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`
`Clinical cancer research
`v.18. no. 14 (July 15 2012)
`
`General Collection
`W1 CL672
`
`2012-07-26 06:48:43
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`NATIONAL
`LIBRARY OF
`MEDICINE
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`
`

`

`
`
`Clinical
`Cancer
`Research
`The Journal of Clinical and Translational Research
`
`July 15, 2012 © Volume 18 * Number 14
`
`Highlights of This Issue
`
`3717
`
`Molecular Pathways
`
`SPECIAL FEATURES
`
`CCR Translations
`
`3719
`
`Mindthe Gap: Potential for Rebounds
`during Antiangiogenic Treatment
`Breaks
`John M.L. Ebos and Roberto Pili
`See article p. 3961
`
`CCR Perspectives in Drug Approval
`
`3722
`
`Vandetanib for the Treatment of
`Symptomatic or Progressive
`Medullary Thyroid Cancerin Patients
`with Unresectable Locally Advanced
`or Metastatic Disease: U.S. Food and
`Drug Administration Drug Approval
`Summary
`Katherine Thornton, Geoffrey Kim,
`V. Ellen Maher, Somesh Chattopadhyay,
`Shenghui Tang, YoungJin Moon, Pengfei Song,
`Anshu Marathe, Suchitra Balakrishnan,
`Hao Zhu, Christine Garnett, Qi Liu,
`Brian Booth, Brenda Gehrke, Robert Dorsam,
`Leigh Verbois, Debasis Ghosh, Wendy Wilson,
`John Duan, Haripada Sarker,
`Sarah Pope Miksinski, Lisa Skarupa,
`Amna Ibrahim, Robert Justice,
`Anthony Murgo, and Richard Pazdur
`
`Statistics in Clinical Cancer Research
`
`3731
`
`Cure Models as a Useful Statistical
`Tool for Analyzing Survival
`Megan Othus, Bart Barlogie,
`Michael L. LeBlanc, and John J. Crowley
`
`CCR Drug Updates
`
`3737
`
`Crizotinib for ALK-Rearranged
`Non-Small Cell Lung Cancer: A New
`Targeted Therapy for a New Target
`Leena Gandhi and Pasi A. Janne
`
`3743
`
`Molecular Pathways: Targeting
`P21-Activated Kinase | Signaling in
`Cancer—Opportunities, Challenges,
`and Limitations
`Jeyanthy Eswaran, Da-Qiang Li, Anil Shah, and
`Rakesh Kumar
`
`Review
`
`3750
`
`Combining Antiangiogenics to
`Overcome Resistance: Rationale
`and Clinical Experience
`Victor Moreno Garcia, Bristi Basu,
`L. Rhoda Molife, and Stan B. Kave
`
`HUMAN CANCER BIOLOGY
`
`3762
`
`3772
`
`3780
`
`3791
`
`Tumor-Associated Macrophages
`in Pediatric Classical Hodgkin
`Lymphoma: Association with
`Epstein-Barr Virus, Lymphocyte
`Subsets, and Prognostic Impact
`Mario Henrique M. Barros, Rocio Hassan, and
`Gerald Niedobitek
`
`Defective Epidermal Innate
`Immunity and Resultant Superficial
`Dermatophytosis in Adult T-cell
`Leukemia/Lymphoma
`Yu Sawada, Motonobu Nakamura,
`Rieko Kabashima-Kubo, Takatoshi Shimauchi,
`Miwa Kobayashi, and Yoshiki Tokura
`
`FGFR4 Blockade Exerts Distinct
`Antitumorigenic Effects in Human
`Embryonal versus Alveolar
`Rhabdomyosarcoma
`Lisa E.S. Crose, Katherine T, Etheridge,
`Candy Chen, Brian Belyea, LindsayJ. Talbot,
`Rex C. Bentley, and Corinne M. Linardic
`
`Integrative Genomic Analysis
`Implicates Gain of PIKSCA at 3q26
