7' 13- L,
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`
` .3. F F I C_.. " JOURNAL OF THE AMEmCAN ASSOCIATION FOR CANCER RESEARCH
`
`HEALTH SBIENBES LIBRAB?
`University of Wisconsin
`
`OCT
`
`7 1993
`
`1305 Linden Drive
`Madison, WI 53706
`
`Ibcfober 1, 1993
`:’V0|Ume 53 - Number 19
`>1!-“P. 4443-4739
`gssw nous-5472 - CNREA 8
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`L
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`LILLY EX. 1005 — 1/1.51
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`LILLY EX. 1005 - 1/11
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`Back Issues and Single Copy Sales of the Journal
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`Copyright 1993 by the American Association for Cancer Research. Inc.
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`LILLY EX. 1005 - 2/11
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`LILLY EX. 1005 - 2/11
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`VOLUME 53 °
`
`Cancer Research
`
`NUMBER 1‘?
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`'
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`CNREA 8 ' PP. ~’l4£13~4739
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`.1443
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`4449
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`4461
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`4466
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`4474
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`44??
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`4481
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`443!»
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`Advances in Brief
`
`CONTENTS
`
`4:889
`
`The Small Heat Shock Protein hsp2.T ls Correlated with Growth
`-.;.u1 Drug Resistance in Human Breast Cancer Cell Lines. Stcfli
`r"h-.-.Icn'cic|i. Chye—Ning Wong. Ming Qiu. Susan G. Hilscnbcck. C.
`ix.-nt Osborne. and Su.r.anne A. W. Fnqua.
`
`in the t(B:2l}
`[Is-tcction of the AMLJ'.v‘ET() Fusion Transcript
`..'\l:1sl-icd Translocation in Acute Myelogenous Leukemia. Furnio
`'*='_ruyuIn.'.I. Peirong ‘fang. Sanford A. Stass. Ann Cork. Emil
`J
`i-rcinsich, Ming-Shcng Lee. and Kun-Sang Chang.
`
`4493
`
`nsmiinant Negative Elfert of a Germ-line Mutant p53: A Step
`rostering Tumorigcnesis. Shiv Srivaslava, Shouwen Wang, Yue An
`"'--rig. Zhcng—Mci Hat). and Esther H. Chang.
`
`Comparison ol’ Loss of Heterozygosily Patterns in Invasive Low-
`-: ‘-ade and High-Grade Epithelial Ovarian Carcinomas. Mark K.
`l)r.uison. Lynn C. Hartmann. William A.C1iby. Karen A. Del.-ucey. Gary
`Keene)». Steve R. Ritland, John Q. Su. Kari C. Podralx. and Robcn
`ii. Jenkins.
`
`EL-tary Fenretinide. a Synthetic Retinoid. Decreases the Tumor
`Incidence and the Tumor Mass of rns+m_vc-induced Carcinomas in
`{ne Mouse Prostate Reconstitution Model System. K. Siawin. D.
`‘(.'1tlrn:)n. S. H. Park. P. T. Scardino. M. Anzano. M. B. Spam, and T. C.
`ihmnpson.
`
`Induction of Protein-DNA Complexes in HeLa S3 Cells by KW-
`.7.l-19. a New Derivative of Mitomycin C. Noboru Fujii. l-£iLoshiAr:1i.
`'iirunnisu Sarto. Masaji Kzisui. and Hirulhmi Nakzmo.
`
`Rcdox Modulation of p53 Conformation and Sequence-specific
`DNA Binding in Vitro. Pierre l-lainaut and Jo Miincr.
`
`Activation of Phospholipase D Participates in Signal Transduction
`P‘athwa_vs Responsive to 7-Radiation. Matias A. Avila. Gabriel
`Olcro. Jose’ Cansudo. Anatoly Dntschilo. Juan A. Vclasco. and Vicente
`?\'ol21rio_
`
`The Incidence of p53 Mutations Increases with Progression of Head
`and Neck Cancer. Jay 0. Boyle. John Hakim. Wayne Koch. Peter van
`dcr Riel. Ralph H. Hruban, R. Amlro Roa. Russell Curran, Yniuncia J.
`Eby. J. Michael Ruppcrt. and David Sidransky.
`
`Human Immunodeficiency Virus Type 1 "Fat Protein Protects Lym-
`phoid. Epithelia], and Neuronal Cell Lines from Death by Apop-
`tosis. Giorgio '/.au1i, Davide Gibellini, Daniela Milani. Meri Mazzoni.
