antibody to the Epidermal
`Growth Factor Receptor in a Human Tumor
`Xenograft Model
`
`Neil I. Goldstein, 1 Marie Prewett, Kazys Zuklys,
`Patricia Rockwell, and John Mendelsohn
`Departm ents of Immu nology/Monoclonal Antibod ies and Protei n
`Chemistry, lmCione Systems, Inc., New York, New York 100 14
`[N. I. G., M. P., K. Z., P. R.], and Depart ment of Medic ine,
`Memorial Sloa n-Ketteri ng Cancer Ce nter and Cornell Uni versity
`Medica l Co ll ege, New York , New York 1002 1 [J . M. ]
`
`ABSTRACT
`The epidermal growth factor receptor (EGFR) is a
`protein tyrosine kinase expressed on many types of tumor
`cells, including breast, ovarian, bladder, head and neck, and
`prostatic carcinoma. There seems to be an association be(cid:173)
`tween up-regulation of the EGFR and poor clinical progno(cid:173)
`sis for a numbet· of human cancers. The 225 antibody is a
`highly specitic murine monoclonal antibody that binds spe(cid:173)
`citically to the human EGFR with an aftinity equal to its
`ligand, competes with the ligand for binding, and blocks
`activation of the receptor tyrosine kinase. In addition, 225
`has been shown to inhibit the growth of human tumor
`xenografts in athymic nude mice.
`The 225 antibody has recently been chimerized with
`human lgG 1 in its constant region to increase its clinical
`utility by decreasing the potential for generation of human
`anti mouse antibodies in recipients. This report compares the
`biological effects of 225 and its chimeric counterpart (des(cid:173)
`ignated C225) against established A431 tumor xenografts in
`nude mice. The results of these experiments indicated that
`C225 was more effective than 225 in inhibiting tumor
`growth in this model. In addition, many of the animals
`treated with C225 were tumor free at the end of each
`treatment protocol. It was determined that the dissociation
`constant of C225 was about 5-fold lower than 225. This
`suggested that the increased capacity of C225 to compete
`with ligand for binding to the EGFR was responsible for its
`enhanced in vivo antitumot· effect.
`
`INTRODUCTION
`The EGFH? is a protein tyrosine kinase encoded by the
`c-erb-B proto-oncogene and expressed on many normal and
`
`Rece ived 5/11 /95; rev ised 6/2/95; accepted 6/23/95.
`1 To w hom requ ests for reprints should be addressed, at Department or
`Immunology/Monoclonal Anti bodies, lmCione Systems, Inc., 180 Ya r(cid:173)
`ick Street, New York, NY IOOL4.
`2 T he abbrev iations used are: EGFR, epidermal growth factor receptor;
`K", dissociat ion co nstant; mAb, monoclonal antibody; C225, ch imeric
`225; Rl , remission index; SPR, surface plasmon resonance; TGF-a ,
`transfo rmi ng growth factor a.
`
`malignant cells (1 , 2). Binding of one of its two ligands (EGF or
`TGF-a) to the EGFR can activate signal tra nsduction pathways
`that regulate cell proliferation. A number of human epithelial
`cancers express high levels of the EGFR and may also produce
`TGF-a in an autocrine manner. Included in this group are
`tumors of the breast, lung, colon, prostate, kidney, bladder, head
`and neck, and ovary (1-4). For many of these cancers, there
`exists an association between up-regulation of receptor expres(cid:173)
`sion and poor clinical prognosis (4).
`Because of the relationship between overexpression of the
`EGFR and clinicall y aggressive disease, a m.Ab directed against
`this receptor may prove to be a useful therapeutic reagent. The
`murine mAb 225 binds only to human EGFR, has a Kd similar
`to its ligand (1 nM), blocks activation of the EGFR by ligand,
`and induces internalization of the receptor. The antibody is able
`to inhibit the growth of cultured EGFR-expressing tumor lines
`and to repress the in vivo growth of these tumors when grown as
`xenografts in nude mice (5-9). More recently, a treatment
`regimen combining 225 plus doxorubicin or cis-platin was
`fo und to show therapeutic synergy against several well-estab(cid:173)
`lished human xenograft models (10, 11).