`and MYC at 8q24 in Chronic
`Lymphocytic Leukemia
`Jennifer R. Brown, Megan Hanna,
`Bethany Tesar, Lillian Werner,
`Nathalie Pochet, John M. Asara,
`Yaoyu E. Wang, Paola dal Cin,
`Stacey M. Fernandes, Christina Thompson,
`Laura MacConaill, Catherine J. Wu,
`Yves Van de Peer, Mick Correll, Aviv Regev,
`Donna Neuberg, and Arnold 5. Freedman
`
`
`
`enLEUUtdUttIttIIISISSSSSSISSI
`
`The Journal of Clinical and Translational Research
`
`www.aacrjournals.org
`
`

`

`CANCER THERAPY: PRECLINICAL
`
`3868
`
`3803
`
`3812
`
`3822
`
`3834
`
`3846
`
`3856
`
`Development and Characterization
`of a Novel CD19CherryLuciferase
`(CDL9CL) Transgenic Mouse for
`the Preclinical Study of B-Cell
`Lymphomas
`Luigi Scotto, Marianna Kruithol-deJulio,
`Luca Paoluzzi, Matko Kalac, Enrica Marchi,
`Jairo Baquero Buitrago, Jennifer Amengual,
`Michael M. Shen, and Owen A. O'Connor
`
`Developmentof a Human
`Monoclonal Antibodyfor Potential
`Therapy of CD27-Expressing
`Lymphoma and Leukemia
`Laura A, Vitale, Li-Zhen He,
`Lawrence J. Thomas, Jennifer Widger,
`Jeffrey Weidlick, Andrea Crocker,
`Thomas O'Neill, James Storey,
`MartinJ. Glennie, Deanna M. Grote,
`Stephen M, Ansell, Henry Marsh, and
`Tibor Keler
`
`Elevation of c-FLIP in Castrate-
`Resistant Prostate Cancer
`Antagonizes Therapeutic Response
`to Androgen Receptor-Targeted
`Therapy
`Clare McCourt, Parnela Maxwell,
`Roberta Mazzuechelli, Rodolfo Montironi,
`MarinaSearpelli, Manuel Salto-Tellez,
`Joe M. O'Sullivan, Daniel B. Longley, and
`David J.J. Waugh
`
`Development of an Fe-Enhanced
`Anti-B7-H3 Monoclonal Antibody
`with Potent Antitumor Activity
`Deryk Loo, Ralph F. Alderson,
`Francine Z. Chen, Ling Hoang,
`Wenjun Zhang, Sergey Gorlatey, Steve Burke,
`Valentina Ciecarone, Hua Li, Vinh Yang,
`‘Tom Son, Yao Chen, Ann N. Easton,
`Jonathan ©.
`Li, JiR. Rillema, Monica Licea,
`Claudia Fieger, Tony W. Liang,
`Jennie P) Mather, Seott Koenig,
`Stanford |. Stewart, Syd Johnson,
`vio Bonvini, and Paul A. Moore
`
`Antitumor Activity of Targeted
`and Cytotoxie Agents in Murine
`Subcutaneous Tumor Models
`Correlates with Clinical Response
`Harvey Wong, Edna F, Choo, Bruno Alicke,
`Xiao Ding, Hank La, Erin MeNamara,
`Frank-Peter Theil, Jay ‘Tibbitts,
`Lori S. Friedman, Cornelis E.C.A, Hop,
`and Stephen E. Gould
`
`Anti-MyelomaEffects of the Novel
`Anthracycline Derivative INNO-206
`Eric Sanchez, Mingjie Li, Cathy Wang,
`
`Cydney M. Nichols, Jennifer Li, He
`and James KR. Berenson
`
`3880
`
`3889
`
`3901
`
`3912
`
`3924
`
`Brachyury, a Driver of the
`Epithelial-Mesenchymal Transition,
`Is Overexpressed in Human Lung
`Tumors: An Opportunity for Novel
`Interventions against Lung Cancer
`Mario Roselli, Romaine L. Fernando,
`Fiorella Guadagni, Antonella Spila,
`Jhessica Alessandroni, Raffaele Palmirotta,
`
`Leopoldo Costarelli, Mary Litzinger,
`Duane Hamilton, Bruce Huang,
`Joanne Tucker, Kwong-Yok T’sang,
`Jeffrey Schlom, and Claudia Palena
