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Journal for ImmunoTherapy of Cancer

Open Access

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016): part two

National Harbor, MD, USA. 9-13 November 2016
  • Casey Ager1Email author,
  • Matthew Reilley2,
  • Courtney Nicholas1,
  • Todd Bartkowiak1,
  • Ashvin Jaiswal1,
  • Michael Curran1,
  • Tina C. Albershardt3Email author,
  • Anshika Bajaj3,
  • Jacob F. Archer3,
  • Rebecca S. Reeves3,
  • Lisa Y. Ngo3,
  • Peter Berglund3,
  • Jan ter Meulen3,
  • Caroline Denis4,
  • Hormas Ghadially5,
  • Thomas Arnoux4,
  • Fabien Chanuc4,
  • Nicolas Fuseri4,
  • Robert W. Wilkinson5,
  • Nicolai Wagtmann4,
  • Yannis Morel4,
  • Pascale Andre4Email author,
  • Michael B. Atkins6Email author,
  • Matteo S. Carlino7,
  • Antoni Ribas8,
  • John A. Thompson9,
  • Toni K. Choueiri10,
  • F. Stephen Hodi10,
  • Wen-Jen Hwu11,
  • David F. McDermott12,
  • Victoria Atkinson13,
  • Jonathan S. Cebon14,
  • Bernie Fitzharris15,
  • Michael B. Jameson16,
  • Catriona McNeil17,
  • Andrew G. Hill18,
  • Eric Mangin19,
  • Malidi Ahamadi19,
  • Marianne van Vugt20,
  • Mariëlle van Zutphen20,
  • Nageatte Ibrahim19,
  • Georgina V. Long21,
  • Robyn Gartrell22,
  • Zoe Blake22Email author,
  • Ines Simoes23,
  • Yichun Fu22,
  • Takuro Saito24,
  • Yingzhi Qian22,
  • Yan Lu22,
  • Yvonne M. Saenger25,
  • Sadna Budhu26Email author,
  • Olivier De Henau26,
  • Roberta Zappasodi26,
  • Kyle Schlunegger27,
  • Bruce Freimark27,
  • Jeff Hutchins27,
  • Christopher A. Barker26,
  • Jedd D. Wolchok26,
  • Taha Merghoub26,
  • Elena Burova28Email author,
  • Omaira Allbritton28,
  • Peter Hong28,
  • Jie Dai28,
  • Jerry Pei28,
  • Matt Liu28,
  • Joel Kantrowitz28,
  • Venus Lai28,
  • William Poueymirou28,
  • Douglas MacDonald28,
  • Ella Ioffe28,
  • Markus Mohrs28,
  • William Olson28,
  • Gavin Thurston28,
  • Cristian Capasso29Email author,
  • Federica Frascaro30,
  • Sara Carpi31,
  • Siri Tähtinen29,
  • Sara Feola32,
  • Manlio Fusciello29,
  • Karita Peltonen29,
  • Beatriz Martins29,
  • Madeleine Sjöberg29,
  • Sari Pesonen33,
  • Tuuli Ranki33,
  • Lukasz Kyruk29,
  • Erkko Ylösmäki29,
  • Vincenzo Cerullo29,
  • Fabio Cerignoli34Email author,
  • Biao Xi34,
  • Garret Guenther34,
  • Naichen Yu34,
  • Lincoln Muir34,
  • Leyna Zhao34,
  • Yama Abassi34,
  • Víctor Cervera-Carrascón35Email author,
  • Mikko Siurala35,
  • João Santos35,
  • Riikka Havunen36,
  • Suvi Parviainen35,
  • Akseli Hemminki35,
  • Angus Dalgleish37Email author,
  • Satvinder Mudan38,
  • Mark DeBenedette39Email author,
  • Ana Plachco39,
  • Alicia Gamble39,
  • Elizabeth W. Grogan39,
  • John Krisko39,
  • Irina Tcherepanova39,
  • Charles Nicolette39,
  • Pooja Dhupkar40Email author,
  • Ling Yu40,
  • Eugenie S. Kleinerman40,
  • Nancy Gordon40,
  • Italia Grenga41,
  • Lauren Lepone41,
  • Sofia Gameiro41,
  • Karin M. Knudson41,
  • Massimo Fantini41,
  • Kwong Tsang41,
  • James Hodge41,
  • Renee Donahue41Email author,
  • Jeffrey Schlom41,
  • Elizabeth Evans42Email author,
  • Holm Bussler42,
  • Crystal Mallow42,
  • Christine Reilly42,
  • Sebold Torno42,
  • Maria Scrivens42,
  • Cathie Foster42,
  • Alan Howell42,
  • Leslie Balch42,
  • Alyssa Knapp42,
  • John E. Leonard42,
  • Mark Paris42,
  • Terry Fisher42,
  • Siwen Hu-Lieskovan43,
  • Antoni Ribas43,
  • Ernest Smith42,
  • Maurice Zauderer42,
  • William Fogler44Email author,
  • Marilyn Franklin45,
  • Matt Thayer45,
  • Dan Saims45,
  • John L. Magnani44,
  • Jian Gong46,
  • Michael Gray46,
  • Jeff Hutchins46,
  • Bruce Freimark46Email author,
  • George Fromm47Email author,
  • Suresh de Silva47,
  • Louise Giffin47,
  • Xin Xu47,
  • Jason Rose47,
  • Taylor H. Schreiber47,
  • Massimo Fantini48,
  • Sofia R. Gameiro48,
  • Karin M. Knudson48,
  • Paul E. Clavijo49,
  • Clint T. Allen49,
  • Renee Donahue48,
  • Lauren Lepone48,
  • Italia Grenga48,
  • James W. Hodge48,
  • Kwong Y. Tsang48,
  • Jeffrey Schlom48,
  • Michael Gray50Email author,
  • Jian Gong50,
  • Jeff Hutchins50,
  • Bruce Freimark50,
  • Jane Grogan51Email author,
  • Nicholas Manieri51,
  • Eugene Chiang51,
  • Patrick Caplazi51,
  • Mahesh Yadav51,
  • Patrick Hagner52Email author,
  • Hsiling Chiu52,
  • Michelle Waldman52,
  • Anke Klippel52,
  • Anjan Thakurta52,
  • Michael Pourdehnad53,
  • Anita Gandhi52,
  • Ian Henrich54Email author,
  • Laura Quick55,
  • Rob Young55,
  • Margaret Chou55,
  • Andrew Hotson56Email author,
  • Stephen Willingham56,
  • Po Ho56,
  • Carmen Choy56,
  • Ginna Laport56,
  • Ian McCaffery56,
  • Richard Miller56,
  • Kimberly A. Tipton57,
  • Kenneth R. Wong57,
  • Victoria Singson57,
  • Chihunt Wong57,
  • Chanty Chan57,
  • Yuanhiu Huang57,
  • Shouchun Liu57,
  • Jennifer H. Richardson57,
  • W. Michael Kavanaugh57,
  • James West57,
  • Bryan A. Irving57Email author,
  • Kimberly A. Tipton58,
  • Kenneth R. Wong58,
  • Victoria Singson58,
  • Chihunt Wong58,
  • Chanty Chan58,
  • Yuanhiu Huang58,
  • Shouchun Liu58,
  • Jennifer H. Richardson58,
  • W. Michael Kavanaugh58,
  • James West58,
  • Bryan A. Irving58Email author,
  • Ritika Jaini59Email author,
  • Matthew Loya59,
  • Charis Eng59,
  • Melissa L. Johnson60Email author,
  • Alex A. Adjei61,
  • Mateusz Opyrchal62,
  • Suresh Ramalingam63,
  • Pasi A. Janne64,
  • George Dominguez65,
  • Dmitry Gabrilovich65,
  • Laura de Leon66,
  • Jeannette Hasapidis66,
  • Scott J. Diede67,
  • Peter Ordentlich66,
  • Scott Cruickshank66,
  • Michael L. Meyers68,
  • Matthew D. Hellmann69,
  • Pawel Kalinski70, 71Email author,
  • Amer Zureikat72,
  • Robert Edwards73,
  • Ravi Muthuswamy73,
  • Nataša Obermajer74,
  • Julie Urban73,
  • Lisa H. Butterfield72,
  • William Gooding72,
  • Herbert Zeh72,
  • David Bartlett74,
  • Olga Zubkova75Email author,
  • Larissa Agapova75,
  • Marina Kapralova75,
  • Liudmila Krasovskaia75,
  • Armen Ovsepyan76,
  • Maxim Lykov76,
  • Artem Eremeev75,
  • Vladimir Bokovanov75,
  • Olga Grigoryeva75,
  • Andrey Karpov75,
  • Sergey Ruchko75,
  • Charles Nicolette77,
  • Alexandr Shuster76,
  • Danny N. Khalil78Email author,
  • Luis Felipe Campesato78,
  • Yanyun Li79,
  • Taha Merghoub79,
  • Jedd D. Wolchok80,
  • Adam S. Lazorchak81Email author,
  • Troy D. Patterson81,
  • Yueyun Ding81,
  • Pottayil Sasikumar82,
  • Naremaddepalli Sudarshan82,
  • Nagaraj Gowda82,
  • Raghuveer Ramachandra82,
  • Dodheri Samiulla82,
  • Sanjeev Giri82,
  • Rajesh Eswarappa82,
  • Murali Ramachandra82,
  • David Tuck81,
  • Timothy Wyant81,
  • Jasmin Leshem83Email author,
  • Xiu-fen Liu83,
  • Tapan Bera83,
  • Masaki Terabe83,
  • Birgit Bossenmaier84,
  • Gerhard Niederfellner84,
  • Yoram Reiter85,
  • Ira Pastan83,
  • Leiming Xia86,
  • Yang Xia86,
  • Yangyang Hu86,
  • Yi Wang87,
  • Yangyi Bao87,
  • Fu Dai87,
  • Shiang Huang88,
  • Elaine Hurt89,
  • Robert E. Hollingsworth89,
  • Lawrence G. Lum90,
  • Alfred E. Chang86,
  • Max S. Wicha91,
  • Qiao Li92Email author,
  • Thomas Mace93Email author,
  • Neil Makhijani93,
  • Erin Talbert93,
  • Gregory Young93,
  • Denis Guttridge93,
  • Darwin Conwell93,
  • Gregory B. Lesinski93,
  • Rodney JM Macedo Gonzales94Email author,
  • Austin P. Huffman94,
  • Ximi K. Wang94,
  • Ran Reshef94,
  • Andy MacKinnon95Email author,
  • Jason Chen95,
  • Matt Gross95,
  • Gisele Marguier95,
  • Peter Shwonek95,
  • Natalija Sotirovska95,
  • Susanne Steggerda95,
  • Francesco Parlati95,
  • Amani Makkouk96Email author,
  • Mark K. Bennett96,
  • Jason Chen96,
  • Ethan Emberley96,
  • Matt Gross96,
  • Tony Huang96,
  • Weiqun Li96,
  • Andy MacKinnon96,
  • Gisele Marguier96,
  • Silinda Neou96,
  • Alison Pan96,
  • Jing Zhang96,
  • Winter Zhang96,
  • Francesco Parlati96,
  • Netonia Marshall97Email author,
  • Thomas U. Marron97,
  • Judith Agudo97,
  • Brian Brown97,
  • Joshua Brody97,
  • Christopher McQuinn98Email author,
  • Thomas Mace98,
  • Matthew Farren98,
  • Hannah Komar98,
  • Reena Shakya98,
  • Gregory Young98,
  • Thomas Ludwug98,
  • Gregory B. Lesinski98,
  • Y. Maurice Morillon99Email author,
  • Scott A. Hammond100,
  • Jeffrey Schlom99,
  • John W. Greiner99,
  • Pulak R. Nath101Email author,
  • Anthony L. Schwartz101,
  • Dragan Maric102,
  • David D. Roberts101,
  • Nataša Obermajer103Email author,
  • David Bartlett103,
  • Pawel Kalinski104, 105,
  • Aung Naing106,
  • Kyriakos P. Papadopoulos107,
  • Karen A. Autio108,
  • Deborah J. Wong109,
  • Manish Patel110,
  • Gerald Falchook111,
  • Shubham Pant112,
  • Patrick A. Ott113,
  • Melinda Whiteside114,
  • Amita Patnaik107,
  • John Mumm114,
  • Filip Janku106,
  • Ivan Chan114,
  • Todd Bauer106, 107,
  • Rivka Colen106,
  • Peter VanVlasselaer114,
  • Gail L. Brown114,
  • Nizar M. Tannir106,
  • Martin Oft114Email author,
  • Jeffrey Infante115,
  • Evan Lipson116Email author,
  • Ajay Gopal117,
  • Sattva S. Neelapu118,
  • Philippe Armand119,
  • Stephen Spurgeon120,
  • John P. Leonard121,
  • F. Stephen Hodi119,
  • Rachel E. Sanborn122,
  • Ignacio Melero123,
  • Thomas F. Gajewski124,
  • Matthew Maurer125,
  • Serena Perna126,
  • Andres A. Gutierrez127,
  • Raphael Clynes126,
  • Priyam Mitra126,
  • Satyendra Suryawanshi126,
  • Douglas Gladstone123,
  • Margaret K. Callahan127,
  • James Crooks128,
  • Sheila Brown128,
  • Audrey Gauthier129,
  • Marc Hillairet de Boisferon129,
  • Andrew MacDonald128,
  • Laura Rosa Brunet130Email author,
  • William T. Rothwell131Email author,
  • Peter Bell131,
  • James M. Wilson131,
  • Fumi Sato-Kaneko132Email author,
  • Shiyin Yao132,
  • Shannon S. Zhang133,
  • Dennis A. Carson132,
  • Cristina Guiducci133,
  • Robert L. Coffman133,
  • Kazutaka Kitaura134,
  • Takaji Matsutani134,
  • Ryuji Suzuki134,
  • Tomoko Hayashi132,
  • Ezra E. W. Cohen132,
  • David Schaer135Email author,
  • Yanxia Li135,
  • Julie Dobkin135,
  • Michael Amatulli135,
  • Gerald Hall135,
  • Thompson Doman136,
  • Jason Manro136,
  • Frank Charles Dorsey136,
  • Lillian Sams136,
  • Rikke Holmgaard135,
  • Krishnadatt Persaud135,
  • Dale Ludwig135,
  • David Surguladze135,
  • John S. Kauh137,
  • Ruslan Novosiadly135,
  • Michael Kalos135,
  • Kyla Driscoll135,
  • Hardev Pandha138,
  • Christy Ralph139,
  • Kevin Harrington140,
  • Brendan Curti141,
  • Rachel E. Sanborn142,
  • Wallace Akerley143,
  • Sumati Gupta143,
  • Alan Melcher144,
  • David Mansfield144,
  • David R. Kaufman145,
  • Emmett Schmidt145,
  • Mark Grose145,
  • Bronwyn Davies146,
  • Roberta Karpathy146,
  • Darren Shafren146Email author,
  • Katerina Shamalov147Email author,
  • Cyrille Cohen147,
  • Naveen Sharma148,
  • James Allison148,
  • Tala Shekarian149Email author,
  • Sandrine Valsesia-Wittmann150,
  • Christophe Caux149,
  • Aurelien Marabelle151,
  • Brian M. Slomovitz152Email author,
  • Kathleen M. Moore153,
  • Hagop Youssoufian154,
  • Marshall Posner155,
  • Poonam Tewary156Email author,
  • Alan D. Brooks157,
  • Ya-Ming Xu158,
  • Kithsiri Wijeratne158,
  • Leslie A. A. Gunatilaka159,
  • Thomas J. Sayers156,
  • John P. Vasilakos160Email author,
  • Tesha Alston160,
  • Simon Dovedi161,
  • James Elvecrog160,
  • Iwen Grigsby160,
  • Ronald Herbst162,
  • Karen Johnson160,
  • Craig Moeckly160,
  • Stefanie Mullins161,
  • Kristen Siebenaler160,
  • Julius SternJohn160,
  • Ashenafi Tilahun160,
  • Mark A. Tomai160,
  • Katharina Vogel161,
  • Robert W. Wilkinson161,
  • Eveline E. Vietsch163,
  • Anton Wellstein163Email author,
  • Martin Wythes164Email author,
  • Stefano Crosignani165,
  • Joseph Tumang164,
  • Shilpa Alekar164,
  • Patrick Bingham164,
  • Sandra Cauwenberghs165,
  • Jenny Chaplin164,
  • Deepak Dalvie164,
  • Sofie Denies165,
  • Coraline De Maeseneire165,
  • JunLi Feng164,
  • Kim Frederix165,
  • Samantha Greasley164,
  • Jie Guo164,
  • James Hardwick164,
  • Stephen Kaiser164,
  • Katti Jessen164,
  • Erick Kindt164,
  • Marie-Claire Letellier165,
  • Wenlin Li164,
  • Karen Maegley164,
  • Reece Marillier165,
  • Nichol Miller164,
  • Brion Murray164,
  • Romain Pirson165,
  • Julie Preillon166,
  • Virginie Rabolli165,
  • Chad Ray164,
  • Kevin Ryan164,
  • Stephanie Scales164,
  • Jay Srirangam164,
  • Jim Solowiej164,
  • Al Stewart164,
  • Nicole Streiner164,
  • Vince Torti164,
  • Konstantinos Tsaparikos164,
  • Xianxian Zheng164,
  • Gregory Driessens165,
  • Bruno Gomes165,
  • Manfred Kraus164,
  • Chunxiao Xu167Email author,
  • Yanping Zhang168,
  • Giorgio Kradjian168,
  • Guozhong Qin168,
  • Jin Qi168,
  • Xiaomei Xu168,
  • Bo Marelli168,
  • Huakui Yu168,
  • Wilson Guzman168,
  • Rober Tighe168,
  • Rachel Salazar168,
  • Kin-Ming Lo168,
  • Jessie English168,
  • Laszlo Radvanyi168,
  • Yan Lan168,
  • Roberta Zappasodi169Email author,
  • Sadna Budhu169,
  • Matthew D. Hellmann170,
  • Michael Postow170,
  • Yasin Senbabaoglu169,
  • Billel Gasmi169,
  • Hong Zhong169,
  • Yanyun Li169,
  • Cailian Liu169,
  • Daniel Hirschhorhn-Cymerman169,
  • Jedd D. Wolchok170,
  • Taha Merghoub169,
  • Yuanyuan Zha171Email author,
  • Gregory Malnassy172,
  • Noreen Fulton172,
  • Jae-Hyun Park172,
  • Wendy Stock173,
  • Yusuke Nakamura172,
  • Thomas F. Gajewski174,
  • Hongtao Liu174,
  • Xiaoming Ju175,
  • Rachelle Kosoff175,
  • Kimberly Ramos175,
  • Brandon Coder175,
  • Robert Petit175,
  • Michael Princiotta175,
  • Kyle Perry175,
  • Jun Zou175Email author,
  • Ainhoa Arina176Email author,
  • Christian Fernandez176,
  • Wenxin Zheng176,
  • Michael A. Beckett176,
  • Helena J. Mauceri176,
  • Yang-Xin Fu177,
  • Ralph R. Weichselbaum176,
  • Mark DeBenedette178Email author,
  • Whitney Lewis178,
  • Alicia Gamble178,
  • Charles Nicolette178,
  • Yanyan Han179Email author,
  • Yeting Wu180,
  • Chou Yang180,
  • Jing Huang180,
  • Dongyun Wu181,
  • Jin Li181,
  • Xiaoling Liang179,
  • Xiangjun Zhou181,
  • Jinlin Hou180,
  • Raffit Hassan182,
  • Thierry Jahan183,
  • Scott J. Antonia184,
  • Hedy L. Kindler185,
  • Evan W. Alley186,
  • Somayeh Honarmand187Email author,
  • Weiqun Liu187,
  • Meredith L. Leong187,
  • Chan C. Whiting187,
  • Nitya Nair187,
  • Amanda Enstrom187,
  • Edward E. Lemmens187,
  • Takahiro Tsujikawa188,
  • Sushil Kumar188,
  • Lisa M. Coussens188,
  • Aimee L. Murphy187,
  • Dirk G. Brockstedt187,
  • Sven D. Koch189Email author,
  • Martin Sebastian190,
  • Christian Weiss191,
  • Martin Früh192,
  • Miklos Pless193,
  • Richard Cathomas194,
  • Wolfgang Hilbe195,
  • Georg Pall196,
  • Thomas Wehler197,
  • Jürgen Alt197,
  • Helge Bischoff198,
  • Michael Geissler199,
  • Frank Griesinger200,
  • Jens Kollmeier201,
  • Alexandros Papachristofilou202,
  • Fatma Doener189,
  • Mariola Fotin-Mleczek189,
  • Madeleine Hipp189,
  • Henoch S. Hong189,
  • Karl-Josef Kallen189,
  • Ute Klinkhardt203,
  • Claudia Stosnach203,
  • Birgit Scheel189,
  • Andreas Schroeder203,
  • Tobias Seibel203,
  • Ulrike Gnad-Vogt203,
  • Alfred Zippelius202,
  • Ha-Ram Park204Email author,
  • Yong-Oon Ahn204,
  • Tae Min Kim205,
  • Soyeon Kim204,
  • Seulki Kim204,
  • Yu Soo Lee204,
  • Bhumsuk Keam205,
  • Dong-Wan Kim205,
  • Dae Seog Heo205,
  • Shari Pilon-Thomas206Email author,
  • Amy Weber206,
  • Jennifer Morse206,
  • Krithika Kodumudi206,
  • Hao Liu206,
  • John Mullinax206,
  • Amod A. Sarnaik206,
  • Luke Pike207,
  • Andrew Bang208,
  • Patrick A. Ott209,
  • Tracy Balboni207,
  • Allison Taylor207,
  • Alexander Spektor207,
  • Tyler Wilhite207,
  • Monica Krishnan207,
  • Daniel Cagney207,
  • Brian Alexander207,
  • Ayal Aizer207,
  • Elizabeth Buchbinder207,
  • Mark Awad207,
  • Leena Ghandi207,
  • F. Stephen Hodi209,
  • Jonathan Schoenfeld207Email author,
  • Anthony L. Schwartz210Email author,
  • Pulak R. Nath210,
  • Elizabeth Lessey-Morillon210,
  • Lisa Ridnour211,
  • David D. Roberts212,
  • Neil H. Segal213Email author,
  • Manish Sharma214,
  • Dung T. Le215,
  • Patrick A. Ott216,
  • Robert L. Ferris217,
  • Andrew D. Zelenetz213,
  • Sattva S. Neelapu218,
  • Ronald Levy219,
  • Izidore S. Lossos220,
  • Caron Jacobson216,
  • Radhakrishnan Ramchandren221,
  • John Godwin222,
  • A. Dimitrios Colevas219,
  • Roland Meier223,
  • Suba Krishnan223,
  • Xuemin Gu223,
  • Jaclyn Neely223,
  • Satyendra Suryawanshi223,
  • John Timmerman224,
  • Claire I. Vanpouille-Box225Email author,
  • Silvia C. Formenti225,
  • Sandra Demaria225,
  • Erik Wennerberg226Email author,
  • Aranzazu Mediero227,
  • Bruce N. Cronstein226,
  • Silvia C. Formenti228,
  • Sandra Demaria228,
  • Michael P. Gustafson229Email author,
  • AriCeli DiCostanzo229,
  • Courtney Wheatley229,
  • Chul-Ho Kim229,
  • Svetlana Bornschlegl229,
  • Dennis A. Gastineau229,
  • Bruce D. Johnson229,
  • Allan B. Dietz229,
  • Cameron MacDonald230Email author,
  • Mark Bucsek230,
  • Guanxi Qiao230,
  • Bonnie Hylander230,
  • Elizabeth Repasky230,
  • William J. Turbitt231Email author,
  • Yitong Xu231,
  • Andrea Mastro231,
  • Connie J. Rogers231,
  • Sita Withers232Email author,
  • Ziming Wang232,
  • Lam T. Khuat232,
  • Cordelia Dunai232,
  • Bruce R. Blazar233,
  • Dan Longo234,
  • Robert Rebhun232,
  • Steven K. Grossenbacher232,
  • Arta Monjazeb232,
  • William J. Murphy232,
  • Scott Rowlinson235,
  • Giulia Agnello235Email author,
  • Susan Alters235,
  • David Lowe235,
  • Nicole Scharping236,
  • Ashley V. Menk237Email author,
  • Ryan Whetstone236,
  • Xue Zeng236,
  • Greg M. Delgoffe236,
  • Patricia M. Santos237Email author,
  • Ashley V. Menk237,
  • Jian Shi237,
  • Greg M. Delgoffe236,
  • Lisa H. Butterfield237,
  • Ryan Whetstone236Email author,
  • Ashley V. Menk237,
  • Nicole Scharping236,
  • Greg Delgoffe236,
  • Misako Nagasaka238Email author,
  • Ammar Sukari238,
  • Miranda Byrne-Steele239Email author,
  • Wenjing Pan239,
  • Xiaohong Hou239,
  • Brittany Brown239,
  • Mary Eisenhower239,
  • Jian Han239,
  • Natalie Collins240Email author,
  • Robert Manguso240,
  • Hans Pope240,
  • Yashaswi Shrestha241,
  • Jesse Boehm241,
  • W. Nicholas Haining240,
  • Kyle R. Cron242Email author,
  • Ayelet Sivan242,
  • Keston Aquino-Michaels242,
  • Thomas F. Gajewski243,
  • Marco Orecchioni244,
  • Davide Bedognetti245,
  • Wouter Hendrickx245,
  • Claudia Fuoco126,
  • Filomena Spada246,
  • Francesco Sgarrella244,
  • Gianni Cesareni246,
  • Francesco Marincola247,
  • Kostas Kostarelos248,
  • Alberto Bianco249,
  • Lucia Delogu244Email author,
  • Wouter Hendrickx250Email author,
  • Jessica Roelands250,
  • Sabri Boughorbel250,
  • Julie Decock251,
  • Scott Presnell252,
  • Ena Wang254,
  • Franco M. Marincola250,
  • Peter Kuppen253,
  • Michele Ceccarelli254,
  • Darawan Rinchai250,
  • Damien Chaussabel250,
  • Lance Miller255,
  • Davide Bedognetti250,
  • Andrew Nguyen256Email author,
  • J. Zachary Sanborn257,
  • Charles Vaske257,
  • Shahrooz Rabizadeh257,
  • Kayvan Niazi257,
  • Steven Benz257,
  • Shashank Patel258Email author,
  • Nicholas Restifo258,
  • James White259,
  • Sam Angiuoli259,
  • Mark Sausen259,
  • Sian Jones259,
  • Maria Sevdali259Email author,
  • John Simmons259,
  • Victor Velculescu259,
  • Luis Diaz259,
  • Theresa Zhang259,
  • Jennifer S. Sims260Email author,
  • Sunjay M. Barton260,
  • Robyn Gartrell260,
  • Angela Kadenhe-Chiweshe260,
  • Filemon Dela Cruz260,
  • Andrew T. Turk260,
  • Yan Lu260,
  • Christopher F. Mazzeo261,
  • Andrew L. Kung260,
  • Jeffrey N. Bruce260,
  • Yvonne M. Saenger262,
  • Darrell J. Yamashiro260,
  • Eileen P. Connolly260,
  • Jason Baird263Email author,
  • Marka Crittenden264,
  • David Friedman263,
  • Hong Xiao265,
  • Rom Leidner263,
  • Bryan Bell263,
  • Kristina Young264,
  • Michael Gough264,
  • Zhen Bian266Email author,
  • Koby Kidder266,
  • Yuan Liu266,
  • Emily Curran267Email author,
  • Xiufen Chen268,
  • Leticia P. Corrales267,
  • Justin Kline269,
  • Cordelia Dunai270Email author,
  • Ethan G. Aguilar270,
  • Lam T. Khuat270,
  • William J. Murphy270,
  • Jennifer Guerriero271Email author,
  • Alaba Sotayo271,
  • Holly Ponichtera272,
  • Alexandra Pourzia271,
  • Sara Schad271,
  • Ruben Carrasco271,
  • Suzan Lazo271,
  • Roderick Bronson273,
  • Anthony Letai271,
  • Richard S. Kornbluth274Email author,
  • Sachin Gupta275,
  • James Termini275,
  • Elizabeth Guirado275,
  • Geoffrey W. Stone275,
  • Christina Meyer276Email author,
  • Laura Helming277,
  • Joseph Tumang278,
  • Nicholas Wilson279,
  • Robert Hofmeister280,
  • Laszlo Radvanyi276,
  • Natalie J. Neubert281,
  • Laure Tillé281,
  • David Barras282,
  • Charlotte Soneson282,
  • Petra Baumgaertner281,
  • Donata Rimoldi281,
  • David Gfeller281,
  • Mauro Delorenzi282,
  • Silvia A. Fuertes Marraco281,
  • Daniel E. Speiser281Email author,
  • Tara S. Abraham283Email author,
  • Bo Xiang283,
  • Michael S. Magee283,
  • Scott A. Waldman283,
  • Adam E. Snook283,
  • Wojciech Blogowski284Email author,
  • Ewa Zuba-Surma285,
  • Marta Budkowska286,
  • Daria Salata286,
  • Barbara Dolegowska287,
  • Teresa Starzynska288,
  • Leo Chan289Email author,
  • Srinivas Somanchi290,
  • Kelsey McCulley289,
  • Dean Lee291,
  • Nico Buettner292,
  • Feng Shi293,
  • Paisley T. Myers294,
  • Stuart Curbishley295,
  • Sarah A. Penny295,
  • Lora Steadman295,
  • David Millar293,
  • Ellen Speers294,
  • Nicola Ruth295,
  • Gabriel Wong295,
  • Robert Thimme292,
  • David Adams295,
  • Mark Cobbold293Email author,
  • Remy Thomas296,
  • Wouter Hendrickx297,
  • Mariam Al-Muftah296,
  • Julie Decock296Email author,
  • Michael KK Wong298,
  • Michael Morse299,
  • David F. McDermott300,
  • Joseph I. Clark301,
  • Howard L. Kaufman302,
  • Gregory A. Daniels303,
  • Hong Hua304Email author,
  • Tharak Rao304,
  • Janice P. Dutcher305,
  • Kai Kang306Email author,
  • Yogen Saunthararajah307,
  • Vamsidhar Velcheti307,
  • Vikas Kumar308Email author,
  • Firoz Anwar309,
  • Amita Verma308,
  • Zinal Chheda310,
  • Gary Kohanbash310,
  • John Sidney311,
  • Kaori Okada310,
  • Shruti Shrivastav310,
  • Diego A. Carrera310,
  • Shuming Liu310,
  • Naznin Jahan310,
  • Sabine Mueller310,
  • Ian F. Pollack312,
  • Angel M. Carcaboso313,