`An obv ious problem with the use of a murine mAb in
`human clinical trials is the potential fo r the generation of human
`antimouse antibody responses (12, 13). Indeed, this was found
`to occur in Phase I clinical trials with 225 in patients with
`advanced squamous carcinoma of the lung. These trials estab(cid:173)
`lished the feasib ility of administering 225 at doses that produced
`receptor-saturating levels in the blood, without inducing toxicity
`( 14). To avoid human anti mouse antibody production, 225 was
`chimerized to the human JgG l constant region. In th is report, we
`compared the in vivo effects of 225 and C225 on established
`A43 1 xenografts in nude mice. A43 1 is a cell line that expresses
`very high levels of the EGFR (about 106/cell) and is autocrine
`for the prod uction of TGF-a (15, 16). In previous animal stud(cid:173)
`ies, A43 1 xenografts treated with 225 beginning on the day of
`tumor challenge, or within 5 days of tumor cell inocul ation,
`were completely inhibited by the antibody alone, whereas 225
`by itself had little or no effect on the growth of established
`tumors (6, 10, 11). The results of the present studies show that
`C225 has an enhanced biological effect on the growth of estab(cid:173)
`lished A431 tumors in nude mice.
`
`MATERIALS AND METHODS
`Cell Lines and Media. A43 1 cells were routinely grown
`in a 1:1 mixture of DMEM and Ham's F-12 medium supple(cid:173)
`mented with 10% fetal bovine serum , 2 mM L-glutamine, and
`antibiotics.
`Prepamtion and Purification of Murine 225 and C225.
`The 225 antibody was grown as ascites in pristane-primed
`BALB/c mice. Ascites fluid was purified by high perfo rmance
`
`LILLY EX. 1008 - 5/12
`
`

`
`1312 Biological Effect of a Chimeric Antibody to EGFR
`
`Table I K"s for 225 and C225 as determined by various methods
`
`K" (nM)
`
`Receptor
`C225
`225
`form
`Method "
`nd"
`A43 I lysates
`l
`Scatchard
`0.39
`M24met cells
`Scatchard
`0.78
`0.147
`Fixed A43 l cells
`ELLS A
`l.l7
`0.201
`SPR
`Soluble receptor
`0.868
`"Scatchard results are expressed asK", SPR results as apparent Kd,
`and ELISA data as apparent affinity; the latter is a relative measure of
`the K". See " Materials and Methods" for description of the generation
`of the ELLSA and SPR data.
`" nd, not done.
`
`Ref.
`8
`22
`
`liquid chromatography (ABX and protein G) and determined to
`be > 95% pure by SDS-PAGE.
`Human clinical grade C225 was produced by growth in
`proprietary se rum-free medium under controlled culture con(cid:173)
`ditions. After cl arification, the concentrated broth was puri (cid:173)
`fied th rough a se ri es of chrom atographi c steps and vialed
`under asceptic conditi ons. Purity was determined by SDS(cid:173)
`PAGE.
`Relative Affinity Measurements Using ELISA. The
`relative binding affinity of the antibodies was determined using
`an ELISA protocol previously described ( 17). Brietly, A431
`cells (104 or 105/well) were grown in 96-well microliter plates
`overnight at 37"C. Cells were fi xed with 3.7% neutral bu ffered
`formalin for 10 min at room temperature. After washing three
`times with PBS, wells were blocked with 1% BSA in HBSS for
`2 h at room temperature. C225 or 225 was added to the we lls at
`various concentrations (seri al dilutions starting at 50 nM). After
`a 2-h incubation at 37°C, plates were extensively washed with
`PBS and either goat antihuman antibocly :horseradish peroxidase
`(1: 1000; Sigma Chemical Co., St. Louis, MO) or goat antimouse
`antibody: horseraclish perox idase (1:5000; Tago, Burlingame,
`CA) were aclclecl for 1 h at 37"C. After washing, the chromogen
`TMB (Kirkegaard and Perry, Gaithersburg, MD) was added fo r
`30 min in the dark. The color reaction was stopped with 1 N
`sul furic acid, and the plates were read at 450 nm in an ELISA
`reader. The relative binding affinity, defin ed as the concentra(cid:173)
`tion giving the half-max imal A, is an approxi mation of an
`antibody 's Kc~ .