`
`Targeting Fibroblast Growth Factor
`ReceptorSignaling Inhibits Prostate
`Cancer Progression
`Shu Feng, Longjiang Shao, Wendong Yu,
`Paul Gavine, and Michael Ittmann
`
`Resistance to TRAIL Is Mediated
`by DARPP-32 in Gastric Cancer
`Abbes Belkhiri, Shoumin Zhu, Zheng Chen,
`Mohammed Soutto, and Wael El-Rifai
`
`GDC-0941, a Novel Class I Selective
`PI3K Inhibitor, Enhances the
`Efficacy of Docetaxel in Human
`Breast Cancer Models by Increasing
`Cell Death Jn Vitro and In Vivo
`Jeffrey J. Wallin, Jane Guan, Wei Wei Prior,
`Leslie fh. Lee, LeanneBerry, Lisa D, Belmont,
`Hartmut Koeppen, Marcia Belvin,
`Lori S, Friedman, and Deepak Sampath
`
`AT13148 Is a Novel, Oral Multi-AGC
`Kinase Inhibitor with Potent
`Pharmacodynamic and Antitumor
`Activity
`Timothy A. Yap, Mike I, Walton,
`Kyla M. Grimshaw, Robert HH. te Poele,
`Paul D. Eve, Melanie R. Valenti,
`Alexis K. de Haven Brandon, Vanessa Martins,
`Anna Zetterlund, Simon P. Heaton,
`Kathrin Heinzmann, Paul S. Jones,
`Ruth E. Feltell, Matthias Reule,
`Steven J. Woodhead, Thomas G. Davies,
`John F. Lyons, Florence |, Raynaud,
`Suzanne A. Eecles, Paul Workman,
`Neil 1. Thompson, and Michelle D. Garrett
`
`Vandetanib, an Inhibitor of VEGF
`Receptor-2 and EGF Receptor,
`Suppresses Tumor Development
`and Improves Prognosis of Liver
`Cancer in Mice
`Kinya Inoue, ‘Takuji Torinura,
`‘Toru Nakamura, Hideki Iwamoto,
`Hiroshi Masuda, Mitsuhiko Abe,
`Osamu Hashimoto, Hironori Koga,
`Takato Ueno, Hirohisa Yano, and Michio Sata
`
`
`
`AR. American Association for CancerResearch
`
`iv
`
`Clinical Cancer Research
`
`

`

`IMAGING, DIAGNOSIS, PROGNOSIS
`
`PREDICTIVE BIOMARKERS AND
`PERSONALIZED MEDICINE
`
`3934
`
`3942
`
`3952
`
`Pyrophosphorolysis-Activated
`Polymerization Detects Circulating
`Tumor DNA in Metastatic Uveal
`Melanoma
`
`Jordan Madic, Sophie Piperno-Neumann,
`Vincent Servois, Aurore Rampanou,
`Maud Milder, Benedicte Trouiller,
`David Gentien, Stéphanie Saada,
`Franck Assayag, Aurélie Thuleau,
`Fariba Nemati, Didier Decaudin,
`Frangois-Clément Bidard,
`Laurence Desjardins, Pascale Mariani,
`Olivier Lantz, and Mare-Henri Stern
`
`A Replication Study and Genome-
`Wide Scan of Single-Nucleotide
`Polymorphisms Associated with
`Pancreatic Cancer Risk and Overall
`Survival
`Jason A, Willis, Sara H. Olson, Irene Orlow,
`Semanti Mukherjee, Robert R. McWilliams,
`Robert C. Kurtz, and Robert J. Klein
`
`Multisite Validation Study to
`Determine Performance
`Characteristics of a 92-Gene
`Molecular Cancer Classifier
`Sarah E. Kerr, Catherine A, Schnabel,
`PeggyS. Sullivan, Yi Zhang, VeenaSingh,
`Brittany Carey, Mark G. Erlander,
`W. Edward Highsmith, Sarah M. Dry,
`and Elena F. Brachtel
`
`CANCER THERAPY:CLINICAL
`
`3961
`
`Rapid Angiogenesis Onset after
`Discontinuation of Sunitinib
`Treatment of Renal Cell Carcinoma
`Patients
`
`Arjan W. Griffioen, Laurie A. Mans,
`Annemarie M.A. de Graaf,