  • Alessandro Sette311,
  • Yafei Hou310,
  • Hideho Okada310,
  • Jessica J. Field314,
  • Weiping Zeng314,
  • Vincent FS Shih314,
  • Che-Leung Law314,
  • Peter D. Senter314,
  • Shyra J. Gardai314,
  • Nicole M. Okeley314Email author,
  • Sarah A. Penny315Email author,
  • Jennifer G. Abelin316,
  • Abu Z. Saeed315,
  • Stacy A. Malaker317,
  • Paisley T. Myers316,
  • Jeffrey Shabanowitz316,
  • Stephen T. Ward315,
  • Donald F. Hunt316,
  • Mark Cobbold318,
  • Pam Profusek319Email author,
  • Laura Wood319,
  • Dale Shepard319,
  • Petros Grivas319,
  • Kerstin Kapp320,
  • Barbara Volz320,
  • Detlef Oswald320,
  • Burghardt Wittig321,
  • Manuel Schmidt320Email author,
  • Julian P. Sefrin322Email author,
  • Lars Hillringhaus322,
  • Valeria Lifke322,
  • Alexander Lifke322,
  • Anna Skaletskaya323,
  • Jose Ponte323,
  • Thomas Chittenden323,
  • Yulius Setiady323Email author,
  • Sandrine Valsesia-Wittmann324Email author,
  • Eva Sivado324,
  • Vincent Thomas325,
  • Meddy El Alaoui324,
  • Sébastien Papot326,
  • Charles Dumontet327,
  • Mike Dyson328,
  • John McCafferty328,
  • Said El Alaoui325,
  • Amita Verma329Email author,
  • Vikas Kumar329,
  • Praveen K. Bommareddy330Email author,
  • Howard L. Kaufman331,
  • Andrew Zloza331,
  • Frederick Kohlhapp330,
  • Ann W. Silk330,
  • Sachin Jhawar330,
  • Tomas Paneque332,
  • Praveen K. Bommareddy333Email author,
  • Frederick Kohlhapp333,
  • Jenna Newman333,
  • Pedro Beltran333,
  • Andrew Zloza334,
  • Howard L. Kaufman334,
  • Felicia Cao335Email author,
  • Bang-Xing Hong335,
  • Tania Rodriguez-Cruz335,
  • Xiao-Tong Song335,
  • Stephen Gottschalk335,
  • Hugo Calderon336,
  • Sam Illingworth336,
  • Alice Brown336,
  • Kerry Fisher336,
  • Len Seymour337,
  • Brian Champion336Email author,
  • Emma Eriksson338Email author,
  • Jessica Wenthe338,
  • Ann-Charlotte Hellström338,
  • Gabriella Paul-Wetterberg338,
  • Angelica Loskog339,
  • Emma Eriksson340Email author,
  • Ioanna Milenova341,
  • Jessica Wenthe340,
  • Magnus Ståhle340,
  • Justyna Jarblad-Leja342,
  • Gustav Ullenhag343,
  • Anna Dimberg340,
  • Rafael Moreno344,
  • Ramon Alemany344,
  • Angelica Loskog345,
  • Emma Eriksson346Email author,
  • Ioanna Milenova347,
  • Rafael Moreno348,
  • Ramon Alemany348,
  • Angelica Loskog349,
  • Sachin Jhawar350Email author,
  • Sharad Goyal351,
  • Praveen K. Bommareddy350,
  • Tomas Paneque352,
  • Howard L. Kaufman351,
  • Andrew Zloza351,
  • Howard L. Kaufman353Email author,
  • Ann Silk353,
  • Janice Mehnert353,
  • Nashat Gabrail354,
  • Jennifer Bryan353,
  • Daniel Medina353,
  • Praveen K. Bommareddy353,
  • Darren Shafren355,
  • Mark Grose355,
  • Andrew Zloza353,
  • Leah Mitchell356Email author,
  • Kader Yagiz356,
  • Fernando Lopez356,
  • Daniel Mendoza356,
  • Anthony Munday356,
  • Harry Gruber356,
  • Douglas Jolly356,
  • Steven Fuhrmann357,
  • Sasa Radoja357,
  • Wei Tan357,
  • Aldo Pourchet358,
  • Alan Frey358,
  • Ian Mohr358,
  • Matthew Mulvey357Email author,
  • Tuuli Ranki359Email author,
  • Sari Pesonen359,
  • Cristian Capasso360,
  • Erkko Ylösmäki360,
  • Vincenzo Cerullo360,
  • Robert H. I. Andtbacka361,
  • Merrick Ross362,
  • Sanjiv Agarwala363,
  • Kenneth Grossmann361,
  • Matthew Taylor364,
  • John Vetto365,
  • Rogerio Neves366,
  • Adil Daud367,
  • Hung Khong361,
  • Stephanie M. Meek368,
  • Richard Ungerleider369,
  • Scott Welden369Email author,
  • Maki Tanaka370,
  • Matthew Williams371,
  • Robert H. I. Andtbacka372,
  • Brendan Curti373,
  • Sigrun Hallmeyer374,
  • Bernard Fox4,
  • Zipei Feng373,
  • Christopher Paustian373,
  • Carlo Bifulco375,
  • Mark Grose376,
  • Darren Shafren376Email author,
  • Sadia Zafar377Email author,
  • Suvi Parviainen377,
  • Mikko Siurala377,
  • Otto Hemminki377,
  • Riikka Havunen377,
  • Siri Tähtinen377,
  • Simona Bramante377,
  • Lotta Vassilev377,
  • Hongjie Wang378,
  • Andre Lieber378,
  • Silvio Hemmi379,
  • Tanja de Gruijl380,
  • Anna Kanerva377,
  • Akseli Hemminki377,
  • Tameem Ansari381Email author,
  • Srividya Sundararaman381,
  • Diana Roen381,
  • Paul Lehmann381,
  • Anja C. Bloom382Email author,
  • Lewis H. Bender383,
  • Ian B. Walters383,
  • Masaki Terabe382,
  • Jay A. Berzofsky382,
  • Fanny Chapelin384Email author,
  • Hideho Okada385,
  • Eric T. Ahrens384,
  • Jeff DeFalco386,
  • Michael Harbell386,
  • Amy Manning-Bog386,
  • Alexander Scholz386,
  • Danhui Zhang386,
  • Gilson Baia386,
  • Yann Chong Tan386,
  • Jeremy Sokolove387,
  • Dongkyoon Kim386,
  • Kevin Williamson386,
  • Xiaomu Chen386,
  • Jillian Colrain386,
  • Gregg Espiritu Santo386,
  • Ngan Nguyen386,
  • Wayne Volkmuth386,
  • Norman Greenberg386Email author,
  • William Robinson2,
  • Daniel Emerling386,
  • Charles G. Drake388Email author,
  • Daniel P. Petrylak389,
  • Emmanuel S. Antonarakis388,
  • Adam S. Kibel390,
  • Nancy N. Chang391,
  • Tuyen Vu391,
  • Dwayne Campogan391,
  • Heather Haynes391,
  • James B. Trager391,
  • Nadeem A. Sheikh391,
  • David I. Quinn392,
  • Peter Kirk394Email author,
  • Murali Addepalli394,
  • Thomas Chang394,
  • Ping Zhang394,
  • Marina Konakova394,
  • Katsunobu Hagihara395,
  • Steven Pai396,
  • Laurie VanderVeen394,
  • Palakshi Obalapur394,
  • Peiwen Kuo394,
  • Phi Quach394,
  • Lawrence Fong396,
  • Deborah H. Charych394,
  • Jonathan Zalevsky394,
  • John L. Langowski397Email author,
  • Murali Addepalli397,
  • Yolanda Kirksey397,
  • Ravi Nutakki397,
  • Shalini Kolarkar397,
  • Rhoneil Pena397,
  • Ute Hoch397,
  • Jonathan Zalevsky397,
  • Stephen K. Doberstein397,
  • Deborah H. Charych397,
  • John Cha398,
  • Zach Mallon398,
  • Myra Perez398,
  • Amanda McDaniel398,
  • Snjezana Anand398,
  • Darrin Uecker398,
  • Richard Nuccitelli398Email author,
  • Amanda McDaniel399,
  • Snjezana Anand399,
  • John Cha399,
  • Darrin Uecker399,
  • Richard Nuccitelli399Email author,
  • Nataša Obermajer400Email author,
  • Julie Urban401,
  • Eva Wieckowski401,
  • Ravikumar Muthuswamy401,
  • Roshni Ravindranathan401,
  • David Bartlett400,
  • Pawel Kalinski402, 403,
  • Ariana N. Renrick404Email author,
  • Menaka Thounaojam405,
  • Portia Thomas404,
  • Samuel Pellom404,
  • Anil Shanker406,
  • Samuel Pellom407,
  • Menaka Thounaojam408,
  • Duafalia Dudimah409,
  • Alan Brooks410,
  • Thomas J. Sayers411,
  • Anil Shanker409Email author,
  • Yu-Lin Su412Email author,
  • Tomasz Adamus413,
  • Qifang Zhang413,
  • Sergey Nechaev413,
  • Marcin Kortylewski413,
  • Spencer Wei414Email author,
  • James Allison414,
  • Clark Anderson415Email author,
  • Chad Tang415,
  • Jonathan Schoenhals415,
  • Efrosini Tsouko415,
  • John Heymach415,
  • Patricia de Groot415,
  • Joe Chang415,
  • Kenneth R. Hess415,
  • Adi Diab415,
  • Padmanee Sharma415,
  • James Allison415,
  • Aung Naing415,
  • David Hong415,
  • James Welsh415,
  • Tina C. Albershardt416Email author,
  • Andrea J. Parsons416,
  • Jardin Leleux416,
  • Rebecca S. Reeves416,
  • Jan ter Meulen416,
  • Peter Berglund416,
  • Stephane Ascarateil417Email author,
  • Marie Eve Koziol418,
  • Sarah A. Penny419,
  • Stacy A. Malaker420,
  • Lora Steadman419,
  • Paisley T. Myers421,
  • Dina Bai421,
  • Jeffrey Shabanowitz421,
  • Donald F. Hunt421,
  • Mark Cobbold422Email author,
  • Peihong Dai423,
  • Weiyi Wang423,
  • Ning Yang423,
  • Stewart Shuman423,
  • Taha Merghoub424,
  • Jedd D. Wolchok425,
  • Liang Deng425Email author,
  • Patrick Dillon426Email author,
  • Gina Petroni426,
  • David Brenin426,
  • Kim Bullock426,
  • Walter Olson426,
  • Mark E. Smolkin427,
  • Kelly Smith426,
  • Carmel Nail426,
  • Craig L. SlingluffJr428,
  • Meenu Sharma429,
  • Faisal Fa’ak430Email author,
  • Louise Janssen429,
  • Hiep Khong429,
  • Zhilan Xiao429,
  • Yared Hailemichael430,
  • Manisha Singh429,
  • Christina Vianden429,
  • Adi Diab429,
  • Jonathan Zalevsky431,
  • Ute Hoch431,
  • Willem W. Overwijk429,
  • Andrea Facciabene432Email author,
  • Pierini Stefano432,
  • Fang Chongyung432,
  • Stavros Rafail432,
  • Yared Hailemichael433Email author,
  • Michael Nielsen433,
  • Faisal Fa’ak433,
  • Peter Vanderslice434,
  • Darren G. Woodside435,
  • Robert V. Market435,
  • Ronald J. Biediger435,
  • Upendra K. Marathi436,
  • Willem W. Overwijk433,
  • Kevin Hollevoet437Email author,
  • Nick Geukens437,
  • Paul Declerck437,
  • Nathalie Joly438,
  • Laura McIntosh438Email author,
  • Eustache Paramithiotis438,
  • Magnus Rizell439,
  • Malin Sternby439,
  • Bengt Andersson440,
  • Alex Karlsson-Parra441Email author,
  • Rui Kuai442Email author,
  • Lukasz Ochyl442,
  • Anna Schwendeman442,
  • James Moon442,
  • Weiwen Deng443,
  • Thomas E. Hudson443,
  • Edward E. Lemmens443,
  • Bill Hanson443,
  • Chris S. Rae443,
  • Joel Burrill443,
  • Justin Skoble443,
  • George Katibah443,
  • Aimee L. Murphy443,
  • Michele deVries443,
  • Dirk G. Brockstedt443,
  • Meredith L. Leong443Email author,
  • Peter Lauer443,
  • Thomas W. Dubensky443,
  • Chan C. Whiting443,
  • Xin Chen444,
  • Yangyang Hu445,
  • Yang Xia445,
  • Li Zhou444,
  • Yangyi Bao446,
  • Shiang Huang447,
  • Xiubao Ren448,
  • Elaine Hurt449,
  • Robert E. Hollingsworth449,
  • Alfred E. Chang445,
  • Max S. Wicha450,
  • Qiao Li451Email author,
  • Charu Aggarwal452,
  • Drishty Mangrolia453Email author,
  • Roger Cohen452,
  • Gregory Weinstein454,
  • Matthew Morrow453,
  • Joshua Bauml452,
  • Kim Kraynyak453,
  • Jean Boyer455,
  • Jian Yan453,
  • Jessica Lee453,
  • Laurent Humeau445,
  • Sandra Oyola453,
  • Susan Duff453,
  • David Weiner456,
  • Zane Yang453,
  • Mark Bagarazzi453,
  • Douglas G. McNeel457Email author,
  • Jens Eickhoff458,
  • Robert Jeraj458,
  • Mary Jane Staab457,
  • Jane Straus457,
  • Brian Rekoske458,
  • Glenn Liu457,
  • Marit Melssen459Email author,
  • Gina Petroni459,
  • William Grosh459,
  • Nikole Varhegyi459,
  • Kim Bullock459,
  • Mark E. Smolkin459,
  • Kelly Smith459,
  • Nadejda Galeassi459,
  • Donna H. Deacon460,
  • Elizabeth Gaughan459,
  • Craig L. SlingluffJr461,
  • Maurizio Ghisoli462,
  • Minal Barve462,
  • Robert Mennel463,
  • Gladice Wallraven464,
  • Luisa Manning465,
  • Neil Senzer466,
  • John Nemunaitis466Email author,
  • Masahiro Ogasawara467Email author,
  • Shuichi Ota467,
  • Kaitlin M. Peace468Email author,
  • Diane F. Hale468,
  • Timothy J. Vreeland469,
  • Doreen O. Jackson468,
  • John S. Berry470,
  • Alfred F. Trappey468,
  • Garth S. Herbert468,
  • Guy T. Clifton468,
  • Mark O. Hardin471,
  • Anne Toms472,
  • Na Qiao472,
  • Jennifer Litton472,
  • George E. Peoples473,
  • Elizabeth A. Mittendorf472,
  • Lila Ghamsari474,
  • Emilio Flano474,
  • Judy Jacques474,
  • Biao Liu474,
  • Jonathan Havel475,
  • Vladimir Makarov475,
  • Taha Merghoub476,
  • Jedd D. Wolchok477,
  • Matthew D. Hellmann477,
  • Timothy A. Chan475,
  • Jessica B. Flechtner474Email author,
  • Pierini Stefano478Email author,
  • Andrea Facciabene478,
  • John Facciponte478,
  • Stefano Ugel478,
  • Francesco De Sanctis478,
  • George Coukos478,
  • Sébastien Paris479,
  • Agnes Pottier479Email author,
  • Laurent Levy479,
  • Bo Lu480,
  • Federica Cappuccini481,
  • Emily Pollock481,
  • Richard Bryant482,
  • Freddie Hamdy482,
  • Adrian Hill481,
  • Irina Redchenko481Email author,
  • Hussein Sultan483Email author,
  • Takumi Kumai483,
  • Valentyna Fesenkova483,
  • Esteban Celis483,
  • Kwong Tsang484Email author,
  • Massimo Fantini484,
  • Ingrid Fernando484,
  • Claudia Palena484,
  • Justin M. David484,
  • James Hodge484,
  • Elizabeth Gabitzsch485,
  • Frank Jones485,
  • James L. Gulley486,
  • Jeffrey Schlom487,
  • Mireia Uribe Herranz488Email author,
  • Stavros Rafail488,
  • Stefano Ugel488,
  • John Facciponte488,
  • Pierini Stefano488,
  • Andrea Facciabene488,
  • Hiroshi Wada489Email author,
  • Atsushi Shimizu489,
  • Toshihiro Osada489,
  • Satoshi Fukaya489,
  • Eiji Sasaki489,
  • Milad Abolhalaj490Email author,
  • David Askmyr491,
  • Kristina Lundberg490,
  • Ann-Sofie Albrekt490,
  • Lennart Greiff491,
  • Malin Lindstedt490,
  • Dallas B. Flies492,
  • Tomoe Higuchi493,
  • Wojciech Ornatowski494,
  • Jaryse Harris495,
  • Sarah F. Adams494Email author,
  • Todd Aguilera496Email author,
  • Marjan Rafat496,
  • Laura Castellini496,
  • Hussein Shehade496,
  • Mihalis Kariolis496,
  • Dadi Jang496,
  • Rie vonEbyen496,
  • Edward Graves496,
  • Lesley Ellies497,
  • Erinn Rankin496,
  • Albert Koong496,
  • Amato Giaccia496,
  • Reham Ajina498Email author,
  • Shangzi Wang498,
  • Jill Smith498,
  • Mariaelena Pierobon498,
  • Sandra Jablonski498,
  • Emanuel PetricoinIII499,
  • Louis M. Weiner498,
  • Lorcan Sherry500Email author,
  • John Waller500,
  • Mark Anderson500,
  • Alison Bigley500,
  • Chantale Bernatchez501,
  • Cara Haymaker501,
  • Nizar M. Tannir501,
  • Harriet Kluger502,
  • Michael Tetzlaff501,
  • Natalie Jackson501,
  • Ivan Gergel503,
  • Mary Tagliaferri503,
  • Jonathan Zalevsky503,
  • Ute Hoch503Email author,
  • Patrick Hwu501,
  • Mario Snzol502,
  • Michael Hurwitz502,
  • Adi Diab501,
  • Theresa Barberi504Email author,
  • Allison Martin504,
  • Rahul Suresh504,
  • David Barakat504,
  • Sarah Harris-Bookman504,
  • Charles Drake504,
  • Alan Friedman504,
  • Sara Berkey505Email author,
  • Stephanie Downs-Canner505,
  • Greg M. Delgoffe506,
  • Robert P. Edwards506,
  • Tyler Curiel507,
  • Kunle Odunsi508,
  • David Bartlett505,
  • Nataša Obermajer505,
  • Tullia C. Bruno509Email author,
  • Brandon Moore510,
  • Olivia Squalls510,
  • Peggy Ebner510,
  • Katherine Waugh510,
  • John Mitchell511,
  • Wilbur Franklin512,
  • Daniel Merrick512,
  • Martin McCarter513,
  • Brent Palmer514,
  • Jeffrey Kern515,
  • Dario Vignali516,
  • Jill Slansky510,
  • Anissa S. H. Chan517Email author,
  • Xiaohong Qiu517,
  • Kathryn Fraser517,
  • Adria Jonas517,
  • Nadine Ottoson517,
  • Keith Gordon517,
  • Takashi O. Kangas517,
  • Steven Leonardo517,
  • Kathleen Ertelt517,
  • Richard Walsh517,
  • Mark Uhlik517,
  • Jeremy Graff517,
  • Nandita Bose517,
  • Ravi Gupta518,
  • Nitin Mandloi518,
  • Kiran Paul518,
  • Ashwini Patil518,
  • Rekha Sathian518,
  • Aparna Mohan518,
  • Malini Manoharan518,
  • Amitabha Chaudhuri518Email author,
  • Yu Chen519Email author,
  • Jing Lin519,
  • Yun-bin Ye519,
  • Chun-wei Xu519,
  • Gang Chen519,
  • Zeng-qing Guo519,
  • Andrey Komarov520Email author,
  • Alex Chenchik520,
  • Michael Makhanov520,
  • Costa Frangou520,
  • Yi Zheng521Email author,
  • Carla Coltharp521,
  • Darryn Unfricht521,
  • Ryan Dilworth521,
  • Leticia Fridman521,
  • Linying Liu521,
  • Milind Rajopadhye521,
  • Peter Miller521,
  • Fernando Concha-Benavente522Email author,
  • Julie Bauman522,
  • Sumita Trivedi522,
  • Raghvendra Srivastava522,
  • James Ohr522,
  • Dwight Heron522,
  • Uma Duvvuri522,
  • Seungwon Kim522,
  • William Gooding522,
  • Robert L. Ferris522,
  • Heather Torrey523,
  • Toshi Mera523,
  • Yoshiaki Okubo523,
  • Eva Vanamee523,
  • Rosemary Foster523,
  • Denise Faustman523Email author,
  • Robyn Gartrell524Email author,
  • Edward Stack525,
  • Yan Lu524,
  • Daisuke Izaki526,
  • Kristen Beck527,
  • Dan Tong Jia527,
  • Paul Armenta527,
  • Ashley White-Stern527,
  • Yichun Fu527,
  • Zoe Blake524,
  • Douglas Marks524,
  • Howard L. Kaufman528,
  • Bret Taback524,
  • Basil Horst524,
  • Yvonne M. Saenger529,
  • Laura Hix Glickman530Email author,
  • David B. Kanne530,
  • Kelsey S. Gauthier530,
  • Anthony L. Desbien530,
  • Brian Francica531,
  • George Katibah530,
  • Leticia P. Corrales532,
  • Justin L. Leong530,
  • Leonard Sung530,
  • Ken Metchette530,
  • Shailaja Kasibhatla533,
  • Anne Marie Pferdekamper533,
  • Lianxing Zheng534,
  • Charles Cho533,
  • Yan Feng534,
  • Jeffery M. McKenna534,
  • John Tallarico534,
  • Steven Bender533,
  • Chudi Ndubaku530,
  • Sarah M. McWhirter530,
  • Charles G. Drake535,
  • Thomas F. Gajewski536,
  • Thomas W. Dubensky530,
  • Elena Gonzalez Gugel537Email author,
  • Charles J. M. Bell538,
  • Adiel Munk537,
  • Luciana Muniz537,
  • Nina Bhardwaj537,
  • Fei Zhao539,
  • Kathy Evans539,
  • Christine Xiao539,
  • Alisha Holtzhausen540,
  • Brent A. Hanks539Email author,
  • Nathalie Scholler541Email author,
  • Catherine Yin541,
  • Pien Van der Meijs542,
  • Andrew M. Prantner543,
  • Cecile M. Krejsa544,
  • Leia Smith544,
  • Brian Johnson545,
  • Daniel Branstetter546,
  • Paul L. Stein541,
  • Juan C. Jaen547Email author,
  • Joanne BL Tan547,
  • Ada Chen547,
  • Yu Chen547,
  • Timothy Park547,
  • Jay P. Powers547,
  • Holly Sexton547,
  • Guifen Xu547,
  • Steve W. Young547,
  • Ulrike Schindler547,
  • Wentao Deng548,
  • David John Klinke548Email author,
  • Hannah M. Komar549Email author,
  • Thomas Mace549,
  • Gregory Serpa549,
  • Omar Elnaggar549,
  • Darwin Conwell549,
  • Philip Hart550,
  • Carl Schmidt550,
  • Mary Dillhoff550,
  • Ming Jin550,
  • Michael C. Ostrowski549,
  • Gregory B. Lesinski549,
  • Madhuri Koti551Email author,
  • Katrina Au551,
  • Nichole Peterson551,
  • Peter Truesdell551,
  • Gillian Reid-Schachter551,
  • Charles Graham551,
  • Andrew Craig551,
  • Julie-Ann Francis551,
  • Beatrix Kotlan552Email author,
  • Timea Balatoni552,
  • Emil Farkas552,
  • Laszlo Toth552,
  • Mihaly Ujhelyi552,
  • Akos Savolt552,
  • Zoltan Doleschall552,
  • Szabolcs Horvath552,
  • Klara Eles552,
  • Judit Olasz552,
  • Orsolya Csuka552,
  • Miklos Kasler552,
  • Gabriella Liszkay552,
  • Eytan Barnea553,
  • Sushil Kumar554,
  • Takahiro Tsujikawa554,
  • Collin Blakely555,
  • Patrick Flynn554,
  • Reid Goodman554,
  • Raphael Bueno556,
  • David Sugarbaker557,
  • David Jablons558,
  • V. Courtney Broaddus555,
  • Brian West559,
  • Lisa M. Coussens554Email author,
  • Paul R. Kunk560Email author,
  • Joseph M. Obeid560,
  • Kevin Winters560,
  • Patcharin Pramoonjago560,
  • Mark E. Smolkin561,
  • Edward B. Stelow560,
  • Todd W. Bauer560,
  • Craig L. SlingluffJr562,
  • Osama E. Rahma563,
  • Adam Lamble564,
  • Yoko Kosaka564,
  • Fei Huang565,
  • Kate A. Saser565,
  • Homer Adams565,
  • Christina E. Tognon564,
  • Ted Laderas564,
  • Shannon McWeeney564,
  • Marc Loriaux564,
  • Jeffery W. Tyner564,
  • Brian J. Druker566,
  • Evan F. Lind564Email author,
  • Zhuqing Liu567Email author,
  • Shanhong Lu567,
  • Lawrence P. Kane568,
  • Robert L. Ferris569,
  • Zhuqing Liu570Email author,
  • Gulidanna Shayan570,
  • Shanhong Lu570,
  • Robert L. Ferris571,
  • Julia Femel572,
  • Takahiro Tsujikawa572,
  • Ryan Lane572,
  • Jamie Booth572,
  • Amanda W. Lund572Email author,
  • Marit Melssen573Email author,
  • Anthony Rodriguez573,
  • Craig L. SlingluffJr574,
  • Victor H. Engelhard573,
  • Alessandra Metelli575Email author,
  • Bill X. Wu575,
  • Caroline W. Fugle575,
  • Rachidi Saleh575,
  • Shaoli Sun575,
  • Jennifer Wu575,
  • Bei Liu575,
  • Zihai Li575,
  • Zachary S. Morris576Email author,
  • Emily I. Guy576,
  • Clinton Heinze576,
  • Jasdeep Kler576,
  • Monica M. Gressett576,
  • Lauryn R. Werner576,
  • Stephen D. Gillies577,
  • Alan J. Korman578,
  • Hans Loibner579,
  • Jacquelyn A. Hank576,
  • Alexander L. Rakhmilevich576,
  • Paul M. Harari576,
  • Paul M. Sondel576,
  • Jenna Newman580Email author,
  • Andrew Zloza581,
  • Erica Huelsmann582,
  • Joseph Broucek582,
  • Howard L. Kaufman581,
  • Dorothee Brech583,
  • Tobias Straub584,
  • Martin Irmler585,
  • Johannes Beckers585,
  • Florian Buettner586,
  • Elke Schaeffeler586,
  • Matthias Schwab587, 588,
  • Elfriede Noessner589Email author,
  • Snjezana Anand590,
  • Amanda McDaniel590,
  • John Cha590,
  • Darrin Uecker590,
  • Richard Nuccitelli590Email author,
  • Peter Ordentlich591Email author,
  • Alison Wolfreys592,
  • Andre Da Costa593,
  • John Silva592,
  • Andrea Crosby592,
  • Ludovicus Staelens593,
  • Graham Craggs592,
  • Annick Cauvin593,
  • Sean Mason592,
  • Alison M. Paterson594,
  • Andrew C. Lake594,
  • Caroline M. Armet594,
  • Rachel W. O’Connor594,
  • Jonathan A. Hill594,
  • Emmanuel Normant594,
  • Ammar Adam594,
  • Detlev M. Biniszkiewicz594,
  • Scott C. Chappel594,
  • Vito J. Palombella594,
  • Pamela M. Holland594,
  • Jay P. Powers595Email author,
  • Annette Becker595,
  • Ada Chen595,
  • Manmohan R. Leleti595,
  • Eric Newcomb595,
  • Holly Sexton595,
  • Ulrike Schindler595,
  • Joanne B. L. Tan595,
  • Steve W. Young595,
  • Juan C. Jaen595,
  • Suthee Rapisuwon596Email author,
  • Arash Radfar597,
  • Kellie Gardner598,
  • Geoffrey Gibney598,
  • Michael Atkins598,
  • Keith R. Rennier599,
  • Robert Crowder599,
  • Ping Wang599,
  • Russell K. Pachynski599,
  • Rosa M. Santana Carrero600Email author,
  • Sarai Rivas601,
  • Figen Beceren-Braun601,
  • Scott Anthony602,
  • Kimberly S. Schluns601,
  • Deepali Sawant603Email author,
  • Maria Chikina603,
  • Hiroshi Yano603,
  • Creg Workman603,
  • Dario Vignali603,
  • Elise Salerno604,
  • Davide Bedognetti605,
  • Ileana Mauldin606,
  • Donna Deacon606,
  • Sofia Shea607,
  • Joel Pinczewski608,
  • Joseph M. Obeid609,
  • George Coukos610,
  • Ena Wang605,
  • Thomas Gajewski611,
  • Franco M. Marincola605,
  • Craig L. SlingluffJr606Email author,
  • Stefani Spranger612Email author,
  • Brendan Horton612,
  • Thomas F. Gajewski613,
  • Akiko Suzuki614Email author,
  • Pamela Leland614,
  • Bharat H. Joshi614,
  • Raj K. Puri614,
  • Randy F. Sweis615Email author,
  • Riyue Bao615,
  • Jason Luke615,
  • Thomas F. Gajewski613,
  • Marie-Nicole Theodoraki616Email author,
  • Frances-Mary Mogundo617,
  • Robert P. Edwards617,
  • Pawel Kalinski618, 619,
  • Haejung Won620Email author,
  • Dayson Moreira620,
  • Chan Gao620,
  • Xingli Zhao620,
  • Priyanka Duttagupta620,
  • Jeremy Jones620,
  • Massimo D’Apuzzo620,
  • Sumanta Pal620 and
  • Marcin Kortylewski620
Journal for ImmunoTherapy of Cancer20164(Suppl 1):73

https://doi.org/10.1186/s40425-016-0173-6

Published: 16 November 2016

Combinations: Immunotherapy/Immunotherapy

P189 Rational combinations of intratumoral T cell and myeloid agonists mobilize abscopal responses in prostate cancer