`Affinity Constants of 225 and C225 Using SPR Tech(cid:173)
`nology. The apparent binding affiniti es of murine 225 and
`C225 were also determined using the BIAcore'" (Pharmacia
`Biosensor, Piscataway NJ; manufacturer's application note 30 I;
`Refs. 18-20). Briefl y, soluble recombinant EG FR (a gift from
`Dr. Joseph Schlessinger, New York University, New York, NY)
`was immobilized on sensor chips via ami no groups as described
`by the manufacturer. Real time binding parameters of 225 and
`C225 to EGFR were established at various antibody concentra(cid:173)
`tions, and the apparent Kc~ was calculated from the binding rate
`constants obtained by analyzing the data using B!Aevaluati on '"
`2.0 software.
`In Vitro Inhibition of Cell Growth with 225 and C225.
`The in vitro inhi bitory activity of 225 and C225 was determined
`as fo llows. A43J cells (I 04 cells/well) were plated in 96-well
`microliter plates in complete growth medium . After adding
`
`C225 or 225 in various concen trations (fou r replicates/concen(cid:173)
`tration, serial dilutions with a starting concent ration of 5 f.Lg/ml),
`plates were incubated fo r 48 h at 37°C, fo ll owed by a 24- h pulse
`with [3 H]thymidine. Cells were harvested, coll ected on filter
`mats, and counted in a Wall ac Microbeta scintillation counter.
`The percentage inh ib ition compares the decrease in [3 H ]thym i(cid:173)
`cl ine incorporation of anti body-treated cells with ce lls grown in
`the absence of antibody or in the presence of an irrelevant
`hum an myeloma lgG l antibody (Tago).
`Phosphorylation Studies. Phosphory lation assays and
`subsequent Western blot analysis were done as prev iously de(cid:173)
`scribed (2 1). Briefl y, A43 1 cells were grow n to 90% con flu ency
`in complete medium and then starved in RPM I and 0.5 % BSA
`for 24 h prior to experimentat ion. Ce lls were stimul ated with 10
`ng/ml EGF in the presence of 10 f.Lg/ml eith er 225 or C225 fo r
`15 min at room temperature. Foll owing st imulation, monolayers
`were washed with icc-cold PBS containing J mM sodium or(cid:173)
`thovanadate. Cell s were lysed and subj ected to SDS- PAGE
`followed by Western blot analysis. The phosphorylation patterns
`were determined by probing the blots with a mAb to phospho(cid:173)
`tyrosine (Upstate Biotechnology, Inc., Lake Pl acid, NY) fo l(cid:173)
`lowed by detection by the enhanced chem ilum inescence method
`(Amersham, Arlington Heights, IL).
`Animal Studies. At hymic nude mi ce (n u/ nu ; 6-8-week(cid:173)
`olcl fe males) were obtained fro m Charles River Laboratories
`(Wilmington, MA) and main ta ined under clean conditions. An(cid:173)
`imals (7-10 mice/treatment group) were inocul ated s.c. on the
`right flank with I 07 A43 1 cells in 0.5 ml HBSS. Antibody
`therapy was begun when tumors reached an ave rage volume
`> 150 mm 3 (7-1.2 days). Treatments consisted of twice weekly
`i.p. injections of 225 or C225 (va rying concent rati ons of anti (cid:173)
`body in 0.5 ml PBS) over 5 weeks. Control animals received
`injections of PBS. Tumors were measu red two times per week,
`and volumes were ca lculated using the fo llowing fo rmul a: TI/6
`X larger diameter X (smaller diameter) 2 (8). Animals were
`followed for at least 2 weeks after the final anti body treatment
`(i.e., 7 weeks after the start of therapy), at which time control
`and test animals with extremely large tu mo rs were euthanized.
`Tumor-free mice and animals with small tumors were fo llowed
`for an additional 2- 3 months. Statistica l analysis of tumor
`growth fo r each of the studies was determined by a Student 's 1
`test using the computer program SigmaStat (Jande!, Sa n Rafael,
`CA). P < 0.05 was considered significant.
`In addition to demonstrat ing growth inh ibitory effects of
`the antibod ies, many anim als were fo und to be in complete
`remission (i. e., tumor free). This biological effect was quantified
`as a RI and defin ed as the num ber of tumor free mice/total
`animals within a treatment group. Animals that died during
`treatment were considered treatment fa ilures and were retained
`in the analysis. For example, one complete remission among 10
`animals would equal a Rl of 0. I.