`Patrycja Nowak-Sliwinska,
`Céline L.M.M. de Hoog, Trees A.M, de Jong,
`Florry A. Vyth-Dreese, Judy R. van Beijnum,
`Axel Bex, and Eric Jonasch
`See commentary p. 3719
`
`3972
`
`3982
`
`3992
`
`Predictive Value of YRCCI Gene
`Polymorphisms on Platinum-Based
`Chemotherapyin Advanced Non-
`Small Cell Lung Cancer Patients:
`A Systematic Review and Meta-
`analysis
`Junjie Wu, Jie Liu, Yuhao Zhou, Jun Ying,
`Houdong Zou, Shicheng Guo, Lei Wang,
`Naiqing Zhao, Jianjun Hu, Daru Lu, Li Jin,
`Qiang Li, and Jiu-Cun Wang
`
`Genetic Polymorphisms in
`MicroRNA-Related Genes as
`Predictors of Clinical Outcomes
`in Colorectal Adenocarcinoma
`Patients
`
`Moubin Lin, Jian Gu, Cathy Eng, Lee M. Ellis,
`Michelle A. Hildebrandt, Jie Lin,
`Maosheng Huang, George A. Calin,
`Dingzhi Wang, Raymond N, DuBois,
`Ernest
`‘T. Hawk, and Xifeng Wu
`
`Serum Insulin-Like Growth Factor-1
`Levels Predict Outcomes of Patients
`with Advanced Hepatocellular
`Carcinoma Receiving
`Antiangiogenic Therapy
`Yu-Yun Shao, Chien-Chung Huang,
`Shiou-Der Lin, Chih-Hung Hsu, and
`Ann-Lii Cheng
`
`CORRECTION
`
`3998
`
`Correction: A Phase I Trial
`of Erlotinib and Concurrent
`Chemoradiotherapy for Stage ILI
`and IV (MO) Squamous Cell
`Carcinoma of the Head and Neck
`
`em
`The Journal of Clinical and Translational Research
`
`www.aacrjournals.org
`
`

`

`
`This material may be protected byCopyrightlaw (Title 17 U.S. Code}
`
`
`
`Cancel Therapy: Preclinical
`
`
`
`Development of an Fc-Enhanced Anti-B7-H3 Monoclonal
`Antibody with Potent AntitumorActivity
`
`Deryk Loo', Ralph F. Alderson*, Francine Z. Chen', Ling Huang’, Wenjun Zhang’, Sergey Gorlatov?,
`Steve Burke®, Valentina Ciccarone”, Hua Li*, Yinhua Yang’, Tom Son', Yan Chen’, Ann N. Easton’,
`Jonathan C.Li’, Jill R. Rillema', Monica Licea’, Claudia Fieger’, Tony W. Liang’, Jennie P. Mather',
`Scott Koenig’, Stanford J. Stewart’, Syd Johnson”, Ezio Bonvini®, and Paul A. Moore”
`
`Abstract
`
`Purpose: The goal ofthis research was to harness a monoclonal antibe dy (mAb) discovery platlorns to
`identify cell-surface antigens highly expressed on cancer and develop, through Fe optimization, potent mAb
`therapies toward these tumor-specific antigens.
`Experimental Design: Villy independent mAbs targeting the cell-surface immunoregulatory B7-113
`protein were obtainedthrough independent intact cell-based immunizations using humantissue progen-
`itor cells, cancercell lines, or cell lines displaying cancer stemcell properties. Binding studies revealed this
`natively reactive B7-113 mAb panelto bind a rangeof independent 87-113 epitopes. IMmunohistochemical
`analyses showedthata subset displayed strongreactivity 10 a broad range of humancancers while exhibiting
`limited binding to normal humantissues. A 7-113 mAb displaying exquisite timor/normal differential
`binding wasselected for humanization andincorporation ofan Fe domain modified to enhanceeffector.