`Paola Bnrgani, Michclc La Placa. and Silvano Capilani.
`
`Refinement of Regional Loss of Hcterozygosity for Chromosome
`l1pl5.5 in Human Breast Tumors. Robert winqvisr. Arm
`MrmnemI'.1:J. Marni Alavaikko. Guillermo Biunco. Penlti J. Taskincn.
`Hcikki Kivinicrni. lrene Newshana, and Webster Cavcnee.
`
`Potential Topoisomerase ll DNA-binding Sites at the Breakpoints
`of a tt9;l1) Chromosome Transloratiun in Acute Myeloid Leuke-
`mia. M. Ncgrini, C. A. Fciix. C. Martin. B. J. Lange, T, N-ukamuru, E_
`Canaam, and C. M. Croce.
`
`Regular Articles
`
`Biochemistry and Biophysics
`
`Secretion or Matrix Melalloproteinascs and Their Inhibitors {Tis-
`sue Inhibitor of Metalloproteinases) by Human Prostate in Explant
`Cultures: Reduced Tissue Inhibitor of Metalloproteinase Secretion
`by Malignant Tissues. Balakrishna L. Lokeshwar, Marie G. Sclzer.
`Norman L. Block and Zccnat Gunja-Smith.
`
`4499
`
`4505
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`4511
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`4518
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`4528
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`4534
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`4542
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`45 50
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`Carcinogenesis
`
`Ability of Aberrant Crypt Foci Characteristics to Predict Colonic
`Tumor Incidence in Rats Fed Cholic Acid. Bcrnadcnc A. Magnuson.
`Ian Carr. and Ranjana P. Bird.
`
`Singlet Oxygen: A Primary Effector in the Ultraviolet A.i'Near-
`Visiblc Light Induction of the Human Heme Oxygenase Gene.
`Sharmila Basu-Modal: and Rex M. "lyrrell.
`
`Human Papillomavirus I6 lmmorlalizalion of Normal Human Ec-
`tocervical Epithelial Cells Alters Retinoic Acid Regulation of Cell
`Growth and Epidermal Growth Factor Receptor Expression.
`Nywana Sizcmore and Ellen A. Rorkc.
`
`Chronic Radiation-induced Alteration in l-Iematopoietic Repair
`during Preclinical Phases of Aplastic Anemia and MyeloproIifera-
`live Disease: Assessing Unscheduled DNA Synthesis Responses.
`Thomas M. Seed and Susan M. Meyers.
`
`Inhibition ol’ Uxidative Stress in HeLa Cells by Chemopreventive
`Agents. Ramesh 5. Bhimani. Walter Troll. Dezider Grunberger. and
`Krystyna Frenkel.
`
`Major Difference in the Hepatocarcinogenicity and DNA Adduct
`Fonning Ability between Toremifene and Tamoxifen in Female
`Crl:CD[BR} Rats. Gordon C. Hard. Michael J. Iatropoulos, Kevin
`Jordan. Leila Radi. Olgicrd P. Kaltcnberg. Anthony R. Irnondi. and
`Gary M. Williams.
`
`Regulation of Protein Kinasc C isozymcs in Kidney Regeneration.
`Liqun Dong. James L Stevens, Doriuno Fabbro. and Susan Jarken.
`
`Clinical in vesligalions
`
`Expression and Prognostic Significance of Platelet-derived Growth
`Factor and its Receptors in Epithelial Ovarian Neoplasms. Rudi
`Henrikscn. Kciko Funa. Erik Wilander. T0rlJ_1i§l'l‘I Biickstrorn. Mona
`Ridderheirn. and Kjcll Ctberg.
`
`LILLY EX. 1005 - 3/11
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`LILLY EX. 1005 - 3/11
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`

`
`4567
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`4573
`
`45 B2
`
`4588
`
`4595
`
`4693
`
`4608
`
`4613
`
`cl-619
`
`4627
`
`4633
`
`Experimental therapeutics
`
`Modulation of Cytotoxicity and Cellular Pharmacology of l,2-Di-
`aminoeyclohexane Platinumtlvl Complexes Mediated by Axial and
`Equatorial Ligands. Yuichiro Kido, Abdul R. Khokhat. Salaarn A]-
`Baker, and Zahid H. Sitidik.
`
`4652
`
`Estrarnustine Depolymerizes Microtuhules by Binding to 'DJbnIin.