`
`RESULTS
`In Vitro Properties of 225 and C225.
`Init iall y, the bio(cid:173)
`logical effects of 225 and C225 were compa red in a series of in
`vitro assays. The apparent Kc~s of the antibod ies were found to
`be 0.1 and 0.201 nM for C225 and 1. 17 and 0.868 nM for 225,
`using the ELISA and SPR methods, respective ly (Table 1).
`
`LILLY EX. 1008 - 6/12
`
`

`
`Clinical Cancer Research 1313
`
`ABCDEF
`
`C225
`- -
`-e- M22.5
`
`250 kD---
`
`Inhibition of EGF-induced phosphorylation of the EGFR by
`Fig . 2
`225 and C225. A431 cells were stimulated with EGF in the presence or
`absence of 225 or C225. Lysatcs were separated by SDS-PAGE and
`analyzed on Western blots using antiphosphotyrosine to probe for phos(cid:173)
`phorylated proteins. A, serum free, no additions; B, EGF stimulated, no
`antibodies; C, EGF stimu lated plus 225; D, EGF stimulated plus C225;
`E, 225 alone, no EGF; F, C225 alone, no EGF.
`
`Table 2 Rls for animals inoculated with A43J cells and treated with
`225 or C225"
`
`Study
`J
`
`2
`
`3
`
`Rl'"
`No. remi ssions/total~>
`Treatment
`0.10
`225
`1/10
`0
`0/10
`PBS
`0.40
`4/JO
`C225
`0
`0/ LO
`PBS
`l.O
`C225, I mg
`7/7
`0.57
`4/7
`C225, 0.5 mg
`0.14
`C225, 0.25 mg
`1/7
`0
`PBS
`017
`"A comparison of complete tumor remissions in athymic nude
`mice carrying established A431 tumors fo llowing treatment with PBS,
`225, or C225 twice weekl y for 5 weeks. Animals were treated with 1 mg
`antibody in 0.5 ml PBS by the i.p. route, except for experiment 4, which
`is a dose-response experiment in which mice were given l , 0.5, or 0.25
`mg/injection. Tumor measurements were done as described in " Mate(cid:173)
`rials and Methods. " This table describes the RI at the time when the
`animals carrying large tumors (in both the PBS and test groups) were
`euthanized. All animals showing complete remissions or small tumors
`were followed for an additional 2- 3 months. Animals that died during
`the course of treatment were considered treatment failures and were
`retained in the analysis.
`h Tumor-free animals/total number of animals. Animal mortality
`was treated as a treatment failure and included in the final analysis.
`'"The Rl is defined as the fraction of mice that were tumor free on
`the day when PBS control mice and test animals with large tumors were
`cuthanized. A complete remission at the 0.25-mg dose level showed a
`subsequent recurrence of tumor (day 47; see also Fig. 4B) and is not
`included in the analysis.
`
`of the studies, large SDs were observed in both the test and
`control groups. This resulted from the random selection of
`tumor-bearing animals for each treatment group. Randomization
`was an attempt to eliminate experimental bias that might have
`occurred from a preponderance of large or small tumors within
`a specific group.
`
`40
`
`35
`
`30 -
`
`25
`
`c .g
`;e 20
`.s:
`.!:
`~ 0
`
`15
`
`10 -
`
`5
`
`0
`
`c.n
`0
`0
`0
`0
`
`1\l
`(n
`0
`8
`
`0
`i:ll
`1\l c.n
`0
`
`" c.n
`8
`Antibody Concentration
`(!J.g/ml)
`
`::: c.n
`
`0)
`0
`
`0
`
`0 .....
`
`Q:l
`0
`
`Fig. 1 Inhibition of the growth or cultured A431 cells in the presence
`of 225 (M225) and C225 . Data arc presented as percentage inhibition.
`The average cpm for cells grown in the absence of antibody was 82,674
`:!:: 4,5 18: There was no inhibitory effect for either the human IgGl or
`mouse IgG I irrelevant control antibodies.
`
`These results (Table 1) were similar to published data for C225
`(K", 0.39 nM; Ref. 22) and 225 (K", 0.79 nM ; Ref. 22; or Kd, 1
`nM ; Ref. 8). Both antibodies were capable of inhibiting the
`proliferation of cultured A431 cells to the same extent (Fig. 1).