`mediated antitumor function via increasedaffinity fortheactivating receptor CD 16A and decreased binding
`to the inhibitory receptor CD32B,
`Results: MGA271,
`the resulting engineered anti-&7-113 mAb, mediates potent antibody-dependent
`cellular cytotoxicity against a broad range of tumorcell types, Furthermore,
`in human CD16A-bearing
`transgenic mice, MGA271 exhibited potent antitumor activity in B7-113-expressing xenograft models of
`renal cell and bladder carcinoma, Toxicology studies carried out
`in cynomolgus monkeys revealed no
`significant test article-related safety findings.
`Conclusions: |his data supports evaluation of MGA271 clinical utility in B7-113-cxpressing cancer,
`while validating a combination of a nontarget biased approach ofintact cell immunizations and immu-
`Hohistochemistry to identily novel cancer antigens with Fe-based mAb engineering to enable potent
`antitumor activity. Clin Ganeer Res:
`I&( 14); 344-45, ©2012 AACR.
`
`Introduction
`
`Antigens that are timo specitie or overexpressed an
`cancer cells represent opportunites for development of
`Lirgebspeciiic antibody based (heripentics with a tanpeof
`possible therapeutic modalities: bor example, unmodified
`pC) monoclonal antibodies (mAb) directed to the PGt
`receptor (EGER) family, neutralize (imor promoting acty
`Hes ofsuch molecules rough antiprolifenative and tamer
`
`'MacroGenios, Inc., South Sar Francises, Calilornin
`Authors’ Affiliation:
`and “MacroGenies, Inc., Rockville, Maryland
`Note: Supplementary data for this article are available at Clinical Ganeor
`Research Online (http://clincancerres, aacrjournals.orq/)
`
`Corresponding Authors: Deryk Loo, MacroGenics, Inc., One Corporate
`Drive, South San Francisco, CA 94080. Phone: 650-624-2636; Fax: 650
`624-2693; E-mail;
`lood@Macrogenics.com and Paul A. Moore, Macro
`Genics, Inc,, 9640 Medical Center Drive, Rockville, MD 20850. Phone: 301-
`354-2692; Fax: 301-251-5321; E-mail: moorep@Macrogenics.com
`doi: 10.1158/1078-0432._CCR-12-0715
`"2012 American Association for Gancer Research,
`
`ididal mechanisms, Although such targeted therapies have
`shown clinical benefit,
`the responses are rarely sustained
`and are often limited to subsets of patients expressing the
`Antigen (ey. amplihed Her 3) metastatic breast cancer
`patients (reated with trastuzumab; rets. 1-3). Morerecently,
`nodiGitions to mAbs have been employed including drug
`conjugation, racionuctice labeling, and glycosylation or
`aminoacid substitutions in bo domains that have shown
`particular therapeutic advantages i preelinicaland clinical
`studies. Tn the case ol the Litter, these Po modifications can
`enhance variable repion dependent signaling properties of
`the mAb and be and imimdne cell-dependent. eflector
`AnCHons such as antibody dependent cellular eytotoxicity
`(ADCC) refs. 4, 4).