`Bjorn l').'iltl|iil'. Anita Fsillstront. Fcntando Cabral. and Beryl Hartley-
`Asp.
`
`Effect of a Template-located 2’,2'-Dilluorodeoxycytidine on the Ki-
`netics and Fidelity of Base Insertion by Klcnow (3'-°5’ExonucIe-
`ase‘l Fragment. William E. Schy, Larry W. llertcl. Julian S. Kroiu.
`Linda El. Bloom. Myron F. Goodman. and Frank C. Richardsoll.
`
`Polyethylene Glycol-modified Chimeric Toxin Contpo-red of Trans-
`forming Growth Factor It and Pseudomanax Exotottin. Qing—cher1g
`Wang. Lee H. Pai_ Waldemar Debinslti. David J. FitzGcrald. and Ira
`Pastan,
`
`in l/Elm and in Vivo Reversal of Multidrug Resistance by
`GFl299l8, an Acridonccarboxamide Derivative. F-‘raneois Hyafll.
`Catherine Vergcly. Pierre Du Vignattd. and Tltierry Gr;itid—P«.3rn:t.
`
`Cardioproteetive Propertiets of 0-tB-Hydroxyethyll-rutosides in
`DoxorulJicin~pretreated BALBic Mice. Saskia A. B. E. van Ackcr.
`Emile ti. Voest. Dolf B. Bcems. llcnk T. Madhttizen. Jan de Jong. Aalt
`Ba.-at. and With J. E van dcr Vijgh.
`
`Main Drug- and Carcinogen-metabolizing Enzyme Systems in Ho-
`man Non-Small Cell Lung Cancer and Peritumoral Tissues.
`Caroline Toussaint. Nicolas Albin, Liliane Massaad. Dontittiquc
`Grunenwaid. Orlando l-’:trise. Jr.. Jackie Monzet. Alain Gouyette. and
`Guy G. Cltahot.
`
`Calfelne Prevents Apoptosis and Cell Cycle Effects Induced by
`Camptotheein or Topolecan in Hi.-60 Cells. Frank ‘l‘mgann.\; Jan
`Kapuscin:-“kl. Jirtnping Gong. Barbara Artlelt. Robert J. U:1rZ._\'I‘lltit:wic.I..
`and Zbignicw Dairrynkicwier.
`
`In l/ivo Antiturnor Activity of 5-Fluorocytosinc on Human Colo-
`rectal Carcinoma Cells Genetically Modified to Express Cytosinc
`Deaminase. Brian E.
`I-luhcr. Elizabeth A. Austin. Steven S. Good.
`Vincent C‘. Knick. Stephen Tihlncls. and Cynthia A. Richards.
`
`Reversible inhibition of Proliferation of Epithelial Cell Lines by
`Agarfcus bisportrs (Edible Mushroom] Lectin. Lugang Yu. David G.
`Fernig. John A. Smith. Jeremy D. Milton. and Jonathan M. Rhodes.
`
`Interaction between 'l‘ira-
`'I'un10r-Specific. Schedule-dependent
`pazamillfl {SR 4233} and (Tisplatin. Mary Jo Doric and J. Martin
`Brown.
`
`4665
`
`4670
`
`4676
`
`4680
`
`4686
`
`469]
`
`4695
`
`Immune Reactions against Hepatitis B Viral Antigens Lead to Ln:
`Rejection of Hepatocellular Carcinoma in BALl3lic Mice. Shu-Hula
`Chen. Cheng—po Hu. Chien—Ktto Lee. and Chungming Chang.
`
`Interferon--gr-induced lntercellular Adhesion Molecule-l: Expres-
`sion on Human Tumor Cells Enhances Bifunctional Antibody-me
`dialed Lysis through a Lymphocyte Function-associated Antigen-I
`Dependent Mechanism. Patrick S. Rantsey. Phillip A. Dean. Alliw
`Tasirnowicz-Alpini, John H. Donohue. and Heidi Nelson.
`
`Molecular Biology and Genetics
`
`Effect ol' Verapamil on Doxorubicin Cardiotoxicity: Altered Muscle
`Gene Expression in Cultured Neonatal Rat Cardiomyocytes.
`Hajimc Alimoto. Nicholas A. Bruno. Dun.» L. Slate, Margaret E.
`Billinghntn. Suzy V, Toni, and Frank M. Torli.