`In addition, both 225 and C225 were able to block EGF-induced
`phosphorylation of the EGFR in A431 cells (Fig. 2). Interest(cid:173)
`ingly, 225 and C225 seemed to block the phosphorylation of
`EGFR in the absence of exogenous ligand [Fig. 2, compare A
`(no antibody) with E (225 alone) and F (C225 alone)]. This
`suggested that the antibodies could inhibit the autocrine activa(cid:173)
`tion of the receptor.
`In Vivo Properties of 225 and C225 against A431 Xe(cid:173)
`nografts. The in. vitro results indicated that chimerization of
`225 did not affect the biological properties of the antibody and
`may have increased the relative binding affinity of C225 for the
`receptor. The capacity of the antibodies to inhibit the growth of
`established A43 1 xenografts in nude mice was then tested. Mice
`were inoculated with A431 cells, and treatments were begun
`when tumors reached an average volume > 150 mm 3
`. Animals
`were then randomly grouped and given injections of PBS and
`225 (study 1) or PBS and C225 (studies 2 and 3). In studies 1
`and 2, animals received inj ections of 1 mg antibody (in 0.5 ml
`PBS) twice weekly over 5 weeks for a total dose of 10 mg
`antibody/animal. In study 3, animals received one of three
`possible doses: 1, 0.5, and 0.25 mg/injection for total closes of
`J 0, 5, and 2.5 mg, respectively. At the end of a treatment
`protocol, tumor-free animals and those with small tumors con(cid:173)
`tinued to be monitored for an additional 2-3 months. In several
`
`LILLY EX. 1008 - 7/12
`
`

`
`1314 Biological Effect of a Chimeric Anti body to EGFR
`
`A
`
`20000
`
`-o- PBS
`- - 225
`
`c 6000
`
`5000
`
`~E
`E 4000
`.5
`Q)
`§ 3000
`0
`>
`0
`§ 2000
`1-
`
`1000
`
`~E 15000
`E
`.s
`Q)
`E
`:::1
`~ 10000
`0
`E
`:::1
`1-
`
`5000
`
`8
`
`~ ,
`.5
`c
`0
`'iii
`
`"' ·e
`Q) a:
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`D 6000
`
`5000
`
`Q)
`
`~E
`E 4000
`.s
`§ 3000
`0
`>
`0
`§ 2000
`1-
`
`1000
`
`10
`
`20
`
`30
`
`40
`
`50
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`Days After Tumor Injection
`
`Fig. 3 Effect of 225 on the growth of established A431 xenogra l'ls in nude mice (stud y l ). Animals were trea ted with PBS or I mg 225 tw ice week ly
`for 5 weeks. Tumors we re measured twice week ly and volumes calcul ated as descri bed in " Materi als and Methods. " T he average tum or vo lumes
`at the start of the study were 383 mm 3 fo r PBS and 5 13 mm 3 for 225. A, average tum or volume fo r each group over the course of the study; B, Rl ;
`C and D, effect of 225 or PBS on tumor growth in individual animals.
`
`Fig. 3 shows the effect of 225 on the growth of A431
`tumors in nude mice (study L, treatments beginning on day 11 ).
`There was no di fference in the average tumor volumes between
`the control and 225 groups over the course of the study (Fig. 3A)
`and only one complete tumor remission was observed (RI, 0.1;
`Fig. 38 and Table 2). When the antitumor responses of225 were
`compared in individual animals, tumor growth was strongly
`inhibited by the antibody in 3 of 10 animals (including the one
`complete remission), and transient regressions were seen in 2
`others (Fig. 3C). These results were similar to data that have
`been previously reported (6, 10, 11).
`On the other hand, C225 alone was found to be extremely
`effective in inhibiting the growth of A431 tumors. As can be
`seen in Fig. 4A (study 2, treatments beginning on day 9), there
`was a significant antitumor effect of C225 beginning on day 33
`(P < 0.02). The Rl for the C225 group was 0.4, indicating that
`4 of LO an imals were tumor free following the treatment regi-
`
`men (Fig. 48 ). Tumor- free animals were fo llowed for an addi (cid:173)
`tional 3 months and remained in complete remission (data not
`shown). The antitumor response of C225 on individual mice was
`very dramatic, with tumor regress ions observed in 9 of 10
`animals, incl uding 3 mice with tumor volumes > 2500 mm3