Casey Ager1, Matthew Reilley2, Courtney Nicholas1, Todd Bartkowiak1, Ashvin Jaiswal1, Michael Curran1

1Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Department of Cancer Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA
Correspondence: Casey Ager (crager@mdanderson.org)

Background

Despite the success of checkpoint blockade immunotherapy in characteristically immunogenic cancers such as melanoma, these antibodies remain ineffective against poorly T cell-infiltrated malignancies including prostate cancer. Sensitizing these “cold” tumors to immunotherapy will require interventions which enhance tumor antigen presentation and T cell priming, while suppressing microenvironmental signals which constrain T cell expansion, survival, and effector function independent of coinhibitory signaling. We investigated whether intratumoral administration of either the STING agonist c-di-GMP (CDG) or dendritic cell (DC) growth factor Flt3-ligand can potentiate the therapeutic effects of T cell checkpoint modulation with αCTLA-4, αPD-1, and α4-1BB in a bilateral subcutaneous model of prostate adenocarcinoma. Additionally, we tested whether intratumoral delivery of low-dose checkpoint modulators with CDG at a single lesion can achieve abscopal control of distal lesions.

Methods

Male C57BL/6 mice were challenged subcutaneously on both flanks with TRAMP-C2 prostate adenocarcinoma, and treatment was administered intraperitoneally and/or intratumorally for 3 doses every 4 days, beginning on day 14 post-implantation for survival experiments or day 31 for flow analysis experiments.

Results

Intratumoral delivery of STING agonist CDG alone potently rejects all injected TRAMP-C2 tumors, but fails to generate systemic control of uninjected lesions. Systemic administration of αCTLA-4, αPD-1, and α4-1BB cures 40 % of mice with bilateral TRAMP-C2, and concurrent administration of CDG at one or both flanks enhances survival to 75 %. Similar effects are observed with intratumoral Flt3L, although administration at both flanks is required for full effect. Intratumoral low-dose αCTLA-4, αPD-1, and α4-1BB at a single flank induces abscopal effects in 20 % of mice, and concurrent administration of CDG enhances systemic immunity to cure up to 50 % of mice. We observe that the level of STING activation required to mediate rejection without inducing ulcerative toxicity is proportional to initial tumor size. Functionally, local STING activation complements intratumoral checkpoint modulation to reduce local MDSC infiltration, enhance CD8:Treg ratios, and downregulate the M2 macrophage marker CD206. In contrast, local Flt3L robustly enhances immune infiltration of injected and distal tumors, but therapeutic benefit is attenuated due to concomitant induction of FoxP3+ Treg.

Conclusions

Intratumoral STING activation via CDG or DC expansion with Flt3L potentiates the therapeutic effects of systemically-delivered αCTLA-4, αPD-1, and α4-1BB against multi-focal TRAMP-C2 prostate cancer. The abscopal potential of CDG alone is weak, in contrast to prior observations, but combining CDG with low-dose checkpoint blockade intratumorally can induce systemic immunity, suggesting an alternative approach for clinical implementation of combination immunotherapies at reduced doses.

P190 Multi-genome reassortant dendritic cell-tropic vector platform (ZVex®-Multi) allows flexible co-expression of multiple antigens and immune modulators for optimal induction of anti-tumor CD8+ T cell responses

Tina C Albershardt, Anshika Bajaj, Jacob F Archer, Rebecca S Reeves, Lisa Y Ngo, Peter Berglund, Jan ter Meulen

Immune Design, Seattle, WA, USA
Correspondence: Tina C Albershardt (tina.albershardt@immunedesign.com)

Background

Induction of immune responses against multiple antigens expressed from conventional vector platforms is often ineffective for reasons not well understood. Common methods of expressing multiple antigens within a single vector construct include the use of fusion proteins, endoprotease cleavage sites, or internal ribosome entry sites. These methods often lead to decreased expression of antigens-of-interest and/or reduced induction of T cell responses against the encoded antigens. Circumventing these limitations, we have developed a novel production process for our integration-deficient, dendritic cell-targeting lentiviral vector platform, ZVex, enabling highly flexible and effective multigene delivery in vivo, making it possibly the most versatile vector platform in the industry.

Methods

Up to five vector genome plasmids, each encoding one full-length antigen or immuno-modulator, were mixed with four constant plasmids, each encoding vector particle proteins, prior to transfection of production cells. Due to the propensity of lentiviruses forming genomic reassortants, the resulting vector preparations hypothetically contain a mix of homozygous and heterozygous vector particles. qRT-PCR was used to determine total and antigen-specific titers of ZVex-Multi vectors, defined as vector genome counts. Mice were immunized with ZVex-Multi vectors or monozygous vectors expressing multiple antigens from the same backbone to compare immunogenicity via intracellular cytokine staining. Two tumor models were used to evaluate therapeutic efficacy: 1) a B16 melanoma model, where tumor cells were inoculated in the flank and measured 2–3 times per week; and 2) a metastatic CT26 colon carcinoma model, where tumor cells were inoculated intravenously, and lung nodules were enumerated 17–19 days post-tumor inoculation.

Results

Titrations by qRT-PCR of multiple ZVex-Multi vector lots demonstrated that production yields of ZVex-Multi expressing up to four different tumor-associated antigens (e.g., NY-ESO-1, MAGE-A3) and two immuno-modulators (e.g., IL-12, anti-CTLA-4 or anti-PD-L1) were highly reproducible. Compared to mice immunized with vectors expressing multiple antigens from the same backbone, mice immunized with ZVex-Multi vectors consistently developed T cells against all targeted TAAs and exhibited improved tumor growth control and survival.

Conclusions

ZVex-Multi is a next generation DC-tropic vector platform designed to overcome limitations of single-genome vector platforms with respect to efficient co-expression of any combination of desired genes. Unlike other vector platforms, ZVex-Multi eliminates multiple cloning steps modifying the vector backbone, which can often result in unpredictable expression patterns of coded gene products. Its versatility and agility makes ZVex-Multi potentially the best-in-class vector platform for co-expression of multiple tumor antigens and immuno-modulators for enhanced cancer immunotherapy against a broad range of tumors.

P191 NK, T cells and IFN-gamma are required for the anti-tumor efficacy of combination-treatment with NKG2A and PD-1/PD-L1 checkpoint inhibitors in preclinical models

Caroline Denis1, Hormas Ghadially2, Thomas Arnoux1, Fabien Chanuc1, Nicolas Fuseri1, Robert W Wilkinson2, Nicolai Wagtmann1, Yannis Morel1, Pascale Andre1

1Innate Pharma, Marseille, Provence-Alpes-Cote d'Azur, France; 2MedImmune, Cambridge, England, UK
Correspondence: Pascale Andre (pascale.andre@innate-pharma.fr)

Background

Monalizumab (IPH2201) is a first-in-class humanized IgG4 targeting NKG2A, which is expressed as heterodimer with CD94 on the surface of NK, γδT and tumor infiltrating CD8+ T cells. This inhibitory receptor binds to HLA-E in humans and to Qa-1b in mice. HLA-E is frequently up-regulated on cancer cells, protecting from killing by NKG2A+ cells. Monalizumab blocks binding of CD94-NKG2A to HLA-E, reducing inhibitory signaling thereby enhancing NK and T cell responses. PD-1/PD-L1 inhibitors are successfully being used to treat patients with a wide variety of cancers. Combined blockade of NKG2A/HLA-E and PD-1/PD-L1 may be a promising strategy to better fight cancer by activating both the adaptive and innate immune systems.

Methods

To assess the effect of combined blockade of NKG2A/HLA-E and PD-1/PD-L1 in vivo, anti-mouse NKG2A and PD-1 antibodies were used in mice engrafted with A20 mouse B lymphoma cell line. For in vitro assays, anti-PD-L1 antibody durvalumab, and monalizumab were tested in human PBMC staphylococcal enterotoxin b assays.

Results

When cultured in vitro, the A20 cells express ligands for PD-1 but not for NKG2A. Exposure to IFN-γ in vitro, or subcutaneous injection into mice, induced expression of Qa-1b, resulting in a tumor model co-expressing PD-L1 and Qa-1b. Monotherapy with PD-1 or NKG2A blockers resulted in moderate anti-tumor efficacy while treatment with combination of NKG2A and PD-1 blockers resulted in a significantly higher anti-tumor immunity, and an increased rate of complete tumor regression. Depletion of either NK, or CD8+ T cells, or IFN-γ was enough to abrogate the efficacy of PD-1 and NKG2A blockade, indicating that both of these effector populations contribute to the efficacy of the combination treatment. To further explore this possibility and to assess the potential therapeutic relevance in humans, well-validated PBMC-based assays were used which showed that blocking both axes with a combination of durvalumab and monalizumab led to increased production of cytokines by both T and NK cells. Furthermore, the magnitude of the increase in cytokine secretion was dependent on the production of high levels of IFN-γ. Since IFN-γ is known to induce HLA-E this suggests that blockade of NKG2A could have a beneficial role in activation of immune cells through the combined blockade of PD-1/PD-L1.

Conclusions

Together, these data indicate that blocking NKG2A in conjunction with PD-1/PD-L1 checkpoint inhibitors provides increased anti-tumor efficacy mediated by IFN-γ and support the rationale for assessing this combination in clinical trials.

P192 Pharmacokinetics and immunogenicity of pembrolizumab when given in combination with ipilimumab: data from KEYNOTE-029

Michael B Atkins1, Matteo S Carlino2, Antoni Ribas3, John A Thompson4, Toni K Choueiri5, F Stephen Hodi5, Wen-Jen Hwu6, David F McDermott7, Victoria Atkinson8, Jonathan S Cebon9, Bernie Fitzharris10, Michael B Jameson11, Catriona McNeil12, Andrew G Hill13, Eric Mangin14, Malidi Ahamadi14, Marianne van Vugt15, Mariëlle van Zutphen15, Nageatte Ibrahim14, Georgina V Long16

1Georgetown-Lombardi Comprehensive Cancer Center, Washington, DC, USA; 2Westmead and Blacktown Hospitals, Melanoma Institute Australia, and the University of Sydney, Westmead, New South Wales, Australia; 3University of California, Los Angeles, CA, USA; 4University of Washington, Seattle, WA, USA; 5Dana-Farber Cancer Institute/Brigham and Women’s Hospital, Harvard University, Boston, MA, USA; 6University of Texas MD Anderson Cancer Center, Houston, TX, USA; 7Beth Israel Deaconess Medical Center, Boston, MA, USA; 8Gallipoli Medical Research Foundation, Greenslopes Private Hospital, and the University of Queensland, Greenslopes, Queensland, Australia; 9Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia; 10Canterbury District Health Board, Christchurch Hospital, Christchurch, New Zealand; 11Waikato Hospital Regional Cancer Centre, Hamilton, New Zealand; 12Royal Prince Alfred Hospital, Melanoma Institute Australia, the University of Sydney, and Chris O’Brien Lifehouse, Camperdown, New South Wales, Australia; 13Tasman Oncology Research, Southport Gold Coast, Queensland, Australia; 14Merck & Co., Inc., Kenilworth, NJ, USA; 15Quantitative Solutions, a Certara company, Oss, Netherlands; 16Melanoma Institute Australia, the University of Sydney, Mater Hospital, and Royal North Shore Hospital, Wollstonecraft, New South Wales, Australia
Correspondence: Michael B Atkins (mba41@georgetown.edu)

Background

The pharmacokinetics of pembrolizumab given as monotherapy are well characterized. Consistent with other monoclonal antibodies, pembrolizumab has low clearance (0.202 L/day), limited central (3.53 L) and peripheral (3.85 L) volume of distribution, and low variability in the central volume of distribution (19 % coefficient of variation). Pembrolizumab monotherapy has low immunogenicity potential, with an observed incidence of treatment-emergent anti-drug antibodies (ADA) of < 1 %. We present data on the pharmacokinetics and immunogenicity of pembrolizumab when given in combination with ipilimumab in the phase I KEYNOTE-029 study.

Methods

KEYNOTE-029 included 2 cohorts that assessed the safety and antitumor activity of pembrolizumab plus ipilimumab: a safety run-in that included patients with advanced melanoma or renal cell carcinoma (RCC) (N = 22) and a melanoma expansion cohort (N = 153). In both cohorts, patients received 4 doses of pembrolizumab 2 mg/kg plus ipilimumab 1 mg/kg Q3W followed by pembrolizumab 2 mg/kg Q3W for up to 2 years. Pembrolizumab serum concentration was quantified with an electrochemiluminescence-based immunoassay (lower limit of quantitation, 10 ng/mL). A validated bridging electrochemiluminescence immunoassay using a standard 3-tiered approach (drug tolerance level, 124 μg/mL) was used to detect ADA in serum.

Results

Across cohorts, 175 patients received pembrolizumab plus ipilimumab: 165 with melanoma and 10 with RCC. At least 1 evaluable sample for pharmacokinetic assessment was available for all 10 patients with RCC and 162 patients with melanoma. The predose serum concentration versus time profiles for pembrolizumab were similar in patients with RCC and melanoma (Fig. 1). Observed serum concentrations were within the range predicted for pembrolizumab 2 mg/kg Q3W given as monotherapy (Fig. 2). Of the 160 patients with melanoma who provided postdose ADA samples, 156 (97.5 %) were negative, 2 (1.3 %) were inconclusive, and 2 (1.3 %) were positive for treatment-emergent ADA. Best overall response in the ADA-positive patients was stable disease in one and progressive disease in the other. No patient with RCC had treatment-emergent ADA.

Conclusions

The addition of ipilimumab does not appear to impact pembrolizumab serum concentration or increase the risk of developing ADA in patients with advanced melanoma or RCC.

Trial Registration

ClinicalTrials.gov identifier NCT02089685.
Fig. 1 (abstract P192).

Arithmetic mean (SE) predose serum concentration-time profile of pembrolizumab following multiple doses of pembrolizumab plus ipilimumab (linear-linear scale)

Fig. 2 (abstract P192).

Observed pembrolizumab serum concentrations from patients with melanoma treated with pembrolizumab plus ipilimumab in relation to the predicted concentration interval (gray) for pembrolizumab 2 mg/kg Q3W monotherapy (log scale)

P193 Establishing a model for successful immunotherapy with T-Vec combined with BRAF inhibition and anti-PD-1 in genetically engineered murine melanoma

Robyn Gartrell1, Zoe Blake1, Ines Simoes2, Yichun Fu1, Takuro Saito3, Yingzhi Qian1, Yan Lu1, Yvonne M Saenger4

1Columbia University Medical Center, New York, NY, USA; 2Institut d'Investigacions Biomediques August Pi i Sunyer, Barcelona, Catalonia, Spain; 3Icahn School of Medicine at Mount Sinai, New York, NY, USA; 4New York Presbyterian/Columbia University Medical Center, New York, NY, USA
Correspondence: Zoe Blake (zb2161@cumc.columbia.edu)

Background

Talimogene laherparepvec (T-Vec) is the first oncolytic virus to be U.S. Food and Drug Administration (FDA) approved for the treatment of cancer. T-Vec, a modified herpes simplex type I (HSV I) virus, has two proposed mechanisms of action: direct cell lysis and immune activation. Combination immunotherapy using T-Vec and checkpoint blockade has shown promise in clinical trials. In preliminary work, our laboratory has shown that T-Vec causes up-regulation of programmed cell death protein 1 (PD-1) on infiltrating T cells in mice, suggesting potential synergy of T-Vec and anti-PD-1 (αPD-1).

Methods

In a temporally and spatially regulated murine model of BRAFCA PTEN−/− spontaneous melanoma [1], tumors are induced on right flank. When tumors reach >5 mm in diameter, mice are randomized into 6 treatment groups comparing combinations of BRAF inhibition (BRAFi), αPD-1, and T–Vec (Table 1). Tumor growth is measured twice a week until end of study. Flow cytometry is performed on tumor, lymph node, and spleen to assess immune microenvironment.

Results

Mean tumor volume and survival was plotted to compare groups (Figs. 3 and 4). Mice treated with triple combination have decreased tumor growth. Mice treated with combination T-Vec + BRAFi with or without αPD-1 have longer survival compared to mice treated with control or single drug arms. Flow cytometry shows increase in percent CD3+/CD45+ cells in tumors of mice treated with combination αPD-1 + T-Vec compared to the control and single drug arms. Percent CD8+/CD3+ cells in tumors treated with immunotherapy appears to be increased compared to the control and BRAFi only group (Fig. 5). Additionally, percent of FOXP3+/CD4+ cells in tumors appears to be decreased in groups receiving T-Vec (Fig. 6) while no change in FOXP3+/CD4+ populations was observed in tumors from groups receiving αPD-1 without T-Vec or in draining lymph node or spleen.

Conclusions

Initial findings show that combination therapy of BRAFi + αPD-1 + T-Vec is more effective than any single treatment. Combination immunotherapy increases infiltration of T cells into tumor. Furthermore, oncolytic virus appears to decrease regulatory T cells infiltrating tumor. This study is ongoing and further analysis will continue as we further evaluate the immune microenvironment using flow cytometry and immunohistochemistry.

Acknowledgements

The study was funded by the Melanoma Research Alliance and Amgen (Amgen-CUMC-MRA Established Investigator Academic-Industry Partnership Award). Reference

1. Dankort, Curley, Cartlidge, et al.: Braf(V600E) cooperates with Pten loss to induce metastatic melanoma. Nature Genetics 2009, 41:544–552.
Table 1 (abstract P193).

Treatment groups

Group

Treatment

Group 1 (Red)

Control Chow + IP 2A3 + IT PBS

Group 2 (Orange)

BRAFi Chow + IP 2A3 + IT PBS

Group 3 (Yellow)

BRAFi Chow + Ip α-PD1 + IT PBS

Group 4 (Green)

BRAFi Chow + IP 2A3 + IT T-Vec

Group 5 (Blue)

BRAFi Chow + IP α-PD1 + IT T-Vec

Group 6 (Purple)

Control Chow + IP α=PD1 + IT T-Vec

IP intraperitoneal, IT intratumoral, BRAFi brief inhibiotor, α-PD1 anti programmed cell death 1, T-Vec talimogene Leherparepvec

Fig. 3 (abstract P193).

Tumor volume comparison of all mice

Fig. 4 (abstract P193).

Survival comparison of treatment groups

Fig. 5 (abstract P193).

Flow cytometry data of CD8+ cells per CD3+ cell populations

Fig. 6 (abstract P193).

Flow cytometry data of CD4+/FOXP3+ cells per CD4+ cell populations

P194 Phosphatidylserine targeting antibody in combination with checkpoint blockade and tumor radiation therapy promotes anti-cancer activity in mouse melanoma

Sadna Budhu1, Olivier De Henau1, Roberta Zappasodi1, Kyle Schlunegger2, Bruce Freimark2, Jeff Hutchins2, Christopher A Barker1, Jedd D Wolchok1, Taha Merghoub1

1Memorial Sloan Kettering Cancer Center, New York, NY, USA; 2Peregrine Pharmaceuticals, Inc., Tustin, CA, USA
Correspondence: Sadna Budhu (budhus@mskcc.org)

Background

Phosphatidylserine (PS) is a phospholipid that is exposed on the surface of apoptotic cells, some tumor cells and tumor endothelium. PS has been shown to promote anti-inflammatory and immunosuppressive signals in the tumor microenvironment. Antibodies that target PS have been shown to reactivate anti-tumor immunity by repolarizing tumor associated macrophages to a M1-like phenotype, reducing the number of MDSCs in tumors and promote the maturation of dendritic cells into functional APCs. In a B16 melanoma model, targeting PS in combination with immune checkpoint blockade has been shown to have a significantly greater anti-cancer effect than either agent alone. This combination was shown to enhance CD4+ and CD8+ T cell infiltration and activation in the tumors of treated animals. Radiation therapy is an effective focal treatment of primary solid tumors, but is less effective in treating metastatic solid tumors as a monotherapy. There is evidence that radiation induces immunogenic tumor cell death and enhances tumor-specific T cell infiltration in irradiated tumors. In addition, the abscopal effect, a phenomenon in which tumor regression occurs outside the site of radiation therapy, has been observed in both preclinical and clinical trials with the combination of radiation therapy and immunotherapy.

Methods

We examined the effects of combining tumor radiation therapy with an antibody that targets PS (1 N11) and an immune checkpoint blockade (anti-PD-1) using the mouse B16 melanoma model. Tumor surface area and overall survival of mice were used to determine efficacy of the combinations.

Results

We examined the expression of PS on immune cells infiltrating B16 melanomas. CD11b + myeloid cells expressed the highest levels of PS on their surface whereas T cells and B16 tumor cells express little to no PS. These data suggest that targeting PS in B16 melanoma would induce a pro-inflammatory myeloid tumor microenvironment. We hypothesize that therapies that induce apoptotic cell death on tumor cells would enhance the activity of PS-targeting antibodies. We therefore examined the effects of combining a PS-targeting antibody with local tumor radiation. We found that the PS-targeting antibody synergizes with both anti-PD-1 and radiation therapy to improve anti-cancer activity and overall survival. In addition, the triple combination of the PS-targeting antibody, tumor radiation and anti-PD-1 treatment displayed even greater anti-cancer and survival benefit.

Conclusions

This finding highlights the potential of combining these three agents to improve outcome in patients with advanced-stage melanoma and may inform the design of future clinical trials with PS targeting in melanoma and other cancers.

P195 A novel anti-human LAG-3 antibody in combination with anti-human PD-1 (REGN2810) shows enhanced anti-tumor activity in PD-1 x LAG-3 dual-humanized mice and favorable pharmacokinetic and safety profiles in cynomolgus monkeys

Elena Burova, Omaira Allbritton, Peter Hong, Jie Dai, Jerry Pei, Matt Liu, Joel Kantrowitz, Venus Lai, William Poueymirou, Douglas MacDonald, Ella Ioffe, Markus Mohrs, William Olson, Gavin Thurston

Regeneron, Tarrytown, NY, USA
Correspondence: Elena Burova (elena.burova@regeneron.com)

Background

In the tumor microenvironment, T cell inhibitory checkpoint receptors trigger signals that suppress T cell effector function, resulting in tumor immune evasion. Clinical antibodies blocking one of these receptors, PD-1, yield positive responses in multiple cancers; however, their efficacy is limited. Simultaneously targeting more than one inhibitory checkpoint receptor has emerged as a promising therapeutic strategy. In support of this concept, mice deficient in PD-1 and LAG-3, an inhibitory checkpoint receptor often co-expressed with PD-1 in the tumor microenvironment, exhibit enhanced anti-tumor activity. Here, we demonstrate increased anti-tumor efficacy of a combined anti–human PD-1 (hPD-1) and anti–human LAG-3 (hLAG-3) therapy using fully human monoclonal antibodies in dual humanized PD-1 x LAG-3 mice. The pharmacokinetics and toxicology of the novel anti-hLAG-3 antibody were assessed in non-human primates to support clinical development.

Methods

REGN2810, a high affinity anti-hPD-1 monoclonal antibody that blocks PD-1 interaction with PD-L1 and PD-L2, and a novel high affinity monoclonal anti–hLAG-3 antibody, which blocks the LAG-3/MHC II interaction were generated. Dual humanized PD-1 x LAG-3 mice were engineered by replacing the extracellular domains of mouse Pdcd1 and Lag3 with the corresponding regions of hPD-1 and hLAG-3 and were used for testing antibody efficacy in a MC38.ova syngeneic tumor model. Expression of humanized PD-1 and LAG-3 were analyzed by flow cytometry. Binding of hLAG-3 to mouse MHC II was confirmed with a cell adhesion assay, and binding of hPD-1 to mouse PD-L1 was confirmed using surface plasmon resonance. The pharmacokinetics of anti-hLAG-3 antibody following a single i.v. dose, and the safety profile in a 4-week weekly i.v. dose regimen of up to 50 mg/kg/dose, were determined in cynomolgus monkeys.

Results

Treatment of MC38.ova tumor-bearing humanized mice with a combination of anti-hPD-1 and anti-hLAG-3 antibodies triggered activation of intratumoral and peripheral T cells. Importantly, the combination treatment exhibited an additive, dose dependent anti-tumor effect compared to the respective monotherapies. Anti-hLAG-3 antibody pharmacokinetics in cynomolgus monkeys followed a standard mean concentration-time profile characterized by an initial brief distribution phase and a linear beta elimination phase. Exposure to anti-hLAG-3 increased in a dose-proportional manner, with elimination half-lives ranging from 10.8 to 11.5 days. Anti-hLAG-3 antibody was well tolerated, and no-observed-adverse-effect level (NOAEL) could be established up to 50 mg/kg.

Conclusions

Preclinical anti-tumor efficacy of combined REGN2810 and anti-hLAG-3 antibody treatment, together with favorable pharmacokinetic and safety data for anti-hLAG-3 antibody in cynomolgus monkeys, support clinical development of this cancer combination immunotherapy.