`
`'[‘ype of Invasive Breast Carci-
`p53 Mutations and Histological
`noma. Antonio Murehetti. Fiatnma Buttitta. Silvia Pellegrini. Daniela
`C ampam. Francesca Diclla. Denise Cecchetti. Robert Callahan. and
`Maria Bisto-t:t:l1i.
`
`Up-Regulation of Lysyl Oxidase in Spontaneous Revcrtants of
`H-ms-transformed Rat Fibroblasts. Alex Hajnal, Roman Klcmcnz.
`and Reinhold Schiifcr.
`
`Clonal Analysis by Study of X Chromosome Inactivation in For-
`rnztlin-ftxed Parallin-embedded Tissue. Robcn D. Masha]. Susan C.
`Lester. and .lcl‘t'rey Sklar.
`
`tumor Biology
`
`Role for Membrane and Secreted insulin-like Growth Factor-hint:l-
`ing Protein-2 in the Regulation of Insulin-like Growth Factor Ac-
`tion in Lung Tumors. Julie G. Reeve. Julie M(,1rgan.Jurg Schwander.
`and Numtan M. Blechen.
`
`Effect of Diets Containing Different Levels of Linoleic Acid on
`Human Breast Cancer Growth and Lung Metastasis in Nude Mice-
`David P. Rose. Mary Ann Halala. Jeanne M. Connolly. and Jodie
`Raybttnt.
`
`Expression of p53 Gene in 184 Unifo-cal l-Iepalocellular Carcino-
`mas: Association with Tumor Growth and lnvasiveness. Hey-Chi
`Hsu.
`l{w:t1'~JtIt'tg Tscng. Po—Lin Lai, Po-Huang Lee. and Shian—‘r'.1n£
`Pong.
`
`Role of Platelet Membrane Glycoproleins ltu"lX and libillla, and
`of Platelet or-Granule Proteins in Platelet Aggregation Induce!‘ l!J’
`Human (lsteosarcoma Cells. Philippe Clemrdin. Jeanne Drouin.
`M:irie~(_‘hristinc Motcl—Kopp. Michel
`Hans.-4.
`Beale
`Kel'|‘°l-
`Claire—Marie Serrc. Cécilc Kaplan. and Piano D. Delmas.
`
`L
`
`LILLY EX. 1005 - 4/11
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`LILLY EX. 1005 - 4/11
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`

`
`470 I
`
`luv,-1-gligflllllfl of the Subcellular Distribution til‘ the bcl-2 0nL'oprn-
`1‘ ;'.
`: Residence in the Nuclear Envelope. Endoplasmic Reticulum.
`and Ouler Mite-ichondrial Mernlmam-s. Slunislziw Krajcwski.
`.‘~.-
`;LL‘lxl
`'I"nii:il-car?“ Shiniehi Tukayaimi. Mantliew .|. Schibler, Wayne
`[-1-nmn. and John C. Reed.
`
`1m-nunodeteclion of Endogenous Opioid Peptides in Human Brain
`‘Tumors and .»\sso-eisilcd Cyst Fluids. Thierry Gustin.
`'l‘hon1as
`B
`licloi. Jean-Marc G. Verna. Laurent
`l-'. Molin,
`.|e:a.n—l‘-‘rairmnis M.
`Brniict.
`|"ran¢oi.s R. Berger. and Alim L. Bcnabid.
`
`4'? 20
`
`4727
`
`A Novel M, 32.000 Nuclear Plwsphoprotein ls Selectively Ex.
`pressed in Cells Competent for Self-Renewal. Loren D. Walensky.
`lfionuld S. C‘olTcy. Tseng-llui Chen. T;r.yy—C‘hoou Wu. and Gary R,
`Pamlerniicl-;_
`
`Expression of Vascular Permeability Faclur {Vascular E[|d(]tI1_gIia]
`Growth Factor} and Its Receptors in Adenocarcinumas 0|‘ the G35-
`Iroimestinal Tract. Lawrence F, Brown, Brygidu Berse. Robert W,
`Jackinaii. Kathi 'l‘ogn:i7.r.i. F.1e:mor _|. Manseau. Donald R. Sanger. and
`Harold F. Dvorak.
`
`.——--—'_ '
`
`4736 A..:1ouncements
`New Schedllle for Annual Meetings of the
`I...-xC.‘R Beginning in I994
`N-_'\\ Privileges and Responsibilities of
`\ urrcspunding Membership
`§—‘gr:i:!Llr:ning of Assoeiiile Metiibership
`(an inn Suggestions for Asmeiuliolt
`<."Il't':cers and Directors
`.-\.«\\'R Special Conferences in Cancer
`l{=:<earcli
`C‘-ulelniar of 1-lveritri
`
`4738 Erratum
`5.’.