`iinproving: effector cell faneton bas
`The rationale lor
`been supported independently by the correlation of claieal
`outcomes Of mAb therapies with the natural polymorph
`isms of Poy receptors (POyYRS) in patients who participated in
`these trials. The allelic boy varbunts show dilferent allinities
`for the he domain of IpG and more favorable clinical
`
`
`3834=Clin Cancer Res; 18(14) July 15, 2012
`AIR Teriehse tition fav Cancer Research
`
`

`

`Translational Relevance
`
`|
`
`Using a monoclonal antibody (mAb)-driven process
`to identify cell-surface antigens highly expressed in can
`cer, we discovered a panel of mAbs against B7-113, a
`mernber of the B7 family of immunomodulatory mole
`cules. To translate this discovery for clinical evaluation,
`we developed MGA271, a humanized B7-113 mAb dis-
`playing broad tumorreactivity but limited normal tissue
`binding that
`incorporates Ve-domain modiheations
`designed to enhance antitumoreffector-mediated func-
`tion, The rationale that potent mAb-mediated effector
`functions will be clinically beneficial
`is supported by
`comparison ofclinical outcomes of mAb therapies with
`natural polymorphisms of FeyRs, which revealed more
`favorable Outcome for patients homozygous for the
`higheraffinity alleles ofthe activating receptors CD16A
`and CID32A. Wehypothesize that the enhancedantitu-
`moreffects of MGA27 1
`in preclinical studies, combined
`with its favorable safety profile in non-human primates,
`as shownin this article, will translate into potent anti-
`tumor activity toward B7-113-positive cancers in the
`|
`| clinic.
`
`
`
`responses were Observed in patients who were homozygous
`for the higher affinity alleles of beyR HA (CD32A) and,
`in
`particular, FeyR WIA (CD16A). These therapeutic mAbs
`include rituximab for
`treatment of
`follicular lymphoma
`(4,5), trastuzumabfor treatment of metastatic breast cancer
`(6), and, albeit controversial, cetuximab for treatment of
`metastatic colorectal caneer (MORC; ref 7). These results
`support the notion that potent immune-mediated effector
`functions can be clinically beneficial, and mAbs with
`enhanced be-mediated activity may favor not only patients
`with Jower affinity allelic variants for PeyRs but also those
`individuals who are homozygous for the higher binding,
`alleles, This strategy can be exploited for cell-surface cancer
`largels irrespective of their causality in the initiauon or
`progression of the tumor.
`‘Yo identify cell-surface cancer antigens suitable for mAb-
`based Lirpeting, we have characterized mAbs generatedina
`larvet-unbiased fashion trom intact cell immunizations of
`serum-free adapted tissue progenitor cells or cancer cell
`lines, including those exhibiting cancer stem cell properties
`(4, 9),
`Immunohistochemical profiling of these mAbs
`reveals those displaying differential expression on human
`cancer tissues compared with normal tissue, whereas bio-
`chemical analyses identily the cell-surface cancer antigen
`recomnized (10). Hlere we describe characterization ofa
`panel
`of 50 independent mAbs
`identified from this
`approach that
`recognize the cell-surface protein B7-H3
`(C1276), a memberof the B7 family of immuneregulators
`(11). Uhe limited normal Gssue binding and broad tumor
`reactivity exhibited by specific B7-113 mAbs prompted
`development of MGA271,
`4
`fully humanized anti-B713
`mAb bearing an engineered Fe domain optimized to
`
`Development of Fe-Enhanced Anti-B7-H3 Monoclonal Antibody
`
`enhance antitumor effector-mediated function through
`combination ol enhanced binding to the activating receptor
`CDIGA and reduced binding to the inhibitory receptor
`(D328. Vo determine the clinical potential of MGA271,
`antitumoractivity was evaltiated in a series of murine
`xenograft models and tts toxicology profile evaluated in
`non-human prirmates.