P196 Combination of PD-L1 blockade with oncolytic vaccines re-shapes the functional state of tumor infiltrating lymphocytes

Cristian Capasso1, Federica Frascaro2, Sara Carpi3, Siri Tähtinen1, Sara Feola4, Manlio Fusciello1, Karita Peltonen1, Beatriz Martins1, Madeleine Sjöberg1, Sari Pesonen5, Tuuli Ranki5, Lukasz Kyruk1, Erkko Ylösmäki1, Vincenzo Cerullo1

1University of Helsinki, Helsinki, Uusimaa, Finland; 2University of Siena, Supersano (LE), Puglia, Italy; 3University of Pisa, Pisa, Toscana, Italy; 4University of Napoli Federico II, Helsinki, Uusimaa, Finland; 5PeptiCRAd Oy, Helsinki, Uusimaa, Finland
Correspondence: Cristian Capasso (cristian.capasso@helsinki.fi)

Background

The immunological escape of tumors represents one of the main obstacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd.

Methods

We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background.

Results

First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy significantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells suggested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM-3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen specific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ratio between activated and exhausted pentamer positive cells (p = 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We observed that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was increased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4+ T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor microenvironment (p < 0.0001).

Conclusions

In conclusion, we demonstrated that the efficacy of immune checkpoint inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaustion, resulting in long-lasting immunological memory and increased median survival.
Fig. 7 (abstract P196).

Survival of C57 mice bearing B16OVA tumors and treated on day 6 post-implantation with either PBS, PDL1 blockade, OVA-targeting PeptiCRAd or the combination of PDL1-blockade and OVA-PeptiCRAd.

P197 In vitro evaluation of immunotherapy protocols through a label-free impedance-based technology allows dynamic monitoring of immune response and reagent efficacy

Fabio Cerignoli, Biao Xi, Garret Guenther, Naichen Yu, Lincoln Muir, Leyna Zhao, Yama Abassi

ACEA Biosciences Inc., San Diego, CA, USA
Correspondence: Fabio Cerignoli (fcerignoli@aceabio.com)

Background

In vitro characterization of reagent efficacy in the context of cancer immunotherapy is a necessary step before moving to more expensive animal models and clinical studies. However, current in vitro assays like Chromium-51, ATP-based luminescence or flow cytometry are either difficult to implement in high throughput environments or are mainly based on endpoint methodologies that are unable to capture the full dynamic of the immune response. Here, we present the adaptation of an impedance-based platform to monitor cytotoxic activity of immune cells activated trough different means.

Methods

Impedance technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. We utilized adherent and B cell leukemia/lymphoma cell lines as well as primary tumor cells as in vitro models for immunotherapy reagent evaluation. We seeded the cells on electrodes coated 96-well plates and monitored cell adhesion and proliferation for 24 hours. The following day effector cells were added at multiple effector:target ratios in presence of BiTEs antibodies and/or anti PD-1/PD-L1 antibodies. Impedance signal was monitored for up to seven days. Control wells were set up with effector cells only or with target plus effector cells but without antibodies. We adapted such adhesion-based technology to monitor non-adherent B-leukemia/lymphoma cells, by developing a strategy where the wells are coated with an anti-CD40 antibody. The coating allows specific adhesion and retention of B cells and measurement of changes in impedance that are proportional to cell number.

Results

Using increasing concentrations of EpCAM/CD3 BiTE, we demonstrated the suitability of an impedance-based approach to quantitatively monitor the efficacy of immune cells-mediated cancer cell killing both under different effector:target ratios and antibody concentrations. Combination treatments with checkpoint reduced timing and increased amount of killed cancer cells. Similar results were also obtained with engineered CAR-T cells against CD19 or NK cell lines, demonstrating specific killing of tumor B cells at very low effector:target ratios. The results were also confirmed by flow cytometry.

Conclusions

Overall, our results demonstrate the value of an impedance-based approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays. Furthermore, the availability of 384-well format and minimal sample handling place the technology in an ideal spot for applications in large reagent validation screening or personalized medicine, like therapeutic protocol validation directly on patient samples.

P198 Tumor necrosis factor alpha and interleukin-2 expressing adenovirus plus PD-1 blockade as a boost for T cell therapy in the context of solid tumor therapies

Víctor Cervera-Carrascón1, Mikko Siurala1, João Santos1, Riikka Havunen2, Suvi Parviainen1, Akseli Hemminki1

1TILT Biotherapeutics, Helsinki, Uusimaa, Finland; 2University of Helsinki, Helsinki, Uusimaa, Finland
Correspondence: Víctor Cervera-Carrascón (victor@tiltbio.com)

Background

Because of the immunosuppressive tumor microenvironment, the immune system is unable to develop effective responses against tumor cells. This phenomenon also acts against the effectiveness of adoptive T cell therapy. In order to overcome this situation in the tumor, an attractive therapeutic combination is the combination of oncolytic viruses and immune checkpoint inhibitors. In this case, besides the last two therapies mentioned above, combinations with T cell therapy were also included. The virus used was engineered to express tumor necrosis factor α (TNFα) and interleukin (IL)-2, two cytokines that will boost the immunogenicity of the virus and thus its antitumor properties. On the other hand, the use of anti-PD-1 will avoid exhaustion on tumor infiltrating T cells and hence remove the barriers that could dampen the desired immune response against the tumor.

Methods

In the study of the antitumor effect of this three armed treatment we used an in vivo model of subcutaneous B16-OVA melanoma-bearing mice. Two experiments were carried out; the first one (n = 47) to establish the differences between the triple, double, and single armed combination therapies and the second experiment (n = 84) was focused on study the differences between the groups that showed the best outcomes in the first one and also optimize viral and anti-PD-1 administration regimes.

Results

Preliminary results show a statistically significant positive effect coming out from the combination of virus therapy and immune checkpoint blockade with regard to both tumor progression and overall survival, with up to 43 % complete tumor regression achieved in some of the groups after 96 days post treatment. On the other hand, the effect of adoptive cell therapy in this combination is not completely clear. More results will be presented after analyzing biological samples collected during both experiments.

Conclusions

Preclinical studies are a key step to detect which combinations are more suitable for success in human trials. In this study we developed a rationale for the combination relying on two concepts: to make silent tumors more visible to the immune system and to counter immunosuppressive mechanisms to unleash the full potential of T cells against the tumor, rendering in a modification of the tumor microenvironment that makes it more susceptible for T cell mediated killing. According to the results displayed from these experiments, the combination of this genetically modified adenovirus and PD-1 blockade is an efficient combination to be considered for future application in humans.

P199 IMM-101 primes for increased complete responses following checkpoint inhibitors in metastatic melanoma; 3 case reports

Angus Dalgleish1, Satvinder Mudan2

1St George's University of London, London, UK; 2The Royal Marsden Hospital and Imperial College London, London, UK
Correspondence: Angus Dalgleish (dalgleis@sgul.ac.uk)

Background

IMM-101, a heat-killed borate-buffered whole cell product of Mycobacterium obuense has been shown to enhance cell mediated cytokine responses and innate immune responses involving NK and gamma delta cells [1]. Complete responses (CR) in patients with melanoma lung metastases demonstrated. Follow up of original publication [2] has shown a 30 % 5-year survival. Combined with gemcitabine in metastatic pancreatic cancer a significant survival advantage over gemcitabine monotherapy is seen [3].

Methods

We present 3 patients with metastatic melanoma, progressed after initial stabilisation with IMM-101, who showed CR after check point inhibitors (CPI) ipilimumab (n = 2), pembrolizumab (n = 1). Patient 1: 2006 46 M melanoma left forearm, BT 3.7 mm, 1 positive lymph node. Recurrent disease treated with surgery, Aldara and low dose IL-2. 2010 pulmonary mets, commenced IMM-101, no response (initial SD). 2011 given Ipilimumab. Patient 2: 2011 50 F axillary lump removed, melanoma (no primary). Concomitant mediastinal, lung, gastric and peritoneal deposits. Gastric surgery, decarbazine. Commenced IMM-101 with cyberknife to lung lesion. 2013 Small bowel obstruction from new disease. Started ipilimumab. Patient 3: 2014 79 M melanoma, left cheek, BT 2.4 mm. Regional lymph node recurrence, treated with a left neck dissection in April 2014. Developed paracardiac nodes, adrenal, lung and multiple large subcutaneous deposits. Commenced IMM-101 with initial shrinkage. However, new large subcutaneous lesions. Commenced pembrolizumab.

Results

Patient 1 - CR on Pet CT, maintained through 2016. Patient 2 - CR maintained for 2 years. Patient 3 - CR of subcutaneous deposits four days after first injection.

Conclusions

The CR rate to CPI’s is disappointing, < 1 % for Ipilimumab. PDL-1 expression is predictive for PD-1 responses and although CPI combinations are clearly needed, most are very toxic. IMM-101 is relatively free of toxicity, enhances PD-1 expression in pre-clinical models but may also prime tumour response to check point inhibitors by its action on macrophage function. Based on these observations, we speculate that IMM-101 primes for CPI’s and propose a trial priming with IMM-101, followed by anti-PD-1 antibodies.

References

1. Fowler D, et al.: Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy. CeII 2012, 61(4):535–547.

2. Stebbing J, et al.: An intra-patient placebo-controlled phase I trial to evaluate the safety and tolerability of intradermal IMM-101 in melanoma. Ann Oncol 2012, 23(5):1314–1319.

3. Dalgleish, et al.: Randomised open-label, phase II study of Gemcitabine with and without IMM-101 for advanced pancreatic cancer (IMAGE-1 Trial). BJC 2016, in press.

P200 Immunological impact of checkpoint blockade on dendritic cell driven T cell responses: a cautionary tale

Mark DeBenedette, Ana Plachco, Alicia Gamble, Elizabeth W Grogan, John Krisko, Irina Tcherepanova, Charles Nicolette

Argos Therapeutics Inc., Durham, NC, USA
Correspondence: Mark DeBenedette (mdebenedette@argostherapeutics.com)

Background

AGS-003 is an individualized, autologous, tumor antigen-loaded, dendritic cell (DC) immunotherapy currently in phase III development for the treatment of metastatic renal cell carcinoma (mRCC) in combination with standard-of-care. Antibodies to PD-1 on activated T cells or PD-L1 expressed on APCs have now been approved for treatment of several cancer indications including RCC. While there is a strong mechanistic rationale for the potential synergy of these agents in combination, data supporting the importance of sequencing the administration of these agents are limited. Since the DC-based immunotherapy, AGS-003, expresses high levels of PD-L1, combinations with checkpoint blockade may remove a critical signal protecting DCs during the early CTL activation phase in vivo. Concurrent administration of checkpoint inhibitors with AGS-003 may, therefore, impede the proposed mechanism of action of AGS-003, which is the induction of tumor-specific CTL responses. Results derived from in vitro modeling of DCs inducing T cell responses can demonstrate how to better mobilize the immune system to overcome the immunosuppressive environment of cancer. Therefore, it was of interest to test anti-PD-1/anti-PD-L1 antibody therapy in vitro in combination with DCs representative of AGS-003, to observe the effects combination therapy would have on antigen-specific CTL proliferation and functional responses.

Methods

DCs derived from monocytes were co-electroporated with MART-1 RNA and CD40 ligand RNA to represent AGS-003 DC products. In vitro co-cultures were set up with autologous CTLs and MART-1/CD40L DCs in the presence of anti-PD-1 or anti-PD-L1 antibodies. In some instances, PD-1 expression was hyper expressed on CTLs by electroporating MART-1-specfic CTLs with PD-1 RNA. Subsequent expansion of MART-1-specific CTLs and multi-functional responses in the presence of checkpoint blockade were mapped using multi-color flow cytometry.

Results

Combination with anti-PD-1 antibody did not did not negatively affect the expansion of MART-1-specific CTL responses; however, if PD-1 was hyper-expressed on previously stimulated MART-1-specific CTLs responses were diminished. Anti-PD-1 antibody blocking restored CTL function in the presence of high levels of PD-1 expression. Interestingly, anti-PD-L1 antibody blocking resulted in suppression of early MART-1-specific CTL expansion and subsequent downstream effector function.

Conclusions

Our results suggest that the sequencing of AGS-003 therapy and checkpoint blockade is important to allow full CTL activation by the DCs prior to anti-PD-1/PD-L1 therapy. Moreover the high expression of PD-L1 on DCs may serve as a “don’t kill the messenger” signal, critical to prevent deletion of the DC prior to full signal delivery during early phases of CTL activation.

P201 Targeting the PD-1/PD-L1 signaling pathway for the treatment of OS lung metastasis

Pooja Dhupkar, Ling Yu, Eugenie S Kleinerman, Nancy Gordon

University of Texas MD Anderson Cancer Center, Houston, TX, USA
Correspondence: Pooja Dhupkar (pmdhupkar@mdanderson.org)

Background

Osteosarcoma (OS) is a primary bone malignancy, commonly culminating into aggressive pulmonary metastasis. Despite chemotherapy advances, the 5-year survival of pulmonary metastatic OS remains 25-30 %. Immunotherapy is one of the promising novel approaches to target minimal residual and relapsed disease. The objective of this study is to determine if blocking the PD-1/PD-L1 immunosuppressive signaling pathway using a PD-1 checkpoint inhibitor will have an effect in OS lung metastasis. Anti-PD-1 and anti-PD-L1 antibodies have exhibited therapeutic benefit in melanoma, and non-small cell lung carcinoma. We hypothesize that disruption of the PD-1/PD-L1 signaling pathway using anti-PD-1 antibody has an effect against OS lung metastasis and improves overall survival.

Methods

Flow cytometry and western blotting were used to analyze PD-L1 expression in 7 different OS cell lines. Immunohistochemistry (IHC) analysis was used to determine PD-L1 expression in OS lung metastases from patients and mice. LM7 human OS mouse model was used to test the effect of blocking murine PD-1 in OS lung metastases. Therapeutic effect of anti-PD-1 treatment was measured by the number of macro and micro-metastases. IHC was used to measure cell proliferation (Ki-67), apoptosis (TUNEL) and cleaved-caspase 3 expression in addition to NK cells and macrophages infiltration. Western blotting was used to address the downstream components of the signaling pathway such as p-Stat3 and p-Erk1/2. The Simple PCI software was used to quantify the IHC data.

Results

Our studies revealed surface and total PD-L1 expression in five out of seven human OS cell lines. Primary and metastatic OS lung tumor samples from patients demonstrated membranous and cytoplasmic PD-L1 expression. Using a human OS mouse model we demonstrated therapeutic effect of anti-PD-1 therapy as the number of macro and micro-metastases decreased in the anti-PD-1 treated group as compared to the untreated. Anti-PD-1 treatment led to a significant increase in the number of NK cells and macrophages in the OS lung tumors suggesting these cells to have a potential therapeutic benefit against OS lung metastases. In addition, anti-PD-1 therapy caused a decrease in PD-L1 expression in the lung tumors, possibly due to a decrease in p-ERK1/2 and p-Stat3 expression.

Conclusions

We conclude that targeting the PD-1/PD-L1 axis could be used to treat OS lung metastasis. Therapeutic efficacy of anti-PD-1 may be due to an increased activity of NK cells and/or macrophages in the lung tumors and that inhibition of the p-Stat3/PD-L1 pathway may be the mechanism implicated in OS lung metastases after anti-PD-1 treatment.

P202 Effect of the class I-HDAC inhibitor entinostat and the pan-HDAC inhibitor vorinostat on peripheral immune cell subsets

Italia Grenga, Lauren Lepone, Sofia Gameiro, Karin M Knudson, Massimo Fantini, Kwong Tsang, James Hodge, Renee Donahue, Jeffrey Schlom

Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
Correspondence: Renee Donahue (renee.donahue@nih.gov)

Background

Cancer immunotherapy requires effective recognition and elimination of tumor cells identified as non-self; however, tumors can evade host immune surveillance through multiple mechanisms, including epigenetic silencing of genes involved in antigen processing and immune recognition. Epigenetic therapy with histone deacetylase (HDAC) inhibitors has shown limited benefit as a monotherapy in patients with solid tumors; however, recent reports suggest the potential for synergy when combined with immunotherapy. Entinostat is a class I-HDAC inhibitor undergoing trials for the treatment of various cancers, while vorinostat is a pan-HDAC inhibitor approved in the United States for the treatment of cutaneous T cell lymphoma. The aim of this study was to extensively evaluate the effects of entinostat and vorinostat on human peripheral immune cell subsets in order to examine the potential for combination of HDAC inhibitors with cancer immunotherapy.

Methods

Peripheral blood mononuclear cells (PBMCs) from metastatic breast cancer patients (n = 7) were exposed in vitro for 48 hours to clinically relevant exposures of entinostat, vorinostat, or vehicle control. PBMCs were then analyzed by multicolor flow cytometry using 27 unique markers to identify 123 immune cell subsets, which included 9 classic cell types [CD4+ and CD8+ T cells, regulatory T cells (Treg), B cells, conventional dendritic cells (cDC), plasmacytoid dendritic cells (pDC), natural killer cells (NK), natural killer T cells (NKT), and myeloid derived suppressor cells (MDSC)], and 114 refined subsets relating to their maturation and function.

Results

Treatment with entinostat and vorinostat induced several notable alterations in peripheral immune cells, suggesting mainly immune activating properties. Exposure to entinostat increased the frequency of activated CD4+ T cells, activated mature NK cells, antigen presenting cells (cDCs), and highly immature MDSCs, as well as decreased total Tregs and those with a suppressive phenotype. Exposure to vorinostat induced fewer changes than entinostat, including increasing the frequency of activated CD4+ T cells, highly immature MDSCs, and NKT cells.

Conclusions

These findings show that while entinostat and vorinostat have overall immune activating properties, entinostat induced a greater number changes than vorinostat. This study supports the combination of HDAC inhibitors with immunotherapy, including therapeutic cancer vaccines and/or checkpoint inhibitors.

P203 Shifting the balance of tumor-mediated immune suppression and augmenting immunotherapy with antibody blockade of semaphorin 4D to facilitate immune-mediated tumor rejection

Elizabeth Evans1, Holm Bussler1, Crystal Mallow1, Christine Reilly1, Sebold Torno1, Maria Scrivens1, Cathie Foster1, Alan Howell1, Leslie Balch1, Alyssa Knapp1, John E Leonard1, Mark Paris1, Terry Fisher1, Siwen Hu-Lieskovan2, Antoni Ribas2, Ernest Smith1, Maurice Zauderer1

1Vaccinex, Rochester, NY, USA; 2University of California, Los Angeles, Los Angeles, CA, USA
Correspondence: Elizabeth Evans (eevans@vaccinex.com)

Background

We report a novel role for semaphorin 4D (SEMA4D, CD100) in modulating the tumor microenvironment (TME) to exclude activated antigen presenting cells and cytotoxic T lymphocytes so as to promote tumor growth. Antibody blockade reduces expansion of MDSC, shifts the balance of M1/M2, T effector/T regulatory cells and associated cytokines and chemokines, and augments tumor rejection with immune checkpoint inhibition.

Methods

Anti-SEMA4D antibodies were evaluated, alone and in combination with immune checkpoint antibodies. Immune response was characterized by immunohistochemistry, flow cytometry, functional assays, and cytokine, chemokine and gene expression analysis. Anti-tumor activity was evaluated in various preclinical models. A phase I trial for single agent VX15/2503 was completed.

Results

SEMA4D restricts migration of macrophages and promotes expansion of suppressive myeloid cells in vitro. Strong expression of SEMA4D at the invasive margins of actively growing tumors in vivo modulates the infiltration and polarization of leukocytes in the TME. Antibody neutralization facilitated recruitment of activated APCs and T lymphocytes into the TME in preclinical models. M-MDSCs were significantly reduced in both tumor and blood following treatment. This was accompanied by a significant shift towards increased Th1 cytokines and CTL-recruiting chemokines, with concurrent reduction in Treg-, MDSC-, and M2-macrophage promoting chemokines (CCL2, CXCL1, CXCL5). Accordingly, an increase in Teff:Treg ratio (3x, p < 0.005) and CTL activity (4x, p < 0.0001) was observed. NanoString gene expression analysis of on-treatment tumors confirms an increase in the gamma-inflammatory gene signature (Ribas, ASCO 2015), including significant increases in CXCL9, Gzmb, CCR5, Stat1, Lag3, Ptprc, Ciita, Pdcd1 (PD-1), and Itga1. These coordinated changes in the tumoral immune context are associated with durable tumor rejection and immunologic memory in preclinical colon, breast, and melanoma models. Importantly, anti-SEMA4D antibody can further enhance activity of immune checkpoint inhibitors and chemotherapy. Strikingly, the combination of anti-SEMA4D with anti-CTLA-4 acts synergistically, with maximal increase in survival (p < 0.01) and complete tumor regression in 100 % of mice, as compared to 22 % with monotherapy (p < 0.01). SEMA4D antibody treatment was well tolerated in nonclinical and clinical studies; including a phase I multiple ascending dose trial in patients with advanced refractory solid tumors. Patients with the longest duration of treatment, 48–55 weeks, included colorectal, breast, and a papillary thyroid patient, who had a partial response by RECIST.

Conclusions

Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. Phase Ib/IIa trials of combination therapy with immune checkpoint inhibition are planned.

P204 Combination of a glycomimetic antagonist to E-selectin and CXCR4, GMI-1359, with an anti-PD-L1 antibody attenuates regulatory T cell infiltration and accelerates time to complete response in the murine CT26 tumor model

William Fogler1, Marilyn Franklin2, Matt Thayer2, Dan Saims2, John L. Magnani1

1GlycoMimetics, Inc., Rockville, MD, USA; 2MI Bioresearch, Ann Arbor, MI, USA
Correspondence: William Fogler (wfogler@glycomimetics.com)

Background

Regulatory T cells (Treg) modulate anti-tumor immunity by suppressing T cell activation. Treg are induced and maintained by immunoregulatory receptors, such as PD-L1, and respond to homing signals within the inflamed tumor microenvironment that include the endothelial cell protein, E-selectin, and the CXCR4 ligand, SDF-1. GMI-1359 is a small molecule glycomimetic beginning clinical evaluation with dual inhibitory activity against E-selectin and SDF-1. The aim of the current study was to determine if GMI-1359 alone or in combination with anti-mPD-L1 antibody affected the in vivo growth of CT26 colon carcinoma and to assess percentages of infiltrative intratumoral cells expressing immune markers.

Methods

Female Balb/c mice were implanted subcutaneously with 5x105 CT26.WT tumor cells. Three days post tumor injection, mice (n = 15/group) were treated with saline, GMI-1359 (40 mg/kg for 12 consecutive days), isotype control antibody (anti-KLH) or anti-mPD-L1 antibody (10 F.9G2, 10 mg/kg on days 3, 6, 10, 13, and 17), or the combination of GMI-1359 and anti-mPD-L1 or anti-KLH. On day 15, tumors and spleens (n = 5/group) were excised and T cells (total CD4+ and CD8+, and CCR7+/CD62L+ subsets of each), regulatory T cells (Treg; CD4/CD25/FoxP3), and myeloid derived suppressor cells (MDSC; CD11b+/Gr1+) were determined by flow cytometry. The remaining mice were followed for tumor response.

Results

Treatments were well tolerated. Mice in control groups and single agent GMI-1359 were all identified with progressive disease. In contrast, treatment with anti-mPD-L1 alone or in combination with GMI-1359 produced a 40 % complete response (CR) rate. The median time to CR was shorter when anti-mPD-L1 was combined with GMI-1359 compared to anti-mPD-L1 alone (14 vs. 23 days, respectively, p < 0.0471). Evaluation of tumor infiltrating cells showed that combination therapy with GMI-1359 and anti-mPD-L1 reduced the percentage of Treg compared to treatment with saline, GMI-1359 or anti-mPD-L1 as single treatments (0.9 % vs. 3.3 %, 2.9 % and 1.9 %, respectively). No other T cell subsets were affected. In spleens, the median percentage of Treg were unaffected by any of the treatments and suggest that the reduction in intratumoral Treg by combined treatment with anti-PD-L1 and GMI-1359 was an attenuated response to maintenance and homing signals in the tumor microenvironment.

Conclusions

In conclusion, these studies demonstrate that the dual E-selectin/CXCR4 antagonist, GMI-1359, in combination with anti-mPD-L1 antibody attenuates the induction and distribution of intratumoral Treg and this reduction in Treg is associated with a more rapid immunotherapeutic anti-tumor response.

P205 Antibody targeting of phosphatidylserine enhances the anti-tumor responses of ibrutinib and anti-PD-1 therapy in a mouse triple negative breast tumor model

Jian Gong, Michael Gray, Jeff Hutchins, Bruce Freimark

Peregrine Pharmaceuticals, Tustin, CA, USA
Correspondence: Bruce Freimark (bfreimark@peregrineinc.com)

Background

Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane that becomes exposed on vascular endothelial cells and tumor cells in the tumor microenvironment, particularly in response to chemotherapy and irradiation. Binding of antibodies targeting PS induces the recruitment of immune cells and engages the immune system to destroy tumor and associated vasculature and by blocking the immunosuppressive action of PS. Recent studies have demonstrated that PS-targeting antibodies enhance the anti-tumor activity of immune checkpoint antibody blockade to CTLA-4 and PD-1 in mouse breast and melanoma tumor models. Ibrutinib is an approved anticancer drug targeting B cell malignancies that is a selective, covalent inhibitor Bruton's tyrosine kinase (BTK) in B cell tumors. Data from recent mouse tumor studies demonstrate that ibrutinib in combination with anti-PD-1 antibody blockade inhibits growth of solid tumors, lacking BTK expression, suggesting that ibrutinib may inhibit interleukin-2 inducible T cell kinase (ITK) and promote Th1 anti-tumor responses.

Methods

The present study was conducted to evaluate a combination therapy including PS-targeting antibody mch1N11, ibrutinib and anti-PD-1 antibody in C57Bl/6 mice bearing triple negative E0771 breast tumors. Tumors were staged to an initial volume of ~100 mm3 and randomized to treatment groups (N = 10) with mch1N11 or isotype control at 10 mg/kg qw, anti-PD-1 at 2.5 mg/kg qw or ibrutinib 6 mg/kg or vehicle qd x 8. Tumor volumes were measured twice per week to determine tumor growth inhibition (TGI) relative to control treated animals. The in vitro sensitivity of E0771 tumor cells to ibrutinib was compared to the drug sensitive Jeko-1 cell line in a 72 hour growth and viability assay.

Results

The E0771 cell line is resistant in vitro to 10 mM ibrutinib. Tumor bearing mice treated with mch1N11, ibrutinib or anti-PD-1 alone had 22.2 %, 23.5 % and 32.6 % TGI respectively. The TGI for mch1N11 and ibrutinib was 30.5 %, ibrutinib and anti-PD-1 was 34.5 %, mch1N11 and anti-PD-1 was 36.1 %. The triple combination therapy had statistically greater TGI compared to control treated mice (59.9 %, p = 0.0084).

Conclusions

Treatment of solid tumors with a combination of inhibitors that target PS, ITK and the PD-1/PD-L1 axis in the tumor microenvironment provides a novel treatment for solid tumors, including triple negative breast cancer.

P206 Gp96-Ig/costimulator (OX40L, ICOSL, or 4-1BBL) combination vaccine improves T cell priming and enhances immunity, memory, and tumor elimination

George Fromm, Suresh de Silva, Louise Giffin, Xin Xu, Jason Rose, Taylor H Schreiber

Heat Biologics, Inc., Durham, NC, USA
Correspondence: George Fromm (gfromm@heatbio.com)

Background

The excitement in the field of immuno-oncology over the last several years, driven largely by the clinical success of the first-wave of checkpoint inhibitors, is tempered by the fact that only 10-40 % of patients respond to these drugs given as monotherapy. It is widely believed that to improve efficacy and patient outcome, new approaches that combine treatments with more than one functionality are needed. Novel approaches that provide combination therapy in a single product, will likely lead the way.

Methods

We have developed a next generation cellular vaccine platform – referred to as ComPACT (COMbination Pan-Antigen Cytotoxic Therapy), that incorporates a tumor antigen chaperone (gp96-Ig) with T cell costimulation (Fc-OX40L), into a single tumor cell line that secretes them both (recently published in Cancer Immunology Research 2016).