`it. el Kouni er at, 5.1’: 3681-36'-J3. 1993.
`
`4739 nuthor Index
`
`Pages ls—26s
`
`Call for Abstracts for I994
`
`AACR Annual Meeting
`
`Includes: Abstract Submission Forms
`Advance Registration and
`Housing Forms
`
`Also available In the back
`or this issue:
`
`Application for Active and
`Corresponding Membership
`
`Application tor Associate
`Membership
`
`Guidelines for Submitting Disks to
`AACR Publications
`
`LILLY EX. 1005 - 5/11
`
`LILLY EX. 1005 - 5/11
`
`

`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`ABSTRA "T
`
`We hm--~ explored the therapeutic effects of anti-epidermal growth
`{mar receptor monoclonal antibodies (MAbs) 225 and 528 on well estab-
`med A43‘ epidermoid carcinoma xenografts, approximately 400 mm’
`[I C,“ in diameter) at the start of treatment.
`in previous reports we
`demoltslril
`‘cl that MAbs 225 and 528 prevented the growth of A431 cell
`mmgrafts in nude Inice when treatment was begun on the day of tumor
`Njl
`inocu itinn. Since anti-epidermal growth factor
`receptor MAb
`mmpy of well established tumors was unable to retard growth, we ex-
`P|ored cot: aination therapy with MAb plus the chemotherapeutic agent
`fl'5.clian1minedichloroplatinunt (cis-DDP). Additive and Ct.|l'lItI.‘I'll.l'l1l.ll]I‘l-lIlE-
`pendent g1 on-th-inhibitory effects of MAb with cis-DDP were observed in
`mum-es ol'A-l3l cells. Neither intensive treatment with 225 MAb (1 mg‘
`muse, i.p. on day 8 after tumor inoculation, and twice weekly for 4 weeks)
`um-a maximally tolerated single dose of cis-DI]? [I50 ugi‘2‘.5 g [6 mglkg)
`mouse weight,
`i.p. on day 81 had significant effects on tumor growth.
`flowever, i‘:c two treatments in combination resulted in substantial xeno-
`grafl growth inhibition, compared with both an untreated control group
`and anima“. treated with a single modality. When a second dose of cis-DDP
`{[59 p.g.l25 g) was added after ll) days, combination therapy with 225 MAI)
`produced
`riking antitumor effects. At the end of 1 month tumor xeno-
`gmtts had disappeared in all but one mouse. and on tumor relapses
`occurred t.'.li‘lI'lg 6 months of observation. identical results were obtained
`with anti-epidermal growth factor receptor MA!) 528 in combination with
`its-DDP. 3 lie results of these studies provide a novel approach to the
`treatment of well established tumor xenografts, which may have applica-
`tion in the therapy of human malignancies.
`
`T?“ ]H-._-,_ ,\|{(‘i l 53, -tn.1'.|4r»4l. Uctobcr l. l0\J3|
`Antitumor Effect of Anti-Epidermal Growth Factor Receptor Monoclonal
`Antibodies plus cis-Diamminedichloroplatinum on Well Established
`A431 Cell Xenografts‘
`EM. Fan, Jose Baseiga, Hideo Masai, and John Mendelsohnz
`.vt-n- York h’)l’i2i
`l’aa-ii.
`.’\’en'
`Uwmmr-V,-4:1 Rt”(‘t'.Dl‘t’)J' !:ti'o.'og_v. Mremm'r'nl' Sltlflrt-KtYlf(.‘l'lFl'_L' t’.'rrm.‘c: C'emrr [2, H, J. B.. H. M.. J. M.) om.’ Comcil Uilll-'(’r.\'iJ'_t' ll-i‘erit'ru! St‘i':rml (J. it-1.}.
`ziutocrinc EGF receptor stimulation for growth in culture. A dose of I
`mg 225 or 528 Math twice weekly, by ip injection, could sustain
`serum lcvcls adequate to saturate EGF receptors (23). Anti—EGF re—
`ceptor MAI) therapy successfully inhibited the growth of Xcnogralted
`tumor cell lines from squamous cancers of the vulva and lung. as well
`as adcnocarcinomns of the breast and colon (I8. 19. 23-27). inhibitory
`activity against xcnografts was also observed with the F(:th')3 frag-
`ment of 225 MAb_. indicating that pharmacological blockade of EGF
`receptor by antibody without the capacity for an immune response can
`also mediate the antilumor effect in vivr) (28). In tliese studies, treaI—
`mcnt with MAb was begun the day of tumor cell inoculation.