`
`Materials and Methods
`
`Cell lines
`Renal (A498, 786-0, and ACHIN), prostate (LnCap), lung
`(SK-MES-1), breast (MDA-MB-468), bladder (SW780 and
`HV-1197), melanoma(UAC 1C-62) cancer cell lines, and Raji
`B-cell lymphoma were obtained from American Type¢ cule
`ture Collection and cultured according, lo recommended
`protocol for fewer than 20 passages
`
`Immunohistochemistry
`OCT-embedded, frozen tissues, ancpositive control B7
`[1 4-expressing, Caki2 and [Is7001 cells were sectioned al 7
`tum. Following drying, sections were incubated in 4°C
`acetone, air dried, then processed in a Dako Autostainer,
`Formalin-hxed paraffin-embedded (VEPE) tissue microar-
`raysections weredeparaffinized, rehyd rated, then processed
`ina Dako Autostainer. Endogenous peroxidase activity was
`quenched with 3%[LO>, then nonspecific binding sites
`were blocked with 5%normal goat serum. Primary mAbs
`(BRCA84D and BRCAGID) were detected using EnVision
`horseradish peroxidase (IRP) anti-mouse polymer(Dako)
`in
`conjunction with
`3,3’ Diaminobenzidine
` (Sigma-
`Aldrich). Slides were counterstained with hematoxylin,
`
`Protein engineering
`chBRCASID was generated by fusing the BRCAS4D VI
`and VII coding sequences to humanKappa or human
`gamma | constant region cDNA, respectively, Po construct
`hHBRCAS4D, humanized BRCASSD VE (ABRCASAD VL)
`and BRCA84D VIL (ABRCAS4D VE) amine acid sequences
`were designed using the CDR sequences from the mouse
`mAb BRCA84D and framework sequences from human
`germline V-kappa or VIL
`segment,
`respectively,
`‘The
`HBRCASED VE and HBRCASAD VIL coding sequences were
`synthesized de novo, fused to the human C-Kappa or human
`gamma 1 constant region CONA, respectively. Single muta-
`tions, L464 in Vi and A93G in VIL, were introducedbysite
`directed mutagenesis to optimize the binding affinity tor
`B7-113,
`MGA271 was generatedfrom hBRCAS4D by exchanging
`its Fe domain lor MGEc0264 (L235V, P2431, R292P, YIOOL,
`and P396L). hBRCASAD-aglycosyl was generated from
`hBRCAS4D by mutagenesis at the N-glycosylation site of
`the WI Fe domain. Antibodies were produced in. stably
`transfected Chinese hamster ovarycells.
`
`ADCC
`Peripheral blood mononuclearcells (PBMC) were iso
`lated from healthy human donor blood(Ficoll Paque Phis;
`GE Healtheare). Target cells, effector cells, and antibody
`
`www.aacrjournals.org
`
`Clin Cancer Res; 18(14) July 15, 2012
`
`3835
`
`

`

`Loo et al.
`Cl
`
`in Dulbecco's modified Eagle's
`were incubated overnight
`bivalent analyte interaction model. The equilibrium dis
`medium/l-12 containing 5% PBS. PBMC effector cells were
`sociation constant (Ky) was calculated as Ky=kyy/kyy,
`added at
`ratios of 25:1 to 30:1, LOI release (Promega
`Corp.) was measuredalter overnight incubation. Cytotox-
`In vivo efficacy
`icity (Yo)=(experimental cell lysis—antibody-independent
`All mouse expermments were carned out under protocols
`cell cytolysis)/(maximum target lysis—spontaneous target
`approved by the MacroGenics Institutional Animal Care
`lysis)
`© 100. Fey receptor genotypes were determined by
`and Use Committee (IACHIC), mCD16
`HhCDIGA'
`sequencing PCR-amplified DNA, To evaluate the ability of
`RAG2 © mice (MCD16 knockout mice expressing the
`murine B7-113 MAbs tosupportADCC, blCylated anti-K7-
`hCDIGA-158P transgene consistent with the distribution
`113 murine MAbs were mixed with a bispecific DART (Dual
`Of CDI6A in human tissues) were bred at MacroGenics.
`Affinity ReTargeting; CD16 FITC) comprising specificity of
`fumorcells (5©10° per mouse) in PBS
`Matrigel were
`human CD16 and Muorescein isothiocyanate (EIPC) and
`implanted subcutancously and antibodies administered
`incubated with cancer target cells together with resting
`intmvenously weekly, beginning approximately 1 week
`human PBMC effector cells (Ee — 30:1).
`following tumor implantation of after tumors of approxi
`mately 200 to 300 mim! had been allowed to form. Tumor
`sizes were monitored twice weekly by orthogonal measure
`ments with electronic calipers. Statistical differences.