Results

The current data extend these findings in additional preclinical settings. Specifically, ComPACT is capable of priming antigen-specific CD8+ T cells (peak: 13.3 % of total CD8+), even more so than a leading OX40 agonist antibody (8.4 %) or vaccine alone (5.6 %), and this is associated with increased CD127 + KLRG-1- memory precursor cells and antigen-specific CD4+ proliferation, with reduced off-target inflammation. Importantly, vaccine-expressed Fc-OX40L stimulated IFNγ+, TNFα+, granzyme-b + and IL-2+ by antigen-specific CD8+ T cells. This pharmacodynamic signature of an anti-tumor immune response predicted enhanced rejection of established MC38, CT26 and B16.F10 tumors. Additionally, tetramer analysis of antigen-specific CD8+ T cells (in all 3 tumor models), identified significant accumulation of tumor infiltrating lymphocytes (TIL), suggesting that ComPACT is not only capable of amplifying antigen-specific T cells, but these T cells can efficiently target and eliminate tumors. We have expanded our repertoire of ‘ComPACT’ vaccines to secrete gp96-Ig along with either Fc-TL1A, Fc-4-1BBL or Fc-ICOSL. Each costimulator/vaccine has a unique functionality, which may be context or tumor dependent. We are currently exploring these mechanistic differences.

Conclusions

Taken together, we show that the magnitude and specificity of vaccination can be enhanced by locally secreted costimulatory molecules when delivered within a single product. This may simplify clinical translation and importantly, provide significant patient benefit by improving safety and lowering costs.

P207 Modulation of antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by the anti-PD-L1 antibody avelumab on human lung and prostate carcinoma cell lines using the HDAC inhibitors vorinostat and entinostat

Massimo Fantini1, Sofia R Gameiro1, Karin M Knudson1, Paul E Clavijo2, Clint T Allen2, Renee Donahue1, Lauren Lepone1, Italia Grenga1, James W Hodge1, Kwong Y Tsang1, Jeffrey Schlom1

1National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 2National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD, USA
Correspondence: Sofia R Gameiro (gameirosr@mail.nih.com)

Background

Chromatin deacetylation is a major determinant in epigenetic silencing of immune-associated genes, a key factor in tumor evasion of host immune surveillance. Deregulation of epigenetic enzymes, including aberrant expression of histone deacetylases (HDACs), has been associated with poor prognosis in several cancer types, including of prostate and lung origin. Vorinostat is a pan-HDAC inhibitor currently approved in the United States for the treatment of cutaneous T cell lymphoma. Entinostat is a class I HDAC inhibitor under clinical investigation for the treatment of various malignancies. HDAC inhibitors have been shown to delete immunosuppressive elements and promote synergistic antitumor effects in combination with various immunotherapies. Checkpoint inhibitors targeting PD-1/PD-L1 interactions are promising immunotherapies shown to elicit objective responses against multiple tumors. Avelumab is a fully human IgG1 mAb monoclonal antibody that inhibits PD-1/PD-L1 interaction by targeting PD-L1, and mediates ADCC against PD-L1-expressing tumor cells in vitro. We examined the sensitivity of human lung and prostate carcinoma cells to avelumab-mediated ADCC following clinically-relevant exposure to vorinostat or entinostat.

Methods

Carcinoma cells were exposed daily to vorinostat (3uM) or DMSO for 4 consecutive days, or to entinostat (500 nM) or DMSO for 72 h, prior to being examined for (a) cell-surface PD-L1 expression or (b) used as target cells lysis assay where NK cells from healthy donors were used as effectors. To examine the effect of HDAC inhibitors on PD-L1 expression in vivo, female nu/nu mice were implanted with NCI-H460 (lung) or PC-3 (prostate) carcinoma cells. When tumors reached 0.5-1 cm3, animals received 4 daily doses of DMSO or vorinostat (150 mg/kg, p.o.). Alternatively, animals received a single dose of entinostat (20 mg/kg, p.o.) or DMSO 72 h prior to tumor excision. Frozen specimens were examined for cell-surface expression of PD-L1 by immunofluorescence.

Results

Our results show that 1) vorinostat and entinostat significantly increase the sensitivity of human lung and prostate carcinoma cells to ADCC mediated by avelumab; 2) the anti-CD16 neutralizing mAb significantly decreases avelumab-mediated lysis of target cells exposed to either HDAC inhibitor; 3) both HDAC inhibitors can enhance tumor PD-L1 expression in vitro and in vivo in prostate and/or lung xenograft models; 4) increased avelumab-mediated ADCC of tumor targets exposed to HDAC inhibitors can occur without increased tumor PD-L1 expression.

Conclusions

These studies provide a rationale for combining vorinostat or entinostat with mAbs targeting PD-L1, including for patients that have failed monotherapy regimens with HDAC or checkpoint inhibitors.

P208 Monoclonal antibodies targeting phosphatidylserine enhance combinational activity of the immune checkpoint targeting agents LAG3 and PD-1 in murine breast tumors

Michael Gray, Jian Gong, Jeff Hutchins, Bruce Freimark

Peregrine Pharmaceuticals, Tustin, CA, USA
Correspondence: Michael Gray (mgray@peregrineinc.com)

Background

Our previous work demonstrated that the addition of phosphatidylserine (PS) targeting antibodies to anti-programmed death ligand 1 (PD-1) therapy in murine triple negative breast cancers (TNBC) significantly enhanced immune system activation and tumor growth inhibition. In these studies, NanoString immune profile analysis showed that intratumoral levels of lymphocyte activation gene 3 (LAG3) mRNA increased in response to PS and PD-1 treatments. This suggests LAG3 may act to attenuate T cell activation in TNBC during I/O therapeutic regimens; however, it is unknown if PD-1 and LAG3 function cooperatively in regulating T cell anergy, and whether adding PS blocking antibodies can further enhance the effectiveness of LAG3 and/or LAG3 + PD-1 therapies.

Methods

Animal studies utilized C57bl/6 mice implanted with the murine TNBC model E0771. Immunoprofiling analysis was performed by flow cytometry and the NanoString nCounter® PanCancer Immune Profiling Panel. Antibody treatments utilized a specific phosphatidylserine targeting antibody (ch1N11), anti-PD-1, or anti-LAG3 alone or in combination. All statistical analysis utilized the student t-test (significant with p < 0.05).

Results

LAG3 and PD-1 were co-expressed on T cells in E0771. Mice treated with antibodies targeting PS, PD-1, and LAG3 alone in combination with each other demonstrated that the addition of PS blocking antibodies to anti-PD-1 therapy or LAG3 had significantly greater anti-tumor activity than either single agent. Comparison of PD-1 + LAG3 combinational therapy vs. single PD-1 or LAG3 treatments showed moderately more anti-tumor activity than single treatments; however, the addition of PS blocking antibodies to either checkpoint inhibitor was as equally effective in inhibiting tumor growth as observed in the combination of LAG3 + PD-1 treatment. Further comparison of PD-1 + LAG3 vs. PS + PD-1 + LAG3 treatments demonstrated that the addition of PS blocking antibodies resulted in a significant decrease in tumor growth accompanied by complete tumor regression in a greater number of animals than observed in the PD-1 + LAG3 treatment group. FACS and NanoString immunoprofiling analysis on each treatment group showed that the addition of PS blocking antibodies to all checkpoint treatment groups, including the combination of PD-1 + LAG3, resulted in enhanced tumor infiltrating lymphocytes (TILs), a reduction of myeloid derived suppressor cells (MDSCs), and enhanced cytokines associated with immune system activation.

Conclusions

Overall, our data demonstrate that while PS, LAG3, and PD-1 therapies each have efficacy in TNBC as single agents, I/O treatments that include PS blocking antibodies offer significantly improved growth inhibition and are capable of increasing TILs compared to single and combinational treatments by T cell checkpoint targeting inhibitors alone.

P209 The immunoreceptor TIGIT regulates anti-tumor immunity

Jane Grogan, Nicholas Manieri, Eugene Chiang, Patrick Caplazi, Mahesh Yadav

Genentech, South San Francisco, CA, USA
Correspondence: Jane Grogan (grogan.jane@gene.com)

Background

Strategies to re-activate exhausted anti-tumor immune responses with antibody blockade of key T cell co-inhibitory receptors such as PD-1/PD-L1 or CTLA-4 have demonstrated transformational potential in the clinic. TIGIT (a PVR-nectin family member) is a dominant immuno-inhibitory receptor on tumor-specific T and NK cells, shown to regulate anti-tumor immunity. Activation of TIGIT on T and NK cells limits proliferation, effector cytokine production, and killing of target tumor cells. The high affinity receptor for TIGIT is PVR, and the counter agonist receptor is CD226, all of which are members of the PVR-nectin family. TIGIT is elevated in the tumor microenvironment in many human tumors and coordinately expressed with other checkpoint immune receptors such as PD-1. However, the spatial and coordinate expression of these receptors and ligands required for these functions, and the cell-types involved in anti-tumor immunity, remains unknown.

Methods

TIGIT, CD226 and PD-L1 blockade will be assessed in preclinical syngeneic tumor model CT26 and MC38. To determine which immune cells are important for allowing tumor progression early and late in disease mice with cell-specific gene ablation for these family members were challenged with tumors. Tumor growth was determined and tumor sections labeled and probed by fluorescence microscopy to assess TIGIT, CD226 and PVR cellular expression.

Results

In mouse models of both cancer, antibody co-blockade of TIGIT and PD-L1 enhanced CD8+ T cell effector function, resulting in significant tumor clearance. TIGIT is expressed on CD8+ T cell, Treg and NK cells. Specific ablation of TIGIT on CD8+ T cells resulted in tumor clearance, and was dependent on PVR in the host tissue. Immunofluorescence studies will be presented.

Conclusions

Therapeutic blockade of TIGIT may result in improved eradication of malignancies when used in conjunction with other anti-cancer therapies including those that modulate anti-tumor immune responses, and is currently being tested in phase I clinical trials. Models indicate that inhibition of TIGIT with a blocking mAb may release CD226 to activate tumor-specific T cells. Another mechanism could involve regulation of T cell suppression by TIGIT on regulatory T cells. A better understanding of the coordinate interaction between these receptors and ligands in tumors will be informative for the appropriate application of checkpoint-therapy combinations.

P210 CC-122 in combination with immune checkpoint blockade synergistically activates T cells and enhances immune mediated killing of HCC cells

Patrick Hagner1, Hsiling Chiu1, Michelle Waldman1, Anke Klippel1, Anjan Thakurta1, Michael Pourdehnad2, Anita Gandhi1

1Celgene Corporation, Summit, NJ, USA; 2Celgene Corporation, San Francisco, CA, USA
Correspondence: Patrick Hagner (phagner@celgene.com)

Background

CC-122 binds the E3 ubiquitin ligase CRL4CRBN resulting in the degradation of the transcription factor Aiolos and activation of T cells. Preclinical and clinical data obtained in hematologic malignancies indicate that CC-122 exerts immunomodulatory activity through enhanced antibody dependent cell-mediated cytotoxicity and a shift in T cell subsets from a naïve to effector and memory subsets. CC-122 is in clinical development in multiple hematologic diseases and in solid tumors such as hepatocellular carcinoma (HCC) as a single agent (NCT01421524) and in combination with nivolumab (nivo). The effects of combining CC-122 with immune checkpoint antibodies in in vitro models of T cell activation and immune co-culture models with HCC cells were examined.

Methods

Carboxyfluorescein succinimidyl ester (CFSE) based proliferation, cytokine production and immune co-culture assays were performed with stimulated peripheral blood mononuclear cells (PBMC) from healthy donors followed by drug treatment. Drug combinations were investigated in mixed lymphocyte reactions (MLR) with monocyte derived dendritic cells and T cells from separate donors. Apoptosis was measured via Annexin V/ToPro3 staining. Synergy calculations were performed with the fractional product method.

Results

In a 3-day CD3-stimulated PBMC assay, CC-122 (1-10 μM) treatment elevated HLA-DR, a marker of T cell activation, by 3.4-5.5 and 3.2-5.3 fold in CD4+ and CD8+ T cells, respectively. Proliferation of CD4+ and CD8+ T cells from CD3-stimulated PBMC treated with vehicle, CC-122 (50nM), nivo (50 μg/ml) or the combination was assessed via CFSE staining. The percentage of proliferating vehicle-treated CD4+ and CD8+ cells was 37 % and 40 %, compared to nivo (45 % and 47 %), CC-122 (54 % and 68 %) and the combination (61 % and 74 %). SEB stimulated PBMC were treated with CC-122 (40nM), and nivo or α-PD-L1 (0.1-100 μg/ml) resulting in secretion of 424, 160 and 154 ng/ml IL-2, respectively. The combination of CC-122 with either nivo or α-PD-L1 (10 μg/ml) resulted in synergistic IL-2 secretion levels of 873 and 813 ng/ml, respectively. In an MLR assay, the combination of CC-122 (100nM) with nivo (10 μg/ml) or α-PD-L1 (10 μg/ml) resulted in synergistic IL-2 and IFNγ secretion. Finally, the combination of CC-122 and nivo or CC-122 and α-PD-L1 significantly increased PBMC-mediated cytotoxicity of HCC cells compared to either single agent or isotype control (p ≤ 0.05).

Conclusions

CC-122 in combination with nivo or anti-PD-L1 antibodies results in synergistic activation of T cells and significantly enhanced immune mediated cytotoxicity against HCC cells. Given the novel mechanism of immunomodulation by CC-122 and synergistic combination with checkpoint blockade, clinical investigation in HCC is currently in progress.

P211 Ubiquitin-specific protease 6 (USP6) oncogene confers dramatic sensitivity of sarcoma cells to the immunostimulatory effects of interferon

Ian Henrich1, Laura Quick2, Rob Young2, Margaret Chou2

1University of Pennsylvania, Philadelphia, PA, USA; 2Children's Hospital of Pennsylvania, Philadelphia, PA, USA
Correspondence: Ian Henrich (ihenrich@mail.med.upenn.edu)

Background

Bone and soft tissue tumors (BSTTs) represent a heterogeneous class of neoplasms that disproportionately affect children. Compared to other malignancies, BSTTs are poorly understood, which has hampered the development of effective therapies. Our lab previously discovered that the oncogenic de-ubiquitylating enzyme USP6 is the key etiologic agent in several benign BSTTs, and is selectively overexpressed in multiple sarcomas, a malignant class of BSTTs [1]. USP6 drives tumorigenesis by directly de-ubiquitylating the Jak1 kinase, leading to its stabilization and activation of STAT transcription factors [2]. Since the Jak1-STAT pathway is a central mediator of interferon (IFN) signaling, we hypothesized that USP6 overexpression in sarcomas would render them hypersensitive to the immune stimulatory effects of IFN, which could be exploited for therapeutic benefit.

Methods

USP6 was expressed in a doxycycline-inducible manner in various patient-derived sarcoma cell lines, including Ewing sarcoma, rhabdomyosarcoma, leiomyosarcoma, and liposarcoma. USP6 expression levels were confirmed to approximate those in primary patient tumor samples.

Results

USP6 conferred exquisite sensitivity of sarcoma cells to the immuno-modulatory effects of IFN. Activation of STAT1 and STAT3 were both enhanced and prolonged in sarcoma cells expressing USP6 upon IFN treatment. RNA-sequencing confirmed that USP6 induces an IFN response signature by itself, and that it synergizes with IFN to dramatically induce interferon-stimulated gene (ISG) expression. The ISGs synergistically induced by USP6 and IFN include a large group of anti-tumor and immunomodulatory genes: the pro-apoptotic ligand TRAIL was dramatically elevated and mediated apoptosis of USP6-expressing sarcoma cells. Immunomodulatory factors synergistically induced by USP6 and IFN included chemokines and cytokines that drive migration and differentiation of T cells.

Conclusions

USP6 overexpression sensitized sarcoma cells to IFN, simultaneously inducing TRAIL-mediated death and stimulating sarcoma cells to produce immune stimulatory/anti-tumorigenic chemokines and cytokines. This dual mechanism of action may position IFN as an extremely effective therapeutic agent for treatment of sarcomas that overexpress USP6.

References

1. Oliveira A, Chou M: The TRE17/USP6 oncogene: a riddle wrapped in a mystery inside an enigma. Front Biosci (Schol Ed) 2012, 4:321–340.

2. Quick L, Young R, Henrich I, Wang X, Asmann Y, Oliveira A, Chou M: Jak1-STAT3 signals are essential effectors of the USP6/TRE17 oncogene in tumorigenesis. Cancer Res 2016, in press.
Fig. 8 (abstract P211).

USP6 Expression in RD-ES Cell Line. USP6 was expressed in a doxycycline-inducible in the patient derived Ewing sarcoma cell line. Clonal lines that express high or medium amounts of USP6 were isolated from the initial pooled population. Expression of USP6 increased Jak1 levels and activation of downstream effectors STAT1 and STAT3 in an USP6 dose dependent manner. Note: Similar lines were/are being created for other sarcomas. RD-ES is used as an example

Fig. 9 (abstract P211).

USP6 Sensitizes Cells to IFN-Induced Death. RD-ES were treated with 1000 U/mL IFNa, IFNB, or IFNy for 24 hours with or without USP6 expression. IFNB was most effective in inducing death (~50%). Death was monitored via trypan blue exclusion

Fig. 10 (abstract P211).

USP6 Expression Determines Sensitivity to IFN-Induced Death. RD-ES, RD-ES/USP6(Med), and RD-ES/USP6(High) were treated with 1000 U/mL IFNB overnight. Higher USP6 increases the sensitivity of the cells to IFNB induced death. Death was monitored by PARP cleavage

Fig. 11 (abstract P211).

USP6 Synergizes with IFN to Massively Upregulate TRAIL. RD-ES were treated with 1000 U/mL IFNa, IFNB, or IFNy for 24 hours with or without USP6 expression. TRAIL was found to be synergistically induced by IFN in the presence of USP6. IFNB was the most potent at nearl5 5000-fold over baseline

Fig. 12 (abstract P211).

USP6 Synergizes with IFNy to Increase Chemokine Expression. RD-ES, RD-ES/USP6(Med), and RD-ES/USP6(High) were treated with 10 U/mL IFNy overnight. The chemokine CXCL10 was synergistically induced in an USP6 dose-dependent manner. Similar expression patterns were seen for other chemokines like CXCL9 and CXCL11

Fig. 13 (abstract P211).

USP6 Induces an IFN-Response In Vitro. RNA-sequencing was performed on RD-ES/USP6(Pooled) were treated with or without dox. The resulting gene expression data was analyzed using Gene Sequencing Enrichment Analysis (GSEA) using the Hallmark dataset (the hallmark dataset contains curated gene sets that are known to be part of key cellular pathways). USP6 induces a strong IFN-response and Jak-STAT gene expression

Fig. 14 (abstract P211).

USP6 Induces an IFN-Response In Vivo (Ewings Sarcoma). The Ewing sarcoma patient microarray dataset (GSE37371) was sorted based on USP6 expression and the top 5 USP6 expressing patient samples were compared to the bottom 5 using GSEA. Similar to the in vitro results, high USP6 expression correlates with activation of Jak-STAT and an IFN-response signature

Fig. 15 (abstract P211).

USP6 Induces an IFN-Response In Vivo (Rhabdomyosarcoma). The Ewing sarcoma patient microarray dataset (GSE66533) was sorted based on USP6 expression and the top 5 USP6 expressing patient samples were compared to the bottom 5 using GSEA. Similar to the in vitro results, high USP6 expression correlates with activation of Jak-STAT and an IFN-response signature

Fig. 16 (abstract P211).

USP6 Potentiates IFN Signaling In Vitro. RD_ES/USP6(Pooled) or RD-ES lines were treated with 1000 U/mL IFNa of IFNB for the indicated time period. USP6 extended the duration and amplified the magnitude of STAT1 and STAT3 activation

P212 CPI-444: a potent and selective inhibitor of adenosine 2A receptor (A2AR) induces anti-tumor responses alone and in combination with anti-PD-L1

Andrew Hotson, Stephen Willingham, Po Ho, Carmen Choy, Ginna Laport, Ian McCaffery, Richard Miller

Corvus Pharmaceuticals, Burlingame, CA, USA
Correspondence: Andrew Hotson (ahotson@corvuspharma.com)

Background

Elevated extracellular adenosine in the tumor microenvironment is immunosuppressive promoting tumor growth and metastasis through signaling via A2AR on immune cells. CPI-444 is a potent, oral, selective A2AR antagonist that has been well tolerated in phase I/Ib studies.

Methods

Preclinical studies were performed with MC38 mouse tumor models and primary human PBMCs. Based on these results, we have initiated a phase Ib trial to examine safety, tolerability, biomarkers, and efficacy of CPI-444 as single agent and in combination with anti-PD-L1 antibody, atezolizumab, in patients with selected solid tumors. Peripheral blood and tumor biopsies are collected pre- and post-treatment for biomarker analysis.

Results

Pre-clinical studies demonstrated cross-talk between adenosine and PD-1/PD-L1 pathways, providing rationale for combination therapy. In MC38, anti-PD-L1 treatment resulted in elevated CD39 and CD73 expression on T cells, consistent with increased capacity to generate adenosine. The adenosine analog NECA inhibited TCR-mediated ERK phosphorylation and production of IL-2 and IFNγ in human PBMCs; these inhibitory effects were blocked by CPI-444. Treatment of MC38 with CPI-444 led to inhibition of tumor growth, with tumor elimination in ~30 % of mice. Combining CPI-444 with anti-PD-L1 synergistically inhibited tumor growth and eliminated tumors in 90 % of mice. When cured mice were re-challenged with MC38, tumors were uniformly rejected, indicating CPI-444 induced systemic anti-tumor memory. CD8+ depletion abrogated efficacy of CPI-444 ± anti-PD-L1 treatment. Biomarker results from the ongoing phase Ib trial demonstrate CPI-444 neutralizes A2AR signaling and activates markers of an immune response. To measure A2AR inhibition, peripheral blood samples were activated with NECA and pCREB quantified using flow cytometry. A2AR signaling was robustly inhibited in 8 of 9 patients in an exposure dependent manner. Of the patients evaluated so far, immune activation was observed by flow cytometry analysis of PD-1/CD8 frequency in all continuously treated patients and a subset of patients on the 14 day schedule. IHC and gene expression of pathway markers in serial tumor biopsies will be discussed.

Conclusions

In total, this shows that CPI-444 exhibits functional inhibition of adenosine signaling, and treatment is associated with activation of markers of anti-tumor immunity. This is the first demonstration of immune modulation in cancer patients receiving an adenosine antagonist.

P213 ProbodyTM therapeutics targeting the PD-1/L1 axis provide preclinical anti-tumor efficacy while minimizing induction of autoimmunity as single agents and in combination with CTLA-4 blockade

Kimberly A Tipton, Kenneth R Wong, Victoria Singson, Chihunt Wong, Chanty Chan, Yuanhiu Huang, Shouchun Liu, Jennifer H Richardson, W Michael Kavanaugh, James West, Bryan A Irving

CytomX Therapeutics, Inc., South San Francisco, CA, USA
Correspondence: Bryan A Irving (birving@cytomx.com)

Background

Immunotherapy has transformed cancer treatment by unleashing potent and durable anti-tumor immunity against many cancers. However, because many of the same mechanisms control anti-tumor immunity and self-tolerance, these therapies can also induce systemic autoimmunity by activating autoreactive T cells in normal tissues. Combinations of checkpoint inhibitors targeting PD-1 and CTLA-4 increase clinical response rates, but similarly increase toxicities, thereby reducing their clinical potential. New approaches are therefore needed that provide anti-tumor activity without dysregulating systemic immunity.

Methods

CytomX has developed Probody therapeutics (Pb-Tx) that are proteolytically-activated antibodies designed to widen the therapeutic index by minimizing drug interaction with normal tissue while retaining anti-tumor activity. Pb-Tx are “masked” to attenuate binding to target in healthy tissue, but can become “unmasked” in the tumor microenvironment by tumor-specific protease activity.

Results

In vitro, the masked PD-1 Pb-Tx had reduced affinity for mouse PD-1 relative to the parental antibody. Binding affinity was completely restored following addition of appropriate proteases. In mice, single-agent antibodies to CTLA-4 and to PD-1, and the PD-1 Pb-Tx induced 10 %, 30 %, and 20 % complete tumor regressions (CRs) against established MC38 tumors, respectively. In combination with anti-CTLA-4, both PD-1 antibody and Pb-Tx induced 80 % CRs and generated effective T cell memory against tumor re-challenge. In 10-week-old NOD mice, a 1 or 10 mpk single dose of anti-PD-1 antibody induced diabetes in 43 % and 57 % of mice, respectively, while a 10 mpk dose of PD-1 Pb-Tx yielded only 14 % disease incidence with delayed onset. In younger NOD mice, the CTLA-4/PD-1 antibody combination induced diabetes in 50 % of mice. In contrast, mice administered the PD-1 Pb-Tx/CTLA-4 antibody combination were completely protected. Similar data were generated with a PD-L1-targeted Pb-Tx.

Conclusions

A PD-1 targeted Pb-Tx provided equivalent anti-tumor efficacy in mice to that of its parental antibody while protecting from anti-PD-1-mediated autoimmunity, both as a single agent and in combination with a CTLA-4 antibody. These results demonstrate that PD-1 Pb-Tx retain anti-tumor efficacy with improved safety profiles preclinically and therefore have promise to enable safer combination immunotherapies.

P214 Enhancement of target expression on breast tumors via hormone receptor antagonism: a novel strategy for enhancing immunotherapeutic efficacy

Ritika Jaini, Matthew Loya, Charis Eng

Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA
Correspondence: Ritika Jain (jainir@ccf.org)

Background

Immunotherapy has historically been successful in highly antigenic tumors but mostly failed in non-antigenic tumors. Our studies in autoimmunity have shown that increased antigen load within a tissue enhances immune reactivity against it. We hypothesize that enhancing protein target expression on breast tumors can increase the efficacy of targeted immunotherapy. Lactation proteins have recently been shown to be effective immunotherapeutic targets on breast tumors. Since lactation proteins are negatively regulated by signaling via the estrogen receptor (ER), we hypothesize that target lactation protein expression on breast tumors can be increased by antagonism of the ER in order to enhance efficacy of antigen specific immunotherapy/vaccination.

Methods

Enhancement of target protein expression in human breast tumors was tested in vitro by treatment of ER+ (MCF7 cells) and ER + PR+ (T47D cells) with different doses of the clinically approved ER modulator, tamoxifen. In vivo modulation of target antigen expression was tested by inoculating 6–7 week old Balb/cJ female mice with 4 T1 breast tumors followed by oral treatment with tamoxifen. Increase in lactation protein expression (e.g. alpha lactalbumin) was assayed by in vitro Luciferase assays followed by confirmation by immunoblotting and immunohistochemistry at different time points post treatment. Effect of increased antigen expression on efficacy of targeted immunotherapy was assessed by antigen specific immunization of 4 T1 tumor bearing mice with or without tamoxifen administration and comparing tumor growth.

Results

Our in vitro studies on human tumors and in vivo murine studies show that antagonism of the ER via tamoxifen treatment can substantially increase expression of target lactation proteins such as alpha lactalbumin on breast tumors. We show that whereas at least a 2–3 fold increased expression of the target protein can be achieved on tumors, normal breast tissue remains unaffected. Tumor progression studies revealed that in spite of increased target expression, no enhancement in efficacy of immunotherapy was achieved via active immunization protocols. However, efficacy of cell based targeted immunotherapies can possibly be enhanced when applied in combination with our proposed strategy to increase target expression.

Conclusions

Singular increase in target antigen expression on tumors is not effective in enhancing efficacy of immunotherapy probably due to associated limiting factors such as DC trafficking, antigen presentation and effective priming. However, the efficacy of cell-based targeted immunotherapeutic strategies that circumvent the limitations around active priming can be enhanced by using our combinatorial strategy of enhancing antigen expression on tumors via hormone receptor antagonism.

Acknowledgements

The PhRMA Foundation for funding support.

P215 Dose escalation/confirmation results of ENCORE 601, a phase Ib/II, open-label study of entinostat (ENT) in combination with pembrolizumab (PEMBRO) in patients with non-small cell lung cancer (NSCLC)

Melissa L Johnson1, Alex A Adjei2, Mateusz Opyrchal3, Suresh Ramalingam4, Pasi A Janne5, George Dominguez6, Dmitry Gabrilovich6, Laura de Leon7, Jeannette Hasapidis7, Scott J Diede8, Peter Ordentlich7, Scott Cruickshank7, Michael L Meyers9, Matthew D Hellmann10

1Sarah Cannon Research Institute, Nashville, TN, USA; 2Mayo Clinic, Rochester, MN, USA; 3Roswell Park Cancer Institute, Buffalo, NY, USA; 4Emory University, Atlanta, GA, USA; 5Dana-Farber Cancer Institute, Boston, MA, USA; 6The Wistar Institute, Philadelphia, PA, USA; 7Syndax Pharmaceuticals, Inc., Waltham, MA, USA; 8Merck Research Laboratories, North Wales, PA, USA; 9Syndax Pharmaceuticals, Inc., New York, NY, USA; 10Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Correspondence: Melissa L Johnson (mjohnson@tnonc.com)

Background

ENT is an oral, class I selective histone deacetylase (HDAC) inhibitor shown in animal models to reduce immunosuppressive myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) and produce synergistic anti-tumor responses when combined with immune checkpoint inhibition. ENCORE 601 is a phase Ib/II study designed to evaluate ENT plus PEMBRO in patients with advanced NSCLC. The objective of the phase Ib dose escalation/confirmation portion was to determine the recommended phase II dose (RP2D).