`To initiate investigation of the clinical application of our anti—EGF
`receptor MAb.-.. we performed a phase [ clinical trial of a single dose
`of '”ln-labeled 225 MAb (29). At MAb doses of 40 mg or higher,
`imaging studies on patients with advanced squamous cell carcinoma
`of the lung visualized each primary tumor and presumed sites of
`metastatic disease with diameter >1 cm. The scrum concentration of
`225 MAh could be maintained at >4li nu (receptor saturating levels}
`for more than 3 days without toxicity. Collectively. these preclinical
`and early clinical studies make :1 strong case for exploring the poten-
`tial therapeutic role of anti-EGF rcccptor MAbs as pharmacoiogicai
`agents that block tyrosine kinasc mediated signal transduction.
`The premise for our approach is that :tnti—EZGF receptor MAbs block
`the access of EGF or TGF-cr to their rcceptors, resulting in thc inhi-
`bition of tumor growth. Studies with cell cultures suggest that block-
`adc of EGF receptors by MAbs results primarily in cylostatic rather
`than cytocidai effects (ll—l4). Furthermore, treatment with MAb has
`not successfully eliminated well established xcnografts (23). An ex-
`ception is the DiFi colon adenocarcinoma cell line which is extremely
`sensitive to MAb treatment, both in culture and in xcnografts (18).
`Therefore. for the treatment of most well established tumors.
`it was
`important to explore methods for enhancing the antitumor effects of
`anti—EGF receptor MAbs.
`There has been interest in Combining cytotoxic chemotherapeutic
`agents with growth-inhibitory bioiogical agents,
`including MAhs
`against the EGF receptor (30, 3]) and against l-iER2/c—erbB-2 protein
`(pl8S”’“”) (32. 33). There is an excellent rationale for exploring
`these combinations. The antiprolifcrativc effect of MAbs targeted
`against receptors on plasma membranes may be limited by competi-
`tion with growth factors produced and released in the cellular cnvi-
`ronment. Chemotherapy can act to reduce the number of malignant
`cells, thereby reducing the concentration of essential aulocrine growth
`factors produced in the ilumcdiatc environment of rcsidual viable
`cells. The potential of cell membrane receptors and signal transduction
`pathways to serve as opportune targets for cancer chemotherapy also
`has received considerable attention (34. 35). Furthermore. recent slud-
`R'5L‘€iVm 6.-'10.-'93: accepted 8.! i9.n"J3.
`ics indicate that both c|1cmothcrapy—induccd cytotoxicity and death of
`The 9051* of publication of lhis article were defrayed in part by the payment of page
`cells deprived of essential growth factors may involve programmed
`
`T|':- article must therefore be hereby marked dlft-'1’l‘ll's1’lll(’Hl‘
`in accordance with
`cell death or apoptosis (36-39). stiggcsting a common cytotoxic path-
`SE-5_ - Section L".-'34 solely to indicate this fact.
`C
`“"5 -
`-rk was supported by The Dmr Friedenherg Foundation rind Nlll Grants
`way lhal might be augmented by combination therapy.
`"33tl6U and Ci-'Li'J’o4l.
`Ci.5'—DDP is an alkylating agent which produces intcrstrand and
`M ‘T'~"“"li
`El FCL|lIC5l5 for reprints should be addressed, at Box 156, 12‘i'S York Avenue.
`intrastrand base cross—linl-ting in DNA and is one of the most active
`°;“1?f!a| tiloan-Kettering Cancer Center. New York. NY lUU2l.
`hi‘ Hhlircviations used arc: EGF. epidermal growth factor: TGF-or.
`transforming
`Elflwlh far. -1' rr: M.-’-\b, monoclonal antibody: c:'.\'-l3l')P. L-is-dinnimincdichloroplritinurn.