`in
`tumor sizes were assessed by 2-way ANOVAs and Bonter-
`roni posttest analyses (GraphPad Prism 5.02).
`
`Mass spectrometry
`GBS antigen was immunoprecipitated from Ad9S8 cell
`membranes using biotinylated antibody andstreptavidin-
`coated resin (Pierce). Alter washing, antigens were eluted
`with low pit buffer and concentrated using Strataclean
`beads (Agilent ‘Technologies, Inc.),
`treated with reducing
`sample buffer, and subjected to SDS-PAGE. Antigen gel
`bands were excised, subjected to enzymatic digestion, fol
`lowed by liquid chromatography tindem mass spectrom.
`etry analysis.
`
`m”pture ELISA
`A MaxiSorp ELISA plate (Nalge Nune Inu.) was coated
`with soluble human 67-113-41g-His (0.3 up/mL) in Buplt
`bicarbonate buffer (Thermo Fisher Scientific) overnight at
`4G. The plate was blocked with PBS Containing 0.5%
`bovine seram albumin (BSA) and 0.1% lween-20 (PRST/
`BSA) for 30 minutes, Antibodies were diluted in PBST BSA
`and applied to the ELISA plate for
`| hour Pollowing wash
`with PBST, PRP conjugated goat anticmouse IpG (HE) b
`dilution b: 10,000 in PBST/BSA, Jackson LinmiunoReseary TD)
`was added for
`| hour, the phite washed and developed with
`80 uL/well of
`IME peroxidase substrate ane terminated
`with 40 pL/well
`[% TSO, Absorbance abd 50 nm (Agu)
`was determined and date analyzed asiag GraphPad Pesan 5
`soltware.
`
`SPRanalysis of human B7-113 binding to selected mAbs
`Binding of
`the BY mAb panel
`to human Be LE
`was analyzed by surbice phistiod resanance (SPRY ina
`BlAcore
`30000) biosensor
`(Miacare AB)
`as previously
`described (12, 13) mAbs were captited on poat anti
`mouse F(ab )) fragment (lackson linmunoResearch) coat
`ed CM-5 sensor chips and: binding: curves obtained (ol
`lowing injection of human B-Cell lis (RED Sys
`tems). Experimental binding curves were also generated
`following injection of BRCARAD and its humanized
`forms to both human and cynomolgus monkey BP-13
`on the CM-5 sensor chip. Data were analyzed using
`BlAevaluation 4.0) software. Kinetic constants,
`heyy ane
`kay) describing the binding of
`first arm of antibody to
`immobilized antigen were estimated by global
`fitting
`analysis af
`the assaciation/dissociation curves to the
`
`Pharmacokinetics/toxicology studies in cynomolgus
`monkeys
`Cynomolgus monkey experiments were conducted at
`SNBL USA, which adheres to the regulations outlined in
`the USDA Animal Welfare Act and the conditions specified
`in the Guide for the Care and Use of Laboratory Animals.
`The study protocols were approved by the ‘Testing Facility
`IACUIC. A single-dose study was conducted with 24 eyno-
`molgus monkeys randomized into 4 groups (3/pender/
`group) receiving vehicle control or MGA2Q71 at
`1, 30, oF
`150 my/ky by 60-minute intravenous infusion, ‘Terminal
`group animals (2/gender/group) were necropsied 7 days
`following doseadministration, Recovery group animals (1/
`gender/proup) were euthanized 40 days following dose
`administration for necropsies. A repeat-dose study was
`conducted with 52 cynomolgus monkeys randomized into
`5 groups receiving vehicle control (6/gender/group) or
`MGA271 (5/zender/proup) weekly lor 4 weeks at
`1, 10,
`10, oF
`150 mp/ke by 60-minute intravenous infusion,
`‘Terminal group animals (4/gender/proup for vehicle con
`trol, 3/gender/group for MGA271) were necropsied 7 days
`following the final dose administration. Recovery group
`animals (2/gender/group) were necropsied 70 days follow
`inp the final dase,
`
`Hesull
`
`Identification of a panel of B7-1