Methods

Patients with stage III/IV NSCLC (previous anti-PD-1/PD-L1 therapy was permitted) were enrolled in a 3 + 3 dose escalation phase. ENT 3 mg and 5 mg QW PO + PEMBRO 200 mg Q3W IV in 21-day cycles were explored to determine the safety and RP2D, followed by a dose-confirmation cohort (n = 9). Pre-treatment biopsies were required. Correlative studies included tumor PD-L1 expression and phenotypic and functional evaluation of immune cell subsets in peripheral blood and tumor tissue.

Results

Twenty-two NSCLC patients (9 of which progressed on prior anti-PD-1/PD-L1 therapy) were treated with ENT plus PEMBRO; 13 in the dose escalation phase (6 at ENT 3 mg and 7 at ENT 5 mg) and 9 in the dose confirmation phase (ENT 5 mg). Of 20 patients with PD-L1 expression results, 7 patients (35 %) had PD-L1 < 1 %, 8 (40 %) had PD-L1 1-49 %, and 5 (25 %) had PD-L1 ≥ 50 %. During dose escalation, 1 patient previously treated with anti-PD-1 therapy experienced a DLT at Cycle 2 Day 15 (ENT 3 mg, Grade 3 immune-mediated hepatitis), and no other DLTs were observed. Among all 22 patients treated, Grade 3/4 treatment-related AEs included hypophosphatemia (9 %), neutropenia (5 %), anemia (5 %), acute respiratory failure (5 %), elevated alkaline phosphatase (5 %) and immune-mediated hepatitis (5 %). Of 6 evaluable patients previously exposed to anti-PD-1/PD-L1, best response includes 3 SD and 3 PD. Of 11 evaluable anti-PD-1/PD-L1 naïve patients, best response includes 1 PR, 1 SD and 9 PD. A reduction in peripheral MDSC levels was observed between pre-treatment and Cycle 2 Day 1 in 7 of 11 patients assessed (median decrease of 40.7 % PMN-MDSCs; 67.3 % M-MDSCs).

Conclusions

5 mg ENT weekly combined with PEMBRO has a manageable safety profile, expected pharmacodynamic effects on reducing MDSCs, and will be further explored in the phase II expansion cohorts including NSCLC and melanoma.

Trial Registration

ClinicalTrials.gov identifier NCT02437136.

P216 Combinatorial reprograming of chemokine environment in colorectal and ovarian cancer patients to promote intratumoral CTL infiltration

Pawel Kalinski1, Amer Zureikat2, Robert Edwards3, Ravi Muthuswamy3, Nataša Obermajer4, Julie Urban3, Lisa H Butterfield2, William Gooding2, Herbert Zeh3, David Bartlett4

1Department of Surgery; University of Pittsburgh Cancer Institute; Department of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, PA, USA; 2University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; 3University of Pittsburgh, Pittsburgh, PA, USA; 4Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA
Correspondence: Pawel Kalinski (kalinskip@upmc.edu)

Background

Since the infiltration of tumor tissues with effector CD8+ T cells (CTLs) is associated with improved clinical outcomes and predicts patients’ responsiveness to checkpoint blockers, we developed a combinatorial approach to selectively enhance the production of CTL chemokines in tumor lesions, while avoiding the activation of healthy tissues. Our preliminary data from human ex vivo tissue culture models demonstrate that a) TLR3-based combinatorial adjuvants selectively induce CTL-attracting chemokines in tumor-associated stromal and myeloid cells, while avoiding undesirable activation of cancer cells and surrounding non-tumor tissues; b) combination of TLR3 ligands with IFNa synergistically amplifies the production of CTL-attracting chemokines and allows to uniformly induce their production in all tumor lesions; and c) that the inclusion of COX2 blockers prevents the induction of Treg-attractants.

Methods

Based on these preclinical data, we developed a phase I/II clinical trial (UPCI 10-131/NCT01545141) to determine the safety and local effectiveness of intravenous infusion of rintatolimod (Ampligen; selective TLR3 ligand) combined with Intron A and oral celecoxib, in patients with resectable recurrent colorectal cancer. Our phase I/II trial UPCI 11–128 (NCT02432378) evaluates the safety, feasibility and local effectiveness of the intraperitoneal delivery of rintatolimod and Intron A (with oral celecoxib) in cisplatin-treated patients with recurrent ovarian cancer.

Results

In the completed phase I of UPCI 10–131, we observed very good safety profile of this combination and, in accordance with our expectations, selective disappearance of CTL-, TH1- and NK cell markers from circulation, lasting 24–48 hours after rintatolimod/Intron A infusion. Comparison of the resected tumors demonstrated enhanced intratumoral ratios of CXCL10 (CTL-attractant) to CCL22 (Treg-attractant) and CD8α (CTL marker) to FoxP3 (Treg marker), in the treatment cohort, compared to patients receiving standard care at our center (non-randomized control). The randomized phase II portion of this clinical study is ongoing. Our recently-implemented study UPCI 11–128 provides preliminary indication of the feasibility and local effectiveness of intraperitoneal modulation of tumor microenvironments in cisplatin-treated ovarian cancer patients.

Conclusions

Our data provide early indications of the safety and feasibility of using combinatorial adjuvants to selectively enhance intratumoral CTL infiltration. Verification of these results in randomized phase II portions of our trials may provide new means to enhance the clinical effectiveness of checkpoint inhibitors, therapeutic vaccines and adoptive T cell therapies (ACT) against “cold tumors”, enhancing the scope of their applications.

Acknowledgements

This project was supported by the NIH grants P01 CA132714 and P50 CA159981. Rintatolimod was provided under MTA by Hemispherx Bio.

Trial Registration

ClinicalTrials.gov identifier NCT01545141.

P217 A model system to characterize the personalized cell immunotherapy, AGS-003, and predict functional activity in combination with PD-1 checkpoint inhibitor and sunitinib

Olga Zubkova1, Larissa Agapova1, Marina Kapralova1, Liudmila Krasovskaia1, Armen Ovsepyan2, Maxim Lykov2, Artem Eremeev1, Vladimir Bokovanov1, Olga Grigoryeva1, Andrey Karpov1, Sergey Ruchko1, Charles Nicolette3, Alexandr Shuster2

1LLC Cellthera Pharm, Volginsky, Vladimir, Russia; 2LLC IBC Generium, Volginsky, Vladimir, Russia; 3Argos Therapeutics Inc., Durham, NC, USA
Correspondence: Andrey Karpov (apkarpov@cellthera.ru)

Background

AGS003 is an immunotherapy consisting of autologous dendritic cells (DCs) electroporated with amplified total tumor RNA plus synthetic CD40L RNA and is currently being tested in combination with standard of care to extend survival of newly diagnosed metastatic RCC patients in the phase III ADAPT clinical trial. We set out to establish an animal model system to more thoroughly study the AGS003 mechanism of action and assess the functionality in combination with other therapeutic agents.

Methods

Mouse DC precursors were processed in a similar manner to how human monocytes are processed to manufacture AGS003. Bone marrow cells from 7–10 week old Balb/c mice were incubated with GMCSF and IL4 and matured with TNFa, IFNg and PGE2. Mature DCs were electroporated with total tumor RNA from RENCA tumor plus synthetic mCD40L RNA and injected i.p. to treat syngeneic BALB/c mice in an orthotopic RCC model. This model system was utilized to test the AGS003-like mouse DCs as a single agent or in combination with the mPD-1 checkpoint inhibitor.

Results

Here we report on successfully developing murine DCs with similar properties to AGS003, including phenotype (CD80, CD83, CD86, MHCI, CCR7), secretion of IL12 induced by CD40L RNA and induction of CD8 + CD28 + CD45RA- memory T cells in vivo. Results showed that, as a single agent, the DC therapy was superior to PBS controls at increasing median survival, slowing tumor growth and decreasing lung metastases. These effects were dependent on inclusion of the amplified total RENCA cell RNA based on comparison with irrelevant RNA controls. In addition, the greatest control of tumor growth rate and median survival occurred when combined with the PD-1 checkpoint inhibitor and sunitinib.

Conclusions

These data demonstrate the importance of amplified total tumor RNA in directing immune responses against the corresponding tumor target and support the strategy of generating and/or augmenting preexisting antitumor immune responses with active immunotherapy to maximize clinical benefit when combined with PD-1 checkpoint inhibition and sunitinib as the standard therapy drug for renal cell carcinoma. This model system may be useful to explore additional combination therapies with other therapeutic agents.
Fig. 17 (abstract P217).

Survival curve of DC-based combination therapy in RCC mouse model. DC monotherapy showed a 50% increase in median OS compared to the PBS group (42 vs 28 days). DC + Sutent combination therapy did not show increased OS compared to monotherapy with DCs or Sutent (40 vs 42 or 43 days). DC + aPD-1 combination therapy showed significantly increased median OS compared to monotherapy with DCs or aPD-1 (67 vs 42 or 34.5 days). DC + Sutent + aPD-1 combination therapy showed significantly increased media OS (>104 days) compared to all other groups tested

P218 Local intratumoral treatment with low-dose CD40 and TLR4 agonists overcomes resistance to PD-1 blockade to control tumors systemically

Danny N. Khalil1, Luis Felipe Campesato1, Yanyun Li2, Taha Merghoub2, Jedd D. Wolchok3

1Memorial Sloan Kettering Cancer Center, New York, NY, USA; 2Ludwig Collaborative Laboratory, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 3Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Correspondence: Danny N. Khalil (khalild@mskcc.org)

Background

Multiple cancer types resistant to immune checkpoint blockade (i.e., anti-PD-1, PD-L1, and/or CTLA-4) also demonstrate impaired antigen presenting cell (APC) activation. We hypothesized that intratumoral administration of agents designed to enforce APC activation would convert a living tumor into a source of immunogenic APCs capable of priming anti-tumor T cells.

Methods

Using a checkpoint-blockade resistant syngeneic B16 murine model with established (0.5 - 1 cm) bilateral tumors, we screened and characterized agents associated with APC activation for their ability to overcome resistance to anti-PD-1 therapy. Such agents were administered either systemically, or intratumorally to one of the two tumors, allowing us to distinguish the effect at the injected tumor from that at the contralateral tumor.

Results

In the setting of PD-1 blockade, we found that intratumoral treatment with the TLR4 agonist monophosphoryl lipid A (MPL) and low-dose CD40 agonist monoclonal antibody (mAb) induces an anti-tumor T cell response. CD8+ T cells subsequently infiltrate and control noninjected tumors at a distant site. Interestingly, locally injected tumors were heavily infiltrated with neutrophils expressing costimulatory markers including CD86 within 3 hours of treatment, and then rapidly regressed. In addition, there was persistence of activated dendritic cells and monocytes in the injected-tumor’s draining lymph node. Within 1 week, distant tumors were infiltrated with activated CD8+ T cells, and showed a marked increase in the T effector to T regulatory ratio. The control of distant tumors was abolished in RAG1-deficient animals lacking lymphocytes. Cured animals fully resisted tumor re-implantation at 90 days, developing fur depigmentation at both the site of initial treatment and the untreated tumor reimplantation site, but not elsewhere, suggesting a highly specific anti-tumor response. Notably, systemic administration of MPL and CD40 at 25-fold higher dose was less effective than intratumoral treatment.

Conclusions

In conclusion, low-dose intratumoral treatment with combined TLR4 and CD40 agonists induces anti-tumor T cells which in turn infiltrate tumors at distant sites and provide durable immunity such that animals are resistant to tumor re-implantation. Given that this regimen relies on agents that are FDA-approved for other indications, or in clinical development, it can readily be translated into clinical trials across a broad range of malignancies that are currently refractory to immunotherapy.

P219 CA-170, a first in class oral small molecule immune checkpoint antagonist, promotes T cell immune activation and inhibits tumor growth in pre-clinical models of cancer

Adam S Lazorchak1, Troy D Patterson1, Yueyun Ding1, Pottayil Sasikumar2, Naremaddepalli Sudarshan2, Nagaraj Gowda2, Raghuveer Ramachandra2, Dodheri Samiulla2, Sanjeev Giri2, Rajesh Eswarappa2, Murali Ramachandra2, David Tuck1, Timothy Wyant1

1Curis, Inc., Lexington, MA, USA; 2Aurigene Discovery Technologies Limited, Bangalore, Karnataka, India
Correspondence: Adam S Lazorchak (alazorchak@curis.com)

Background

Antibody-mediated immune checkpoint blockade has transformed cancer therapy. However, the majority of patients fail to respond to antibody therapies targeting single immune checkpoint pathways and antibodies exhibit a long in vivo half-life which may contribute to the emergence of immune-related adverse events. Additionally, antibody therapies must be administered by intravenous infusion in a hospital or clinic setting, which places additional burden on patients who may have mobility challenges. CA-170 is a small molecule, orally bioavailable antagonist of the PD-L1, PD-L2 and VISTA/PD-1H immune checkpoint pathways, currently undergoing phase I clinical testing. CA-170 was developed through a rational design and a screening strategy which identified small molecules that could antagonize T cell suppression independently mediated by PD-L1, PD-L2 and VISTA/PD-1H in functional assays.

Methods

CA-170 inhibition of the PD-1/PD-L1/2 or VISTA/PD-1H signaling has been inferred though in vitro T cell effector function rescue studies using human, monkey or mouse cells stimulated in the presence of inhibitory PD-L1, PD-L2 or VISTA/PD-1H proteins. CA-170 selectivity was tested against the related inhibitory immune checkpoint pathways CTLA-4, LAG-3, BTLA or the immune co-stimulatory B7/CD28 pathway in functional assays. CA-170 in vivo antitumor activity and immune stimulatory activity was tested in multiple syngeneic mouse tumor models. Definitive toxicology and pharmacokinetic profiling studies were performed in mouse and cynomolgus monkey.

Results

CA-170 exhibits potent immune rescue activity, comparable to that of blocking PD-1 or VISTA/PD-1H antibodies when tested in cell culture assays that measure the proliferation or IFN-γ secretion of T lymphocytes stimulated in the presence of inhibitory PD-L1, PD-L2 or VISTA/PD-1H proteins. CA-170 does not exhibit off target activity against CTLA-4, LAG-3, BTLA pathways or the B7/CD28 pathway in functional assays. In immune competent mice, orally administered CA-170 inhibits the growth of syngeneic tumors, enhances peripheral T cell activation, and promotes the immune activation of tumor infiltrating CD8+ T cells in a dose dependent manner. In preclinical safety studies conducted in rodents and non-human primates, orally administered CA-170 shows no signs of toxicity when dosed up to 1000 mg/kg for 28 consecutive days. CA-170 exhibits an oral bioavailability of approximately 40 % and <10 % in mouse and monkey, respectively, and the plasma half-life ranges from approximately 0.5 hours for mouse to approximately 3.25-4.0 hours for cynomolgus monkey.

Conclusions

These non-clinical data provide a strong rationale for the continued clinical development of CA-170, the first oral, small molecule immune checkpoint antagonist for the treatment of advanced cancers.

P220 Combination of local immunotoxins with CTLA-4 blockade eradicates murine tumors by promoting anti-cancer immunity

Jasmin Leshem1, Xiu-fen Liu1, Tapan Bera1, Masaki Terabe1, Birgit Bossenmaier2, Gerhard Niederfellner2, Yoram Reiter3, Ira Pastan1

1National Cancer Institute, NIH, Bethesda, MD, USA; 2Roche Pharmaceutical Research &Early Development, Discovery Oncology, Innovation Center Penzberg, Roche Diagnostics GmbH, Penzberg, Germany; 3Technion Institute, Haifa, Iceland
Correspondence: Jasmin Leshem (jasmin.leshem@nih.gov)

Background

Immune check point blockade therapy using antibodies to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) benefits only a limited number of cancer patients. Combination therapies are being pursued to augment the immune activation and drug efficacy. SS1P and RG7787 are immunotoxins that consist of an anti-mesothelin antibody fragment genetically fused to a portion of Pseudomonas exotoxin A. We previously observed in patients delayed onset of responses to SS1P treatment that persisted long after discontinuation of the drug. This observation led us to hypothesize that immunotoxins elicit anti-tumor immunity that can be further potentiated by adding anti-CTLA-4 antibodies (aCTLA-4).

Methods

To test our hypothesis, we constructed 66C14-M murine breast cancer cell line expressing human mesothelin on its cell surface. The cells were grown in BALB/c mice transgenic for human mesothelin, because they were rejected by wild type mice. RG7787 or SS1P were injected directly into established tumors (average size >80 mm3) and aCTLA-4 was administered IP.

Results

We found that the combination of aCTLA-4 with RG7787 or SS1P induced complete remissions in 23 out of 38 mice treated (60 %) providing a significant survival benefit compared to mono-therapy (P < 0.001). No cures were obtained when aCTLA-4, RG7787, or SS1P were given separately. In addition, we found that responding mice treated with aCTLA-4 and SS1P had more abundant tumor-infiltrating CD8+ T cells compared to mice treated with aCTLA-4 or SS1P alone (P < 0.05) and that the response was blocked when mice were treated with anti-CD8 antibodies. Furthermore, 22 out of the 23 surviving mice rejected an additional tumor challenge with the same number of 66C14-M or the parental cells (no human mesothelin) implanted 45 days after the mice were cured. These findings point to immune mediated tumor regression. To explore the mechanism responsible for the anti-tumor effect, we combined aCTLA-4 with a mutant RG7787 that is unable to kill 66C14-M cells and found that the survival of mice was not significantly better than that achieved with aCTLA-4 monotherapy. Some bacterial products activate the immune system by receptors that directly recognize microbe associated molecular patterns (MAMPs). However, our result indicates that MAMP recognition does not explain our findings.

Conclusions

Combining intra-tumoral injection of immunotoxins with systemic administration of aCTLA-4 induced a high rate of immune mediated tumor regression. Our findings provide the first preclinical evidence to support use of this combination in patients.

P221 Adjuvant effect of anti-PD-L1 in boosting HER2-targeted T cell adoptive immunotherapy

Leiming Xia1, Yang Xia1, Yangyang Hu1, Yi Wang2, Yangyi Bao2, Fu Dai2, Shiang Huang3, Elaine Hurt4, Robert E Hollingsworth4, Lawrence G Lum5, Alfred E Chang1, Max S Wicha6, Qiao Li7

1University of Michigan, Ann Arbor, MI, USA; 2The No.1 People’s Hospital of Hefei, Hefei, Anhui, People’s Republic of China; 3Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 4MedImmune Inc., Gaithersburg, MD, USA; 5University of Virginia Cancer Center, Charlottesville, VA, USA; 6University of Michigan Medical School, Ann Arbor, MI, USA; 7University of Michigan Medical Center, Ann Arbor, MI, USA
Correspondence: Qiao Li (qiaoli@med.umich.edu)

Background

Adoptive immunotherapy utilizing anti-CD3 x anti-HER2 bispecific antibody (HER2Bi)-armed T cells benefited both HER2+ patients and patients with 1 or 2+ HER2 expression, ones that would be considered “HER2-negative” by classical criteria. We have also shown that the level of cancer stem cell (CSC) marker ALDH in HER2+ breast cancer cells (ALDHhighHER2+) is much higher than that in HER2 breast cancer cells (ALDHlowHER2), and that in luminal breast cancers that are considered HER2, HER2 is actually selectively expressed in the ALDHhigh CSC population. These observations might account for the surprising result that HER2Bi-armed T cells, while intended to target HER2, seemed to benefit HER2 patients after adoptive transfer.

Methods

We tested the “mouse HER2” (neu) expression on ALDHhigh vs. ALDHlow 4 T1 cells (mouse TNBC). For mHER2 targeting in animal models, we generated anti-mouse HER2-CD3 bispecific (mHER2Bi) that binds to mouse HER2 and mouse CD3.

Results

HER2Bi-armed T cells used in the clinical trial killed ALDHhigh human breast CSCs isolated from MCF7 (HER2) tumor significantly more than ALDHlow MCF7 cells in vitro, while the same HER2Bi-armed T cells killed ALDHhigh human breast CSCs (ALDHhighHER2+) isolated from BT474 (HER2+) tumor equally to ALDHlow BT474 cells (ALDHlowHER2+). We also found that mHER2 was selectively expressed in the ALDHhigh 4 T1 CSC population. These results replicated our findings in human breast cancers that HER2 is selectively expressed on CSCs, even in HER2 murine tumors, such as 4 T1. In vitro, the mHER2Bi-armed T cells killed ALDHhigh 4 T1 CSCs significantly more than ALDHlow 4 T1 cells. In vivo, adoptive transfer of mHER2Bi-armed T cells for HER2-targeted therapy showed antitumor effect in mHER2 4 T1-bearing host. Administration of anti-mouse PD-L1 during mHER2Bi-armed T cell adoptive transfer decreased metastases significantly more than the use of either strategy alone.

Conclusions

These studies have generated evidence providing proof of principle that due to the selective expression of HER2 on CSCs, HER2-targeted T cell therapy could benefit HER2 hosts as well as HER2+ hosts via immune destruction of HER2+ CSCs, and use of anti-PD-L1 could significantly boost the efficacy of HER2-targeted T cell therapy.

P222 Combining IL-6 blockade with novel targeted therapeutics in pancreatic cancer

Thomas Mace, Neil Makhijani, Erin Talbert, Gregory Young, Denis Guttridge, Darwin Conwell, Gregory B Lesinski

The Ohio State University, Columbus, OH, USA
Correspondence: Thomas Mace (thomas.mace@osumc.edu)

Background

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer in America with few efficacious therapeutic options other than surgery. PDAC is characterized by dense and heterogeneous stroma that secretes elevated levels of the proinflammatory cytokine interleukin-6 (IL-6). Our laboratory has previously reported that higher IL-6 in PDAC patients is strongly associated with poor overall survival. Additionally, patients with pancreatic and gastrointestinal cancers have the highest incidence of cachexia. This syndrome, characterized by the loss of skeletal muscle and adipose tissue, cannot be reversed by nutritional intervention and is mediated impart by IL-6 signaling. Further, work completed by our group and others have also shown that IL-6 and other factors can promote cross-talk between the STAT3 and MEK pathways. Thus, we hypothesized that IL-6 blockade can be utilized to enhance the efficacy of novel immune or targeted therapeutics (anti-PD-L1 and cobimetinib) in pancreatic cancer.

Methods

In vivo efficacy studies were conducted with antibodies (Ab) blocking IL-6, in combination with checkpoint immunotherapy (anti-PD-L1) or MEK inhibition (cobimetinib). Experiments were conducted in mice bearing subcutaneous KPC-derived MT5 tumors; orthotopically injected KPC-luciferase expressing tumor cells in the pancreas; and Colon26 tumor bearing CD2F1 mice to determine effects on cancer cachexia.

Results

IL-6 blockade combined with anti-PD-L1 (p < 0.02) or cobimetinib (p = 0.007) elicited anti-tumor efficacy in mice bearing subcutaneous KPC derived MT5 tumors, compared to vehicle controls. IL-6 blockade in combination with anti-PD-L1 antibodies limited tumor growth of orthotopic KPC-luciferase expressing tumor cells compared to isotype controls (p = 0.05). As a pancreatic cachexia model is not currently available, we tested IL-6 blockade in combination with cobimetinib on a classically accepted tumor cachexia model (CD2F1 mice bearing Colon26 tumors). Only mice treated with cobimetinib or the combination of IL-6 plus cobimetinib resulted in significant tumor inhibition compared to IL-6 alone or vehicle controls (p < 0.0001). Furthermore, mice administered IL-6 alone or in combination with cobimetinib prevented tumor-induced body weight loss (p < 0.005) and protected lean mass and hind limb muscles as compared to vehicle-treated mice (p < 0.05).

Conclusions

These pre-clinical results indicate that inhibition of IL-6 may affect the efficacy of novel targeted therapeutics on tumor progression, immunosuppression, and cachexia in pancreatic cancer.

P223 Distinct chemokines and chemokine receptors control the trafficking of effector and regulatory cells into melanoma tumors in the setting of combined PD-1 and CTLA-4 blockade

Rodney JM Macedo Gonzales, Austin P Huffman, Ximi K Wang, Ran Reshef

Columbia Center for Translational Immunology, Columbia University Medical Center, New York, NY, New York, NY, USA
Correspondence: Rodney JM Macedo Gonzales (rjm2198@cumc.columbia.edu)

Background

Pharmacologic blockade of the CTLA-4 and PD-1 immune checkpoint molecules is an effective approach for cancer immunotherapy especially in melanoma, but only a subset of patients respond. Trafficking of immune regulatory cells into the tumor microenvironment creates an immunosuppressive environment which dampens the anti-tumor response. Thus, identifying the mechanisms involved in the trafficking of effector and regulatory cells is critical for the development of strategies that increase effectiveness of checkpoint blockade. We aimed to determine which trafficking molecules are involved in anti-tumor responses by studying both human and murine melanoma.

Methods

RNA sequencing data were obtained from 475 melanoma patients (The Cancer Genome Atlas database). Additionally, C57BL/6 mice were subcutaneously injected with 100,000 B16-F10 cells in the flank and sacrificed at day 14 for flow cytometry analysis. Anti-PD-1 + anti-CTLA-4 blocking antibodies or PBS were injected intraperitoneally at days 5, 8 and 11 after tumor inoculation.

Results

Analysis of RNA-seq data showed that inflammatory chemokines (CCL2, CCL5, CXCL9, CXCL10) and their receptors (CCR2, CCR5, CXCR3) were overexpressed in human melanoma tumors. Interestingly, unsupervised clustering demonstrated that CCR2, CCR5 and CCL2 were associated with CD68 and CD14 genes while CXCR3, CCL5, CXCL9 and CXCL10 were associated with CD8A, CD8B and T-bet genes. Moreover, immunophenotyping of tumor-infiltrating CD45+ cells from B16-F10 tumor-bearing mice revealed higher levels of CCR2. Interestingly, monocytic myeloid-derived suppressor cells (M-MDSCs) and inflammatory dendritic cells had the highest expression of these receptors. When B16-F10 tumor-bearing mice were treated with anti-PD-1/anti-CTLA-4 antibodies, we observed a significant reduction of tumor size and increased levels of CD45+ cells (p < 0.05), CD8+ T cells (p < 0.05) and increased CD8/Treg ratio (p < 0.01) in comparison to controls; however, the numbers of M-MDSC were not reduced. More importantly, CCR2 and CCR5 were still high within total CD45+ cells (26-30 %) and M-MDSCs (54-71 %) in both treated and control mice. Additionally, dual checkpoint blockade significantly increased the expression of CCR1 (p < 0.05) and CXCR3 (p < 0.05) in CD8+ T cells, without increasing levels of CCR2 and CCR5.

Conclusions

Our data suggest that dual checkpoint blockade increases the trafficking of CD8+ T cells into the tumor using the CXCL9/CXCL10-CXCR3 axis but does not affect the CCL2-CCR2 and CCL5-CCR5 axis that are critical for M-MDSCs trafficking into the melanoma microenvironment. These results are important for the development of novel immunotherapy combinations that harness trafficking mechanisms to improve the efficacy of immunotherapies.

P224 Targeting tumor glutamine metabolism with CB-839 enhances the efficacy of immune checkpoint inhibitors

Andy MacKinnon, Jason Chen, Matt Gross, Gisele Marguier, Peter Shwonek, Natalija Sotirovska, Susanne Steggerda, Francesco Parlati

Calithera Biosciences, South San Francisco, CA, USA
Correspondence: Andy MacKinnon (amackinnon@calithera.com)

Background

T cell activation and proliferation are metabolically demanding processes that require essential nutrients such as glucose and glutamine. Within the tumor microenvironment, competition between tumor cells and immune cells for limited nutrients can lead to poor T cell activation and suppression of an anti-tumor immune response. Engagement of immune checkpoints such as PD-1 further suppresses T cell activation. While therapeutic blockade of immune checkpoints may partially relieve T cell suppression, low nutrient availability in the tumor microenvironment is expected to limit an optimal immune response. CB-839 is a glutaminase inhibitor currently in phase I oncology trials. CB-839 blocks glutamine consumption by tumors leading to elevated glutamine levels in the tumor microenvironment. Based on the high demand of T cells for glutamine, we hypothesized that CB-839 might synergize with immune checkpoint inhibitors to relieve immune suppression and lead to enhanced anti-tumor immune responses.