`drugs against the human epithelial tumors that express high levels of
`4637
`
`INTRODUCTION
`
`High expression of EGF“ receptors and the presence of a potential
`TGF-oi rt. _diated autocrinc stimulation pathway in many human can-
`cers have stimulated investigation of therapy with MAbs that block
`binding L1‘. ligand to EGF receptors (I-10). We have produced and
`cl1:iractcri=cd two MAbs against the EGF receptor, 225 IgG] and 528
`igG2,, which bind to the receptor with affinity similar to EGF and
`TGF—n, t
`.rnpcie with these ligands for receptor binding, and block
`EGF or TGF—ct
`induced activation of EGF receptor tyrosine phos-
`|Jl10ryiation(ll—l4). These MAbs inhibit proliferation of ‘:1 variety of
`Cultured malignant human cell lines which express EGF receptors and
`TGF-or. including vulva (11-13), breast (15, 16), colon (I7, 18), lung
`(19). ran: = (20). and prostate cancers (21). MAI"; 455, which binds to
`EGF receptors without inhibiting the binding of ligand, had no effect
`011 prolift ration of cultured cell lines (1 1. 22).
`Additional experiments demonstrated the capacity of these anti-
`BGF receptor MAbs to produce concentration-dependent inhibition of
`W10graf' of human tumor cell lines. which appeared to depend upon
`
`LILLY EX. 1005 - 6/11
`
`LILLY EX. 1005 - 6/11
`
`

`
`tcm3) I'D
`
`TumorSize
`
`0
`
`5
`
`15
`Days
`
`25
`
`35
`
`Ewitiili 1'33“
`Fig. l. The inliiliition ol A-13] cell xcttogralts by anti-I-IUF receptor
`dependent upon tumor wine. MAb 225 treatment (1 rng_.-'tttou<e.
`i.p.. twice :<. week] m
`5“'“‘-‘l' 0" ‘J33 3 f.1v 5 (‘iv “F 5 ll} film-‘F bvc. tumor ittocttlatinn and uric.-.| on claw 32
`(at-mm). The control group was treated with phttsphtiie-l\utt'cred saline [0] The datit an
`expressed as the meilll tumor size 1 SE (7 mice per groitpj.
`
`(41). cit;-DDP (platinol) was 21 gift front the Bristol Myers—Squilib Ci':mpanv.
`Cell Culture and Growth Assay A431 cells were ntatntained in 1:1 Dul-
`hecctfs modified Eagles'.~:
`incdiutnt’Ham's F-l2 mixture tvlv) supplentented
`with ltlfito fetal call‘ serum. Cells were distributed into ti—wt:|| plates. and
`treatment was started the next day.
`(-is—DDl-‘ was freshly clissolvetl
`in phos-
`pltatc—btIffercd saline and added to appropriate wells, with or without anli—EGF
`receptor MAb at concentrations indicated in the figure legends. After 24 It. the
`drug was removed by washittg the cells twice. followed by addition of cell
`culture medium and Mfitb. The medium and M.-Kb were replenished every" 2-3
`ilays. Alter ti days of culture. cells were liarve-stud by iryp5iniy_;irion and
`counted with a (.'.:ntitcr counter.
`
`A43] Cell Xenngrnlts. B.r‘tI.Bt'e nude mice were implanted sac. with It)’
`A433 cells.-"niouse. Tumors were nieasurud every 3-4 d:1_vs with vcrnicr cali-
`pers. Tumor size was calculated by the formula:
`
`-.
`77
`)
`F ah‘
`
`I CDDP
`
`7
`
`O‘!
`
`I 225 Mm
`225 Matt + CDDP
`[DJ -_tg.-'tn|x2-tnt
`
`wllerc at is the length and I7 is the width. and is =: b, Ar-iinmls with cslithilfihtjtl
`tumor xenograI’t.s' were divided into groups with comparable tumor size and
`treated. as dcserihctl
`in the text and figure legends. Briefly.
`tor anti—EGF
`receptor Mfith treatment, mice received 1
`tug of MN:
`rip.
`tn ph0!~tp|t2tlC-
`buffered saline twice in week. slurliltg on day 3. S, or it after tumor cell
`inoculation and ending on day 32. Previous studies have Cslubiisltcd this as the
`dose of MAE) required for prevention of tumor growth (23). Variotts doses and
`schedules of i.p. ci.a'—DDP were explored in tLn'nor—bearing nude mice. begin—
`ning 8 (lays after tumor cell
`inoculation. The mice were followed for the
`observation of xcnograft growth rate. body weight eltangcs. and life rsputl.
`
`"£5
`‘E4
`§
`
`$2
`0
`
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`
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`
`RESULTS
`
`0
`
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`~34
`§
`
`32
`O
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`1
`
`Effects of Anti-EGF Receptor MAD 225 upon Well Established
`Thtnor Xenografts. The data in Fig.
`1 show that inhibition of A43l
`cell xenograft:-. by 225 MAb is dependent upon tumor size. We dem-
`onstrated previously that anti—ECiF receptor Mfiibs 225 and 528 pre-
`vented the formation of s.c,
`tumor xcnogratts when the nude mice
`werc treated with l
`ing MAL) i.p. beginning on tltc day of tumor cell
`inoculation and that treatment with an irrelevant [gt] antibody had no
`inhibitory effect (23, 24}. Treatment of nude mice with 225 ll/[Ab
`beginning 3 days after tumor cell inocttlatinn. when the mean tumor
`treatment conditions, the exposure time of A431 cells to cr'_v-l)DPtt't’~‘
`size was about 150 lIl1‘l'1’-5. also was able to inhibit the formation of
`only for the first 24 h, and the cells were maintained in cute re foratl
`A431 cell xenografts (Fig.
`I]. However,
`it‘ MAb treatment was de-
`additional 5 days after removal of the drug. rt":-DD!’ inhibited A431
`layed until 5 days after tumor cell inoculation, when tumors reached
`cell proliferation in a dose-dependent fashion. The continttous p1’€5'
`a mean size of about 20!! mm", 225 MAb could only retard tumor
`citce of 20 ntvr 225 MAb [a saturating concentration) ti-r 5 day?
`xettograft growth. If the treatment was started 8 days after tumor cell
`produced additive inhibitorv effects on cell growth. Converscl,V'.- ""5
`ittoeulatiori, when the tumor was well established with a rncait size of
`additive effect was also observed when the concentration o I" 135 MA”
`about 400 mm” (approximately I cm in diameter), 225 MAI-t had
`varied from 0.2 to 200 mi, in cultures treated for 24 h with it] t'«£';""‘|
`almost no effect on xcnografi growth. The experiment in Fig.
`l
`is
`[tl.33 mt-it r.-is-DDP (Fig. 21?).
`identical results were obtained W119”
`representative of three independent studies. showing limited response
`225 M/\b was replaced by 528 M/\b. which also blocks liitttllllg "f
`to MAb tlterapy at this and higher doses. Similar results also were
`EGl~‘tTGF—tx: however. no additive effect was found when 325 MN’
`observed with 528 MAI“: treatment (data not shown).
`was replaced by 455 MM). which binds to EGF receptors ht. does I19‘
`Additive Cytotoxieily of cis-DDP and Anti-EGF Receptor MAI)
`block binding of EGl*‘,-’I“GF—ct (data not shown).
`in Cell Culture. Experiments were carried out to determine the etlect
`Toxicity and Antittimor Activity of cis-DDP on xet..»gralf°"
`of c.'i.t'—DDP at subtoxic done. both alone aitd combined with 225 or 528
`Nude Mice.
`In preliminary studies. we found that ntide mire beating
`MAb. on the growth ofA43I Cetl cultures. Fig. 2:1 shows the response
`tumor xettogratts were more vulrier-able in toxicity front Ciltllltllhflapy
`to inct'easiitg concentrations c-is-DDP. To more closely mimic in t'r'vo
`than non—tunmr-bearing micc. Therefore. we explored th=
`dost! ‘’
`aiofiti
`
`LILLY EX. 1005 - 7/11
`
`cool: + 225 M.Ab tannin;
`
`
`
`
`
`-
`
`V
`
`/
`r
`
`5:
`
`0
`
`is
`200
`
`z
`a
`20
`2
`0.2
`0.3
`0.1
`0.03
`o
`225 lltlfitb tnlirll
`CDDP lugtrnl rt 24 hours)
`Fig. 2. Additive c_\.*10toxicit_v oi co-I)[)P in combination with 22-3 .\-inn .n A-13! mil
`cultur
`. A-'13! cells were seeded onto 6 well plates at 2 >4
`It)" l.'I’.‘iiKt"V\'CIl. In .4. A43] CCU’
`were treziicd with u'.s-I)l'}P at indicated cortccnlrzttions on the first day for it Ala] of 14]‘-
`in the continuoits presence [E] or 2-tit:xI;1'I€t: (I) of Zti HM 225 -.\"l.r\b for (1 tItI_\-‘R. In BA-531
`cells were treated with inilic.-ited coitceittralions of 215 M.-Kb for ft darn. plus .
`0! mini‘
`(I!