Methods

Ex vivo T cell activation was performed with anti-CD3/CD28 on CD3+ cells isolated from human PBMCs. Changes in mRNA expression after T cell activation was monitored by NanoString analysis. In vivo efficacy studies were conducted in syngeneic CT-26 or B16 tumor models.

Results

T cell activation in the absence of glutamine inhibited cell proliferation and the expression of cell surface activation markers. Analysis of mRNA expression also showed suppression of normal activation markers and induction of T cell exhaustion markers including PD-1, CTLA-4 and BTLA, suggesting that T cell activation in the absence of glutamine may be sufficient to induce an exhausted phenotype. Previous work showed that CB-839 blocks glutamine consumption in tumors leading to reduced cell proliferation. Surprisingly, CB-839 had only minimal impact on T cell proliferation, highlighting differences in glutamine utilization pathways between tumor cells and T cells. In mouse tumor models, administration of CB-839 elevated tumor glutamine levels, consistent with inhibition of tumor glutaminase. Combination of CB-839 with anti PD-1 or anti PD-L1 in the syngeneic CT-26 colon model augmented tumor regressions relative to checkpoint inhibition alone. CB-839 also enhanced the anti-tumor activity of checkpoint inhibitors in the B16 melanoma model. Depletion of CD8+ T cells from tumor-bearing animals reversed the anti-tumor effects of the combination, confirming an immune-mediated mechanism of action.

Conclusions

These data highlight a novel therapeutic approach to treat cancer by selectively targeting tumor metabolism as a means of enhancing the efficacy of checkpoint blockade. Our data provide a rationale for combining CB-839 with immune checkpoint inhibitors in the clinic.

P225 Arginase inhibitor CB-1158 alleviates immunosuppression and enhances anti-tumor responses as a single agent and in combination with other immunotherapies

Amani Makkouk, Mark K Bennett, Jason Chen, Ethan Emberley, Matt Gross, Tony Huang, Weiqun Li, Andy MacKinnon, Gisele Marguier, Silinda Neou, Alison Pan, Jing Zhang, Winter Zhang, Francesco Parlati

Calithera Biosciences, South San Francisco, CA, USA
Correspondence: Amani Makkouk (amakkouk@calithera.com)

Background

T cells and natural killer (NK) cells require L-arginine for proliferation. Arginine depletion by arginase in the tumor microenvironment induces immunosuppression and is associated with tumor immune evasion. Arginase is expressed by myeloid-derived suppressor cells (MDSCs) and polymorphonuclear cells (PMNs), and its pharmacological inhibition is expected to restore arginine levels and relieve immunosuppression, leading to anti-tumor immune responses.

Methods

We developed CB-1158, a potent and selective small molecule inhibitor of arginase (IC50 = 98 nM). The activity of CB-1158 was examined ex vivo using immune cells isolated from healthy volunteers or cancer patients, and in vivo using murine syngeneic tumor models. Arginase abundance in cancer patient plasma and in tumor tissue microarrays was also examined.

Results

In a co-culture system of T cells with PMNs or MDSCs, CB-1158 reverses PMN- or MDSC-mediated immunosuppression by blocking arginine depletion, thereby allowing T cells to proliferate. T cells activated in the presence of PMN-conditioned media show suppressed production of cytokines involved in Th1-type adaptive immunity, and this effect is reversed by the addition of CB-1158. In vivo, CB-1158 has high oral bioavailability and is very well tolerated. In tumor-bearing mice, twice daily dosing of CB-1158 causes dose-dependent pharmacodynamic increases in plasma and tumor arginine levels associated with single agent anti-tumor efficacy in multiple syngeneic models. The anti-tumor efficacy of CB-1158 is abrogated in immunocompromised mice or via depletion of either CD8+ T cells or NK cells, confirming an immune-mediated mechanism of action. Moreover, CB-1158 enhances CD8+ T cell infiltration into tumors and increases expression of Th1 cytokines, T cell and NK cell activation markers, and interferon-inducible genes in the tumor. The immunomodulatory activity of CB-1158 supports the potential of its combination with other immunotherapies and/or standard-of-care therapies. CB-1158 enhances the anti-tumor efficacy of checkpoint inhibitors, including anti-PD-L1 and epacadostat in the B16F10 model. Moreover, CB-1158 enhances the anti-tumor efficacy of standard-of-care therapies such as chemotherapy. To assess the clinical potential of CB-1158, the abundance of arginase in tumors and plasma from cancer patients across multiple cancer histotypes was surveyed. Arginase-expressing PMN infiltrates are abundant in multiple tumor types. Plasma arginase levels are elevated in cancer patients compared to healthy controls, and are associated with decreased plasma arginine.

Conclusions

These results support the clinical development of CB-1158, a first-in-class arginase inhibitor, as a novel immunomodulatory agent antagonizing myeloid-mediated immunosuppression. A phase I clinical trial testing the clinical activity of CB-1158 in cancer patients has been initiated.

P226 Revealing how adoptive T cell transfer into lymphodepleted host and checkpoint blockade therapy work together to treat blood cancers

Netonia Marshall, Thomas U Marron, Judith Agudo, Brian Brown, Joshua Brody

Icahn School of Medicine at Mount Sinai, New York, NY, USA
Correspondence: Netonia Marshall (Netonia.marshall@mssm.edu)

Background

Blood cancers, with an estimated 160,000 new cases, account for nearly 10 % of all cancer diagnoses and 9.4 % of all cancer deaths this year in the United States. Unfortunately, despite therapeutic advances, the mortality rate still continues to rise. Thus, novel, mechanistically distinct therapies, such as immunotherapy, may have a significant impact particularly in addressing aggressive lymphoma subtypes such as those being modeled in our transplant-based approach. Two classes of immunotherapies that have had great success in treating a wide array of cancers are: checkpoint blockade (e.g., anti-CTLA-4 antibody-based treatments and anti-PD-1 antibody-based treatments) and adoptive T cell (e.g., TIL) transfer lymphocytes into lymphodepleted hosts.

Methods

We have developed a novel therapy combining these approaches into 'checkpoint-blockade-primed immunotransplant' comprised of: treatment of tumor-bearing host with anti-CTLA-4 and/or anti-PD-1 antibodies, and splenocyte harvest and transfer to lymphodepleted recipient.

Results

Our results show that this combined therapy results in superior anti-tumor immunity compared to either individually as seen by increased production of IFN γ positive T cells. Treatment of both tumor-bearing donor and recipient with anti-PD-1 and anti-CTLA-4 antibodies induces cure of the majority of recipients, in a CD8, NK, and IFNγ-dependent manner, despite the finding that antibody therapy alone (without transplantation and T cell transfer) induces minimal anti-tumor effect. Furthermore, we have demonstrated that T cells exposed to checkpoint blockade and transfer into the lymphopenic hosts demonstrate: greater in vivo serum levels of IL-15 and IL-7, higher ex vivo levels of IL-15R and IL-7 -receptors expression on CD8s, in vitro STAT5 phosphorylation in response to common γ-chain cytokines, in vivo proliferation in response to exposure to cognate tumor antigen, and in vitro production of IFNγ and TNF production in response to exposure to cognate tumor antigen.

Conclusions

Ongoing studies will seek to assess the dependence of the above observations (cytokine production, proliferation, anti-tumor effect) on specific common γ-chain cytokines, the role of the lymphopenia in inducing T cell trafficking to tumor versus organs, and guide development of the immunotransplant model to optimize the amplification of anti-tumor immunity observed.

P227 Immunomodulatory cytokine blockade in combination with CTLA-4 blockade in murine models of pancreatic cancer

Christopher McQuinn, Thomas Mace, Matthew Farren, Hannah Komar, Reena Shakya, Gregory Young, Thomas Ludwug, Gregory B Lesinski

The Ohio State University, Columbus, OH, USA
Correspondence: Christopher McQuinn (Christopher.mcquinn@osumc.edu)

Background

Pancreatic cancer remains a significant challenge with 5 year survival rates of less than 7 %. This devastating malignancy is expected to become the second leading cause of cancer death in the United States by 2030. Although effective in other malignancies, there has been a relative paucity of efficacy when immune checkpoint blockade has been applied in pancreatic cancer. We hypothesize this limited efficacy is due to local and systemic alterations in cytokine expression that shape the immune contexture in these patients. Although dysregulated cytokines represent attractive targets in pancreatic cancer, there are limited data to help prioritize among them for future translation. Prior studies from our group demonstrated that plasma interleukin-6 (IL-6), interleukin-10 (IL-10) and circulating CD8 + CTLA-4+ cells were correlated with overall survival in a population of n = 71 treatment naïve metastatic pancreatic cancer patients. We hypothesized that targeting IL-10 and IL-6 would augment the efficacy of antibodies targeting CTLA-4.

Methods

In vivo efficacy of blocking antibodies against IL-6, IL-10, and CTLA-4 were evaluated in C57BL/6 mice bearing syngeneic, subcutaneous murine pancreatic tumor cells derived from LSL-Kras G12D ;LSL- p53 R172H ;Pdx1-Cr and in a highly aggressive genetically engineered mouse model, harboring Kras G12D ; p53 R172H and Brca2 mutation (KPC-BRCA2). Relevant immune biomarkers were analyzed using flow cytometry or IHC, as appropriate.

Results

In vivo studies demonstrated that combined blockade of IL-6 and CTLA-4 significantly decreased the rate of tumor growth in comparison to both isotype control (P = 0.0001) and anti-CTLA-4 alone (P = 0.0207). Treatment with antibody against IL-10, or IL-10 blockade in combination with anti-CTLA-4 slowed tumor growth in comparison to isotype control but were inferior to single agent anti-CTLA-4. FACS analysis of splenocytes from these mice revealed that combined IL-6 and CTLA-4 blockade increased the proportion of circulating CD4+ central memory cells (CD62L + CD44+). Blockade of IL-6 and CTLA-4 in combination, and as single agents, resulted in an increase in circulating Th1 cells while both isotype control and anti-IL-6 had significantly more naïve systemic T cells (CD4+/CD8 + CD62L + CD44-). IHC analysis revealed increased infiltrating CD3+ cells throughout the tumor foci of the combination group in comparison to both single agents and isotype control (all P’s < 0.01). Ongoing analysis will further delineate the proportion and detailed phenotype of infiltrating and systemic immune cells.

Conclusions

Antibodies targeting IL-6 but not IL-10 augment the efficacy of anti-CTLA-4 in murine models of pancreatic cancer, modulate T cell infiltration and immune biomarkers to promote Th1 immune responses.

P228 Improvement of a therapeutic cancer vaccine in mice with the addition of a GITR-ligand fusion protein

Y Maurice Morillon1, Scott A Hammond2, Jeffrey Schlom1, John W Greiner1

1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA; 2MedImmune LLC, Gaithersburg, MD, USA
Correspondence:Y Maurice Morillon (yves.morillon@nih.gov)

Background

Breaking tolerance mechanisms to mount a durable adaptive immune response within tumors remains one of the preeminent challenges of immuno-oncology. Immunosuppressive hurdles include: 1) suppression from immunoregulatory and tumor cells, 2) insufficient intratumoral “immune space” and survival signals, and 3) exhaustion of tumor specific effector T cells (Teff). GITR, an activating receptor belonging to the tumor necrosis factor receptor (TNFR) super family, is constitutively expressed on FoxP3+ regulatory T cells (Tregs) and to a lesser extent on quiescent Teff. Activation upregulates GITR on Teff and Tregs, which however remains highest among Tregs. Thus, selectively targeting GITR can deliver activating signals to Teff cells, while depleting high-GITR-expressing Tregs, and may improve efficacy of a therapeutic cancer vaccine.

Methods

Recombinant poxviruses [modified vaccinia Ankara (rMVA-), fowlpox (rF-)] were engineered to express human CEA and murine costimulatory molecules, B7.1, ICAM-1 and LFA-3 (TRICOM); termed rMVA- or rF-CEA-TRICOM. A prime-boost strategy was utilized; the priming vaccine utilized rMVA-CEA-TRICOM while rF-CEA-TRICOM provided the boost. The diversified prime-boost vaccine regimen can break tolerance in transgenic mice expressing human CEA. GITR was targeted with a fusion protein (GITRL-FP) consisting of the extracellular domain of murine GITR-ligand molecularly fused to a trimerization domain and murine IgG2a-Fc. Murine colon adenocarcinoma cells expressing human CEA (MC32A) were implanted subcutaneously in CEA. Tg mice and treated with control IgG2a, GITRL-FP, rMVA/rF-CEA-TRICOM, or rMVA/rF-CEA-TRICOM + GITRL-FP.

Results

Initial studies paired twice weekly dosing of GITRL-FP concurrent with MVA/rF-CEA-TRICOM and modest improvements in antitumor effects resulted. GITRL-FP targets GITR expressing cells, delivering activating signals, while the IgG2a-Fc depletes via Fc-mediated effector functions. Investigation into mechanism revealed depletion of Tregs and Teff. To circumvent the problem of depleting vaccine induced Teff, administration of GITRL-FP was switched to a single dose given 2 days prior to vaccine. The short half-life of the fusion protein allowed for temporal intratumoral depletion of both Tregs and Teff. Single dose GITRL-FP abrogated the immunosuppressive constraints of Tregs and created a lymphopenic intratumoral T cell compartment. These events allowed for expansion of Teff in response to MVA-CEA-TRICOM as shown by a 20 % increase of proliferating intratumoral CD4+ Teff compared to GITRL-FP monotherapy, and a 2-fold increase in activated peripheral CD8+ Teff. Reduced tumor growth and improved survival was observed comparing combination to GITRL-FP monotherapy. Tumor-free mice were also protected against tumor rechallenge.

Conclusions

These data demonstrate the increased efficacy of utilizing targeted depletion of immunosuppression in combination with an immune boosting cancer vaccine.

P229 Elucidating the role of CD47 in innate lymphoid cell-mediated tumor therapy

Pulak R. Nath1, Anthony L. Schwartz1, Dragan Maric2, David D. Roberts1

1National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 2National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA
Correspondence: Pulak R. Nath (pulak.nath@nih.gov)

Background

CD47 is a ubiquitous cell surface receptor that interacts with the secreted protein thrombospondin-1 and its counter-receptor SIRPα on phagocytes and antigen presenting cells (APCs). CD47 is highly expressed across many cancer types, hence, representing a potential target for therapeutic intervention. We recently reported a direct role for thrombospondin-1/CD47 signaling on cytotoxic T lymphocytes (CTL) to limit target tumor cell killing [1]. Many tumors are not sufficiently immunogenic to induce protective adaptive immunity. However, innate lymphoid cells (ILCs) may also play functional roles in tumor regression [2]. Here we evaluated the role of CD47 in NK and other ILCs homeostasis within the lymphoid organs as well as among tumor infiltrating lymphocytes.

Methods

We analyzed tumor infiltration of NK and other ILCs following antisense suppression of CD47 alone or in combination with anti-CTLA-4 blockade. C57Bl/6 mice were injected with B16F10 melanoma in the hind limb, and once the tumors reached an average of 100 mm3, mice were treated with CD47-morpholino, anti-CTLA-4, or combined treatments.

Results

Treatment of mice with CD47-morpholino increased the frequencies of splenic LinCD3NK1.1+ and LinCD3CD127+ populations. Studies using CD47-null mice further validated this result. We observed higher granzyme B and perforin mRNA expression in the CD3CD4CD8 cells compared to the CD8+ cells from spleens of CD47-null mice. Image cytometric analysis revealed that these are mononuclear lymphocytes. These cells express higher eomes and T-bet, but lower Gata3 and Rorγt as compared to their CD4+ counterparts, suggesting that they fall within the NK and ILC1 lineages. Indeed, lineage-depleted splenocytes from CD47-null mice showed higher frequencies of NK1.1+ and CD127+ cells compared to wildtype littermate controls. These cells infiltrated into tumors of B16F10-bearing mice, and their numbers further increased following treatment with a combination of CD47-morpholino and anti-CTLA-4 antibody, which resulted in enhanced therapeutic benefits.

Conclusions

Our data suggest that deficiency of CD47 in the tumor microenvironment or therapeutic blockade increases subtypes of ILCs with potent anti-tumor properties. The mechanism by which CD47 controls the homeostatic balance of ILCs or their development remains to be determined.

References

1. Soto-Pantoja DR, et al.: CD47 in the tumor microenvironment limits cooperation between antitumor T-cell immunity and radiotherapy. Cancer Res 2014, 74:6771–6783.

2. Dadi S, et al.: Cancer immunosurveillance by tissue-resident innate lymphoid cells and innate-like T cells. Cell 2016, 164:365–377.

P230 Restoration of antitumor effectiveness of PD-1 inhibition in immunotherapy-resistant “cold” tumors by combinatorial treatment enhancing the numbers of tumor-specific CTLs in tumor tissues

Nataša Obermajer1, David Bartlett1, Pawel Kalinski2

1Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA; 2Department of Surgery; University of Pittsburgh Cancer Institute; Department of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, PA, USA
Correspondence: Nataša Obermajer (obermajern2@upmc.edu)

Background

Intratumoral accumulation of effector type-1 T cells (CTLs) is an independent prognostic factor of survival of patients with many cancer types and is required for the effectiveness of checkpoint blockade therapies.

Methods

In this study, we have tested whether the enhancement of the numbers of tumor-infiltrating CTLs by DC vaccines together with combinatorial reprogramming of tumor-associated chemokines, can be used to convert the nominally checkpoint-resistant “cold” tumors into PD-1-sensitive ones.

Results

In colorectal and ovarian cancer (transplantable MC38 and ID8 models) bearing mice, we observed only marginal therapeutic effect of PD-1 inhibition alone or combined with DC vaccine. However, combinatorial reprogramming of tumor-associated chemokines, using TLR3 ligand polyI:polyC12U, interferon-a and COX2 blockers, resulted in a striking increase in the numbers of tumor-infiltrating CTLs recognizing cancer-associated antigens and allowed for the conversion of these immunotherapy-resistant tumors into sensitive ones, resulting in high numbers of long-term surviving animals.

Conclusions

This combinatorial DC-based vaccination approach may be used to induce specific immune cells against different tumor-relevant antigens and may be included as a component of anti-tumor therapeutic approaches that, by themselves do not induce new effector cells, nor promote their intratumoral accumulation.

P231 Immune activation by PEGylated human IL-10 (AM0010) and anti-tumor activity in renal cancer alone and in combination with anti-PD-1

Aung Naing1, Kyriakos P Papadopoulos2, Karen A Autio3, Deborah J Wong4, Manish Patel5, Gerald Falchook6, Shubham Pant7, Patrick A Ott8, Melinda Whiteside9, Amita Patnaik2, John Mumm9, Filip Janku1, Ivan Chan9, Todd Bauer12, Rivka Colen1, Peter VanVlasselaer9, Gail L Brown9, Nizar M Tannir1, Martin Oft9, Jeffrey Infante10

1University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2South Texas Accelerated Research Therapeutics, LLC, San Antonio, TX, USA; 3Memorial Sloan Kettering Cancer Center, New York, NY, USA; 4UCLA, Los Angeles, CA, USA; 5Sarah Cannon Research Institute/Florida Cancer Specialists, Sarasota, FL, USA; 6SCRI at HealthONE, Denver, CO, USA; 7Oklahoma University, Oklahoma Citu, OK, USA; 8Dana-Farber Cancer Institute, Boston, MA, USA; 9ARMO BioSciences, Redwood City, CA, USA; 10Sarah Cannon Research Institute, Nashville, TN, USA
Correspondence: Martin Oft (martin.oft@armobio.com)

Background

IL-10 is regarded as an anti-inflammatory cytokine, but it is at least equally important for the cytotoxicity and proliferation of antigen activated CD8+ T cells. Activation of CD8+ T cells through the T cell receptor elevates IL-10 receptors and PD-1 on the cells. This provides the mechanistic rationale for combining AM0010 and anti-PD-1 for the treatment of cancer patients. A phase I clinical trial investigated the tolerability and anti-tumor activity of AM0010 alone and in combination with anti-PD-1 immune checkpoint inhibitors.

Methods

Patients with advanced RCC were treated with AM0010 (daily SC) alone or in combination with pembrolizumab (q3wk IV) or nivolumab (q2wk IV). Tumor responses were monitored following irRC. Immune responses were measured by analysis of serum cytokines, and the activation and clonality of T cells in peripheral blood mononuclear cells. Nineteen patients with RCC (15 evaluable), were treated with AMO010 alone (20 mg/kg). Eight patients were treated in combination with pembrolizumab (2 mg/kg) and 15 patients with nivolumab (3 mg/kg).

Results

AM0010 alone or in combination with anti-PD-1 was tolerated well (observation periods exceeding 16 months). All TrAEs were transient and TrAEs leading to study discontinuation were not observed. There was no colitis, pneumonitis, or endocrine disruptions. G3/4 TrAEs in monotherapy included anemia (9), hypertriglyceridemia (3), thrombocytopenia (2), ALT/AST increase (2) and fatigue (2). AM0010 combination with anti-PD-1 did not increase TrAEs. Objective responses (PR/CR) were observed in 4 of 15 evaluable RCC patients in monotherapy (27 %), in 4 of 8 patients in AM0010/pembrolizumab (50 %). Progression-free survival (PFS) was 3 and 9.4 months, respectively. The AM0010/nivolumab cohort is currently in progress. AM0010 alone and also in combination with anti-PD-1 increased Th1 cytokines (IL-18, IFNg, TNFa), CD8+ T cell associated effector molecules such as FasL and LymphotoxinB as well as cytokines stimulating T cell proliferation (IL-4, IL-7). As a result, the number and proliferation of activated, PD-1+/LAG-3+ CD8+ T cells in the blood of patients were increased on AM0010. In contrast, the proliferation of FoxP3+ Tregs and TGFb was decreased. AM0010 alone or with anti-PD-1 induced oligoclonal expansion of T cell clones in the blood without affecting total lymphocyte counts. In particular, selected T cells clones previously not detected in the blood of patients before treatment were strongly expanded (de novo amplification).

Conclusions

AM0010 alone or in combination with anti-PD-1 is well-tolerated. The clinical activity and the observed CD8+ T cell activation encourages the continued exploration of AM0010 in phase III studies.

Trial Registration

ClinicalTrials.gov identifier NCT02009449.
Fig. 18 (abstract P231).

See text for description

Fig. 19 (abstract P231).

See text for description

P232 Initial experience administering BMS-986016, a monoclonal antibody that targets lymphocyte activation gene (LAG)-3, alone and in combination with nivolumab to patients with hematologic and solid malignancies

Evan Lipson1, Ajay Gopal2, Sattva S Neelapu3, Philippe Armand4, Stephen Spurgeon5, John P Leonard6, F Stephen Hodi4, Rachel E Sanborn7, Ignacio Melero8, Thomas F Gajewski9, Matthew Maurer10, Serena Perna10, Andres A Gutierrez11, Raphael Clynes10, Priyam Mitra10, Satyendra Suryawanshi10, Douglas Gladstone1, Margaret K Callahan12

1Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Seattle Cancer Care Alliance, University of Washington, Seattle, WA, USA; 3University of Texas MD Anderson Cancer Center, Houston, TX, USA; 4Dana-Farber Cancer Institute, Harvard University, Boston, MA, USA; 5Center for Hematologic Malignancies, Oregon Health and Sciences University, Portland, OR, USA; 6New York Presbyterian Hospital, Weill Cornell Medical College, New York, NY, USA; 7Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Cancer Center, Portland, Oregon, USA, Portland, OR, USA; 8Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Navarra, Spain; 9University of Chicago Medical Center, Chicago, IL, USA; 10Bristol-Myers Squibb, Princeton, NJ, USA; 11Bristol-Myers Squibb, Lawrence Township, NJ, USA; 12Memorial Sloan Kettering Cancer Center, New York, NY, USA
Correspondence: Evan Lipson (evanlipson@jhmi.edu)

Background

LAG-3 is a transmembrane receptor that negatively regulates T cell activation. Signaling through LAG-3 and other T cell inhibitory receptors, including programmed death-1 (PD-1), can lead to T cell exhaustion and is a mechanism of immune escape for tumors. Preclinical data suggest that simultaneous blockade of LAG-3 and PD-1 may function synergistically to restore T cell activation and mediate tumor regressions. Here, we describe preliminary first-in-human phase I/IIa data for BMS-986016, a fully human IgG4 monoclonal antibody that targets LAG-3, alone and in combination with nivolumab (anti-PD-1) in patients with advanced B cell malignancies or solid tumors.

Methods

Sequential cohorts received BMS-986016 ± nivolumab every 14 days in 56-day cycles during dose escalation or expansion until disease progression, completion of 12 cycles, or prohibitive toxicity. Primary objectives included safety and tolerability.

Results

As of May 2016, 89 patients had received BMS-986016 alone (20 mg [n = 8], 80 mg [n = 13], 240 mg [n = 24], or 800 mg [n = 15]) or with nivolumab (BMS-986016/nivolumab; 20/80 mg [n = 7], 20/240 mg [n = 9], 80/240 mg [n = 9], or 240/240 mg [n = 4]). The pharmacokinetic characteristics of BMS-986016 were assessed across dose levels in patients treated with monotherapy and combination therapy. Anti-drug antibody assessments suggested low immunogenicity. Increases in peripheral blood T cell LAG-3 receptor occupancy (RO; 74-99 %) were observed with escalating BMS-986016 dose and exposure. The maximum tolerated dose (MTD) was not reached with BMS-986016 monotherapy; evaluations to determine the MTD for the combination are ongoing. Infrequent and manageable treatment-related adverse events (TRAEs) were observed across monotherapy doses (Fig. 20), and included toxicities typically associated with immune checkpoint blocking agents. DLTs among patients receiving combination therapy included grade (G)3 mucositis, G4 ventricular fibrillation, G4 elevated lipase, and G4 myocarditis. Most TRAEs were grade 1–2. TRAEs leading to discontinuation of therapy were reported in 3 % (BMS-986016) and 14 % (BMS-986016 + nivolumab) of patients. There were no treatment-related deaths. Objective tumor regression was observed with LAG-3 monotherapy, and with combination therapy in PD-1-naive patients and in patients with disease progression on nivolumab monotherapy.

Conclusions

BMS-986016 monotherapy was well tolerated at the dose levels tested. Emerging data characterizing the safety of the combination will be presented. BMS-986016 ± nivolumab demonstrated biological activity as evidenced by toxicities characteristic of immune checkpoint blockers and objective tumor regressions. These preliminary data support the ongoing evaluation of this combination in patients with solid tumors and hematologic malignancies.

Trial Registration

ClinicalTrials.gov identifier NCT02061761 and NCT01968109.
Fig. 20 (abstract P232).

TRAEs reported in > 2 patients or any TRAE ≥ grade 3 reported in patients treated with BMS-986016 ± nivolumab

P233 The effects of combination treatment of IMM-101, a heat-killed whole cell preparation of Mycobacterium obuense (NCTC 13365) with checkpoint inhibitors in pre-clinical models

James Crooks1, Sheila Brown1, Audrey Gauthier2, Marc Hillairet de Boisferon2, Andrew MacDonald1, Laura Rosa Brunet3

1MCCIR, University of Manchester, Manchester, England, UK; 2Oncodesign, Dijon, Bourgogne, France; 3Immodulon Therapeutics Ltd, Uxbridge, England, UK
Correspondence: Rosa Brunet (lrb@immodulon.com)

Background

While harnessing the power of the immune system to control cancer is becoming established as an effective way of treating patients, it has become increasingly clear that transformed cells exploit a number of mechanisms to escape such control. Hence, while the clinical use of checkpoint inhibitors (CPI) has yielded significant success, there is mounting evidence to suggest that combination treatment of CPI with immunomodulating therapies may further benefit cancer patients. Immodulon Therapeutics is developing IMM-101, an immunotherapeutic agent based on a heat-killed whole cell preparation of Mycobacterium obuense (NCTC 13365), which modulates systemic immune responses, as an adjunctive immunotherapy for cancer. Based on exposure data in over 300 patients, alone and in combination, IMM-101 is well-tolerated. Additionally, extended overall survival and progression-free survival were observed in IMAGE-1, a randomized open-label, phase II, first-line, proof of concept study (NCT01303172), in combination with gemcitabine in advanced pancreatic ductal adenocarcinoma.

Methods

We found that in vitro exposure of IMM-101 primes in vitro generated murine dendritic cells (DC) and human monocyte-derived DC in a dose dependent manner and functionally affects DC by enhancing their ability to process and present antigen. Moreover, IMM-101 activated DC promote T cell secretion of IFN-γ following re-stimulation of draining lymph node cell preparations, 7 days after adoptive transfer of IMM-101 primed DC into naïve recipient mice. We also investigated whether the effects of IMM-101 on innate and adaptive immune responses indeed improve on the therapeutic benefit of CPI treatment (anti-CTLA-4 or anti-PD-1) in two murine xenograft models using B16-F10, a mouse melanoma cell line, and EMT6, a mouse breast cell line.

Results

We assessed effects on tumor burden and local and systemic immunological bias in treated mice. We report a significant benefit from combination treatment of CPI and IMM-101 on tumor burden. We also observed significant change to the CD8+/Treg ratio at the tumor site. We performed in vitro stimulation (antigenic as well as polyclonal) of immune cells present at the tumor site, in the draining lymph nodes and in the spleen. We report results at different time points over the course of the disease.

Conclusions

On the basis of these promising results, formal clinical evaluation of IMM-101 in combination treatment with anti-PD-1 treatment is being undertaken (EudraCT identifier 2016-001459-28).

P234 Intrathecal AAV9.trastuzumab for both tumor prophylaxis and treatment extends survival in a murine xenograft model of HER2+ human breast cancer brain metastasis

William T. Rothwell, Peter Bell, James M. Wilson

University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
Correspondence: William T. Rothwell (rothw@mail.med.upenn.edu)

Background

Breast cancer brain metastases (BCBM) occur in up to 14.3 % of patients with human epidermal growth factor receptor 2 positive (HER2+) primary tumors [1]. Intravenous trastuzumab (anti-HER2 monoclonal antibody (mAb), Herceptin®) extends survival in patients with HER2+ systemic disease but does not cross the blood brain barrier (BBB) to treat HER2+ BCBM effectively [2]. Intrathecal (IT) trastuzumab can extend survival in patients with HER2+ BCBM [3] but requires regular IT infusions which carry risks and can compromise quality of life. Gene therapy offers a one-shot solution for mAb delivery across the BBB. Adeno-associated viral vectors, particularly serotype 9 (AAV9), can safely and efficiently deliver exogenous genes (transgenes) to central nervous system tissues after a single IT administration, resulting in constitutive, long-term expression of the transgene product [4].

Methods

We characterize a xenograft model of HER2+ BCBM using BT474.M1 human ductal carcinoma cells injected stereotaxically into the brain parenchyma of Rag1−/− mice. AAV9.trastuzumab is delivered IT as tumor prophylaxis (at least 21 days before tumor administration) or as tumor treatment (3 days post tumor administration).

Results

Median survival (MS) of Rag1−/− mice receiving IT AAV9.trastuzumab tumor prophylaxis (MS = 111 days, n = 7) is significantly greater after tumor administration than mice receiving vehicle (MS = 48.5 days, n = 8, p = 0.0012*), AAV9 expressing an irrelevant antibody (MS = 54.5 days, n = 10, p = 0.0027*), or AAV9 without a transgene (MS = 50 days, n = 4, p = 0.0069*). MS of mice bearing tumors treated with IT AAV9.trastuzumab (MS = 82 days, n = 6) is significantly greater than controls receiving vehicle (MS = 61 days, n = 7, p = 0.002*). *Log-rank (Mantel-Cox) test.

Conclusions

IT AAV9.trastuzumab as both tumor prophylaxis and treatment increases survival in a murine xenograft model of HER2+ BCBM, thus showing promise as HER2+ BCBM treatment and, more broadly, as a prophylactic measure for patients with HER2+ primary disease to extend survival in the case of BCBM.

References

1. Kennecke H, et al.: Metastatic behavior of breast cancer subtypes. J Clin Oncol 2010, 28:3271–3277.

2. Koo T, Kim I: Brain metastasis in human epidermal growth factor receptor 2-positive breast cancer: from biology to treatment. Radiat Oncol J 2016, 34:1–9.

3. Zagouri F, Sergentanis T: Intrathecal administration of trastuzumab for the treatment of meningeal carcinomatosis in HER2-positive metastatic breast cancer: a systematic review and pooled analysis. Breast Cancer Res 2013, 139:13–22.

4. Hinderer C, et al.: Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna. Mol Ther Methods Clin Dev 2014, 1:14051.

P235 Immunotherapy of head and neck squamous cell cancers with synthetic TLR agonists and checkpoint inhibitors in preclinical models

Fumi Sato-Kaneko1, Shiyin Yao1, Shannon S. Zhang2, Dennis A. Carson1, Cristina Guiducci2, Robert L. Coffman2, Kazutaka Kitaura3, Takaji Matsutani3, Ryuji Suzuki3, Tomoko Hayashi1, Ezra E.W. Cohen1

1Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA; 2Dynavax Technologies, Berkeley, CA, USA, Berkeley, CA, USA; 3Repertoire Genesis Incorporation, Osaka, Japan, Ibaraki, Osaka, Japan
Correspondence: Fumi Sato-Kaneko (fukaneko@ucsd.edu)

Background

Head and neck squamous cell cancers (HNSCC) constitute the sixth leading cancer by incidence worldwide. Though PD-1/PD-L1 blockade is effective in some patients, the majority do not benefit. We examined combination therapy with anti-PD-1 and synthetic agonists of toll-like receptors (TLR)7 and TLR9 in mouse models representing human papilloma virus (HPV)-positive and HPV-negative HNSCC, respectively. We hypothesized that the intratumoral treatment with TLR agonists could activate innate immune cells in the tumor microenvironment and enhance tumor specific adaptive immunity. Furthermore, this would be synergistic with checkpoint inhibitors that release negative signals on tumor infiltrating CD8+ T cells.

Methods

Syngeneic tumor mouse models, SCC-7 cells (HPV-negative)/C3H background and MEER cells (HPV-positive)/C57BL/6 background, were used. Mice were implanted with tumor cells subcutaneously into opposite flanks. Treatments were started with intratumoral injections into only the right side with TLR7 or TLR9 agonists with or without intraperitoneal injections of anti-PD-1 mAb. Lymphocytes were isolated from tumors and spleens on days 13 and 21 post tumor implantation, and were analyzed using flow cytometry. The T cell receptor (TCR) repertoire of CD8+ T cells in the tumor and the spleen was evaluated by unbiased high throughput quantitative sequencing.

Results

In both HPV-negative and HPV-positive models, the combination therapies of intratumoral TLR7 or TLR9 agonists with anti-PD-1 suppressed tumor progression both at agonist-injected and uninjected sites (abscopal-like effect) (Fig. 21). In the HPV-negative model, the combination treatment with TLR7 agonists and anti-PD-1 increased the M1/M2 ratio in CD11b+F4/80+ tumor infiltrating macrophages (Fig. 22). Ex vivo treatment with TLR7 agonist upregulated the expression of costimulatory molecules CD40, CD80, and decreased the expression level of CD206 (M2-macrophage marker). The combination therapy with TLR7 agonist increased the frequency of CD8+ T cells in both sides of tumors and spleen. Elevated IFNγ+ activated T cell population was observed in mice treated with the TLR7 ligand and anti-PD-1 therapy (Fig. 23). TCR repertoire analysis showed anti-PD-1 increased clonal expansion of splenic CD8+ T cells (Fig. 24).

Conclusions

The combination therapy with TLR agonists and anti-PD-1 suppressed progression of tumors in both injected and distant sites by two different mechanisms of action; clonal expansion of low frequency CD8+T cell population by anti-PD-1, and recruitment and activation of tumor specific T cells by intratumoral treatment with TLR ligands.

Acknowledgements

We thank Dr. John Lee in Sanford Research, who kindly provided us with HNC cells. This work was supported by the Fernanda and Ralph Whitworth Immunotherapy Foundation.
Fig. 21 (abstract P235).

Therapeutic effect of intratumoral TLR7 and TLR9 agonist and anti-PD-1 in HPV negative HNC

Fig. 22 (abstract P235).

Increased M1/M2 ratio by combination therapy with TLR7 agonist and anti-PD-1 in HPV negative HNC

Fig. 23 (abstract P235).

Increased IFNγ+ activated CD8+ T cell population by combination therapy

Fig. 24 (abstract P235).

Anti-PD-1 increased clonal expansion of splenic CD8+ T cells

P236 Modulating the intra-tumor immune balance through combinatorial blockade of CSF-1R and PD-L1 enhances anti-tumor efficacy

David Schaer1, Yanxia Li1, Julie Dobkin1, Michael Amatulli1, Gerald Hall1, Thompson Doman2, Jason Manro2, Frank Charles Dorsey2, Lillian Sams2, Rikke Holmgaard1, Krishnadatt Persaud1, Dale Ludwig1, David Surguladze1, John S Kauh3, Ruslan Novosiadly1, Michael Kalos1, Kyla Driscoll1

1Eli Lilly and Company, New York, NY, USA, 2Eli Lilly and Company, Indianapolis, IN, USA; 3Eli Lilly and Company, Bridgewater, NJ, USA
Correspondence: David Schaer (schaer_david@lilly.com)

Background

Multiple mechanisms are involved in establishing an immunosuppressive tumor microenvironment. Although blockade of the PD-1/L1 axis alone has led to durable clinical responses in multiple malignancies, the majority of patients do not receive or maintain clinical benefit from monotherapy. Colony stimulating factor receptor 1 (CSF-1R) expressing tumor associated macrophages (TAMs) have been implicated as a poor prognostic factor in many cancers. TAMs and other suppressive myeloid cells may represent an additional suppressive axis present in many malignancies where PD-1/L1 blockade has shown some or little activity. CSF-1R has been implicated for maintaining TAM function and viability in tumor tissues, making it an attractive target to modulate TAM and possibly myeloid mediated suppression in cancer. As the CSF-1R Inhibitor LY3022855 has recently entered clinical testing in combination with PD-L1 blockade, it is important to understand how inhibiting two immune suppressive mechanisms will alter immune function to help guide rational clinical development.

Methods

To study and understand the immune modulating effects of CSF-1R inhibition, we developed an anti-mouse CSF-1R surrogate antibody CS7. CS7 blocks CSF-1 binding to CSF-1R and inhibits in vitro proliferation and differentiation of macrophages and depletes tissue resident macrophages in vivo.

Results

Monotherapy treatment with CS7 causes intra-tumor depletion of ~50-60 % of F4/80+ TAMs leading to a modest delay in tumor growth. This reduction was associated with an increased intra-tumor immune inflammation signature and reduced inhibitory metabolites, highlighting the role TAMs play suppressing the immune response inside the tumor microenvironment. Combining CSF-1R blockade with anti-PD-L1 enhances the control of tumor growth, displaying a late combinatorial effect leading to complete regressions in the majority of mice (~60 %). Mice achieving complete regressions developed immunologic memory resisting rechallenge over 60 days after cessation of therapy. Intra-tumor gene expression analysis demonstrated a synergistic increase in T cell activation and reduction of immune suppression late in the response, correlating with the time point of increased efficacy. Effects of CS7 were dose-dependent, suggesting that while lower doses of CS7 are able to cause TAM depletion, modulation of the microenvironment requires more complete block of CSF-1R.

Conclusions

Our results demonstrate that combination of CSF-1R blockade with PD-L1 checkpoint inhibition alters the tumor microenvironment in favor of enhanced immune activation. In addition, our data imply that the mechanism of CSF-1R blockade immunotherapy may extend beyond reduction of intra-tumor macrophages.

P237 A combination study of an intravenously delivered oncolytic virus, coxsackievirus A21 in combination with pembrolizumab in advanced cancer patients: phase Ib KEYNOTE 200 (STORM study)

Hardev Pandha1, Christy Ralph2, Kevin Harrington3, Brendan Curti4, Rachel E Sanborn5, Wallace Akerley6, Sumati Gupta6, Alan Melcher7, David Mansfield7, David R Kaufman8, Emmett Schmidt8, Mark Grose9, Bronwyn Davies9, Roberta Karpathy9, Darren Shafren9

1University of Surrey, Guildford, England, UK; 2St James University Hospital, Leeds, England, UK; 3Institute for Cancer Research, London, England, UK; 4Providence Cancer Center, Portland, OR, USA; 5Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Cancer Center, Portland, Oregon, USA; 6Huntsman Cancer Institute, Salt Lake City, UT, USA; 7Institute for Cancer Research, London, England, UK; 8Merck & Co., Inc., Kenilworth, NJ, USA; 9Viralytics Ltd., Sydney, New South Wales, Australia
Correspondence: Darren Shafren (darren.shafren@viralytics.com)

Background

Coxsackievirus A21 (CVA21, CAVATAK) is a naturally-occurring ICAM-1 targeted oncolytic immunotherapeutic virus. Pembrolizumab is a human programmed death receptor-1 (PD-1) blocking antibody that has yielded significant solid tumor responses via reversal of tumor induced T cell suppression. Preclinical studies in immune-competent mouse models of non-small cell lung cancer (NSCLC) and melanoma confirmed that combinations of i.v. CVA21 + anti-PD-1 mAbs mediated significantly greater antitumor activity compared to use of either agent alone. We postulate that the combination of CVA21 + pembrolizumab may translate to a similar benefit in the clinic. We describe a phase Ib study assessing safety and efficacy of IV CVA21 ± pembrolizumab in advanced cancer patients (pts).

Methods

The phase I STORM (systemic treatment of resistant malignancies; KEYNOTE 200) primary objectives are to assess dose-limiting toxicities (DLT) of CVA21 ± pembrolizumab. Secondary objectives are to assess ORR by irRECIST 1.1 criteria, PFS, and OS. Treatment Part A: pts were infused with CVA21 in 100 mL saline in Cohort 1 (n = 3), at a dose of 1 x 108 TCID50, in Cohort 2 (n = 3) at a dose of 3 x 108 TCID50 and in Cohort 3 (n = 10) at a dose of 1 x 109 TCID50 on study days 1, 3, 5, 22 and Q3W for 6 additional infusions. Part A enrollment is complete. Treatment Part B: pts are infused with CVA21 in 100 mL saline + pembrolizumab. In Cohort 1 (n = 3), CVA21 is administered at a dose of 1 x 108 TCID50, in Cohort 2 (n = 3) at a dose of 3 x 108 TCID50 and in Cohort 3 (n = ~80) at a dose of 1 x 109 TCID50 on study days 1, 3, 5, 8, 29, and Q3W for 6 additional infusions. Pembrolizumab is given in all cohorts at 200 mg IV Q3W from Day 8 for up to 2 years. Treatment with CVA21 ± pembrolizumab will continue until confirmed CR or PD (whichever comes first) per irRECIST 1.1 or DLT. Part B, Cohort 1 enrollment is complete.

Results

IV delivery of CVA21 to all patients in Part A was generally well tolerated, with no Grade 3 or 4 product-related AE’s.

Conclusions

CVA21 tumor targeting in patients with melanoma, NSCLC, and bladder cancer patients in Part A Cohort 3 was confirmed by detection of CVA21 viral RNA in tumor biopsies at study Day 8.

Trial Registration

ClinicalTrials.gov identifier NCT02043665.

P238 Mutational status of p53 can influence its recognition by human T cells

Katerina Shamalov, Cyrille Cohen

Bar Ilan University, Ramat Gan, HaMerkaz, Iceland
Correspondence: Katerina Shamalov (kate.shamalov@gmail.com)

Background

p53 was reported to be an attractive immunotherapy target because it is mutated in approximately half of human cancers, resulting in its inactivation and often accumulation in tumor cells. Peptides derived from p53 are presented by class I MHC molecules and may act as tumor-associated epitopes which could be targeted by p53-specific T cells. Interestingly, it was recently shown that there is a lack of significant correlation between p53 expression levels in tumors and their recognition by p53-TCR transduced T cells.

Methods

To better understand the influence of the mutational status of p53 on its presentation by the MHC system and on T cell anti-tumor reactivity, we generated several mutant p53 constructs and expressed them in HLA-A2+/p53- cells. Upon co-culture with p53-specific T cells, we measured the specific recognition of p53-expressing target cells by means of cytokine secretion, marker upregulation and cytotoxicity, and in parallel determined p53 expression levels by intracellular staining. We also examined the impact of mutant p53 expression on cell cycle dynamics and on the expression levels of the pro-apoptotic protein caspase-3.

Results

Our results show that selected p53 mutations altering protein stability can modulate p53 presentation to T cells, leading to a differential immune reactivity inversely correlated to measured p53 protein levels.

Conclusions

Thus, p53 may behave differently than other classical tumor antigens and its mutational status should therefore be taken into account when elaborating immunotherapy treatments of cancer patients targeting p53.

P239 Fc gamma receptor IV mediated depletion of tumor infiltrating regulatory T cells by anti-CTLA-4 antibody is promoted by TLR1/2 agonist and hence its efficacy in combination treatment of melanoma

Naveen Sharma, James Allison

University of Texas MD Anderson Cancer Center, Houston, TX, USA
Correspondence: Naveen Sharma (nsharma1@mdanderson.org)

Background

Immune checkpoint blockade therapies have been successfully employed clinically to treat melanoma. Ipilimumab, which blocks inhibitory receptor CTLA-4 was one of the first checkpoint blockade therapies to get FDA approval for treating melanoma patients. Despite the effectiveness of these drugs, a significant number of cancer patients do not respond, and durable responses are only observed in a fraction of patients across tumor types. Therefore, combination therapies including checkpoint blockade antibodies are being studied to improve the outcome from immunotherapy treatment. Pattern recognition receptors like TLRs have been shown to have anti-tumor effects in various tumor models, through their effect on innate immunity.

Methods

In this study, we set out to combine the innate immune arm like TLR ligand with the adaptive immune arm for treatment of melanoma in a mouse model. We identified TLR1/2 ligand Pam3CSK4 as innate immune system modulator to combine with anti-CTLA-4 antibody in this combination therapy. Mice were injected intradermal (i.d.) on the right flank with 3 × 105 B16/F10 and considered day 0. Initial B16/F10 challenge was doubled to 6 × 105 in experiments where mice would be sacrificed on day 14. Mice were then treated with intraperitoneal (i.p) injection of 100 μg anti-CTLA-4 antibody and intratumoral injection with TLR1/2 ligand on every third after initial tumor challenge till day 12. The dose of anti-CTLA-4 antibody was doubled on day 3. In experiments where mice would be sacrificed on day 14, only two doses of anti-CTLA-4 antibody and TLR ligands were given on Day 9 and Day 12 after injection. These mice were either sacrificed on day 14 for obtaining lymphoid organs and tumors for phenotypic and functional analysis or tumor growth was analyzed.

Results

Our studies show that combining TLR1/2 ligand Pam3CSK4 with anti-CTLA-4 antibody decreases tumor burden and increases survival significantly, compared to anti-CTLA-4 antibody treatment alone. In our studies we found both CD4+ and CD8+ T cells to be important for this combination treatment efficacy. Most interestingly, we found that the mechanism of efficacy of combination treatment is due to an increased depletion of regulatory T cells modulated by enhanced FcγRIV expression on macrophages in combination therapy.

Conclusions

Our findings suggest that combining TLR1/2 ligand with anti-CTLA-4 antibody will be an interesting prospect for treatment of cancer and it also suggest that TLR1/2 ligand modulate FcγRIV expression, which can be used to modulate the efficacy of other antibody-based immunomodulatory therapies.

P240 Immunostimulatory and oncolytic properties of rotavirus can overcome resistance to immune checkpoint blockade therapy

Tala Shekarian1, Sandrine Valsesia-Wittmann2, Christophe Caux1, Aurelien Marabelle3

1Université Claude Bernard Lyon 1, Centre Leon Berard, Lyon, Rhone-Alpes, France; 2Centre Leon Berard, Innovations in Immunotherapy Platform, Lyon, Rhone-Alpes, France; 3Institue Goustave Roussy, Université Claude Bernard Lyon 1,Centre Leon Berard, Villejuif, Ile-de-France, France
Correspondence: Tala Shekarian (talashekarian@yahoo.com)

Background

Immune checkpoint targeted therapies against PD-1, PD-L1, and CTLA-4 are currently revolutionizing cancer care. However, a minority of patients generate objective tumor responses with these treatments. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors in order to overcome the resistance to immune checkpoint blockade therapy. Pattern recognition receptors (PRR) such as toll-like receptor agonists have been shown to overcome resistance to immune checkpoint targeted therapy in pre-clinical models. Besides their intrinsic ability to stimulate PRR, the oncolytic properties of common viruses can be exploited also for the priming of anti-tumor immune responses. Hypothesis: Can anti-infectious vaccines be used as a source or PRR agonists and/or oncolytic viruses?

Methods

We tested a TLR-Luc transgenic cell line for screening of the TLR agonist activity of anti-infectious vaccines. Cytotoxic activity induced by the vaccines was determined by SRB test and IncuCyte imaging. Flow cytometry was performed to identify the type of cell death and to characterize different population of infiltrated immunity cells in the tumors. In vivo effect of immune checkpoints blockade were determined in monotherapy and in combination with the vaccines on tumor growth.

Results

We confirmed that commercially available anti-infectious vaccines do have PRR agonist properties. Interestingly, we discovered that rotavirus vaccines also have oncolytic properties. These attenuated viruses can directly kill cancer cells with features of immunogenic cell death such as upregulation of calreticulin on dying cancer cells. Moreover, they have pro-inflammatory properties and can activate the NF-Kb pathway in a TLR and IRF3 independent manner. These in vitro biological properties translate into in vivo anti-tumor activity. Intra-tumoral rotavirus therapy has anti-tumor effects which are partly immune mediated as demonstrated by their activity in NSG xenograft models of human tumors. Interestingly, in immunocompetent syngeneic murine tumor models of neuroblastoma and lymphoma, intra-tumoral rotavirus therapy can overcome resistance and synergize with immune checkpoint targeted therapy. This therapeutic effect relied on specific modifications of tumor immune infiltrates and immune activation pathways. Intratumoral rotavirus vaccines was associated to an increase of leukocytes in the tumor microenvironment and upregulation of activation markers such as OX40/CD137 and CD86 on T cells and APC, respectively.

Conclusions

Rotavirus vaccines are clinical grade products. Therefore, in situ immunization strategies with intra-tumoral attenuated rotavirus can be implemented quickly in the clinic. Intra-tumoral priming of the anti-tumor immunity with oncolytic and immunostimulatory rotavirus vaccines could be a feasible strategy to overcome resistance to anti-PD-1/anti-CTLA-4 therapy in patients with cancer.

P241 A phase I/II study of durvalumab alone or in combination with AXAL in recurrent/persistent or metastatic cervical or human papillomavirus (HPV) + squamous cell cancer of the head and neck (SCCHN): preliminary phase I results

Brian M Slomovitz1, Kathleen M Moore2, Hagop Youssoufian3, Marshall Posner4

1Sylvester Comprehensive Cancer Center/University of Miami, Miami, FL, USA; 2Stephenson Oklahoma Cancer Center, Oklahoma City, OK, USA; 3Advaxis Immunotherapies, Princeton, NJ, USA; 4Icahn School of Medicine at Mount Sinai, Mount Sinai Medical Center, New York, NY, USA
Correspondence: Brian M Slomovitz (bslomovitz@med.miami.edu)

Background

The success of immunotherapy for cervical cancer and HPV+ head and neck cancer may be enhanced by a combination of immune checkpoint blockade and tumor-selective vaccination. Axalimogene filolisbac (AXAL or ADXS11-001) is an irreversibly attenuated Listeria monocytogenes-listeriolysin O (Lm-LLO) immunotherapy bioengineered to secrete an HPV E7 tLLO fusion protein that induces HPV-specific cytotoxic T cells and reduces tumor-associated immune tolerance. Durvalumab is a selective, high-affinity human IgG1 mAb that blocks PD-L1 binding to PD-1 (IC50 0.1 nM) and CD80 (B7.1; IC50 0.04 nM). The PD-1/PD-L1 pathway is an important checkpoint used by tumor cells to inhibit antitumor responses. Preclinical mouse models demonstrate combination AXAL/anti–PD-1 treatment significantly reduces tumor growth and prolongs survival.

Methods

This is a phase I/II study (NCT02291055) of AXAL + durvalumab in patients (≥18 years) with either recurrent/metastatic cervical cancer or metastatic HPV+ SCCHN, who progressed on ≥1 platinum-based therapy. The primary objectives of the phase I, Part A dose escalation are to determine the safety/tolerability and establish the combination recommended phase II dose (RP2D) of AXAL (1 × 109 colony-forming units q4wk) and durvalumab (3 mg/kg or 10 mg/kg q2wk) following a 3 + 3 design. Part A includes a SCCHN expansion cohort (N = 20) at the RP2D to evaluate efficacy. Part B will evaluate tumor response (RECIST and immune-related RECIST) of durvalumab monotherapy and AXAL + durvalumab combination therapy at the RP2D in recurrent/metastatic cervical cancer. Preliminary results of phase I dose escalation are reported.

Results

To date, 11 patients are enrolled in phase I (AXAL + durvalumab 3 mg/kg: N = 5; AXAL + durvalumab 10 mg/kg: N = 6); 91 % had ECOG performance status 0, 73 % had cervical cancer, of which 75 % received prior bevacizumab. No dose-limiting toxicities have been observed. The following adverse events (AEs) were reported (3 vs. 10 mg/kg): 100 % vs. 83 % of patients experienced AEs; 20 % vs. 50 % experienced SAEs; 60 % vs. 50 % experienced Grade 1 and Grade 2 treatment related AEs (TRAEs); Grade 3 TRAEs occurred in n = 1 (rigors) and n = 2 (rigors and neutropenia, respectively) patients. In the AXAL + durvalumab 3 mg/kg cohort, 2 patients with cervical cancer obtained an objective response; 1 CR that is ongoing (9 months follow-up) and 1 PR with subsequent disease progression. Tumor assessments from the AXAL + durvalumab 10 mg/kg cohort are not yet available. The RP2D was declared at durvalumab 10 mg/kg q2wk + AXAL 1 × 109 colony-forming units q4wk.

Conclusions

The combination of AXAL/durvalumab appears safe and tolerable. Preliminary data indicate encouraging antitumor activity of the combination immunotherapy regimen.

Trial Registration

ClinicalTrials.gov identifier NCT02291055.

P242 A specific 17-beta-hydroxywithanolide (LG-02) sensitizes cancer cells to apoptosis in response to TRAIL and toll-like receptor (TLR) ligands

Poonam Tewary1, Alan D Brooks2, Ya-Ming Xu3, Kithsiri Wijeratne3, Leslie AA Gunatilaka4, Thomas J Sayers1

1CIP, Center for Cancer Research, BSP, Leidos Biomed Research Inc, National Cancer Institute-Frederick, Frederick, MD, USA; 2CIP, Center for Cancer Research, Leidos Biomed Research Inc, National Cancer Institute-Frederick, Frederick, MD, USA; 3University of Arizona, Southwest Center for Natural Products Research and Commercialization, Tucson, AZ, USA; 4University of Arizona, Tucson, AZ, USA
Correspondence: Poonam Tewary (tewaryp@mail.nih.gov)

Background

Despite many therapeutic successes, cancer is the second-most frequent cause of mortality in the United States. Strategies for cancer therapy aim to overcome excessive proliferation and avoidance of apoptosis. Therefore, methods of inducing apoptosis have become an important approach in the design of effective cancer therapies. Among these tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has shown considerable promise as a nontoxic apoptotic inducer in cancer immunotherapy. However, many primary tumors are inherently resistant to TRAIL-mediated apoptosis and require additional sensitization. Therefore, there is an underlying interest in identifying agents that can be combined with TRAIL to improve its efficacy. Recent studies have also described a role of TLR3 signaling for initiating apoptosis in malignant cells and thus promote anticancer immune responses. We have previously shown that, withanolide E (WE), a 17β-hydroxywithanolide (17-BHW) and a natural product derived from the medicinal plant Physalis peruviana was capable of sensitizing tumor cells to TRAIL-mediated apoptosis by reducing cellular levels of the anti-apoptotic protein cFLIP.

Methods

Encouraged by this, we screened a small library of 17-BHWs and have identified several that are more potent than WE for their ability to promote death ligand-mediated cancer cell death.

Results

Among the 30 compounds tested, LG-02 was found to be 4–5 fold more potent than WE in sensitizing tumor cells to apoptotic signaling in response TRAIL as well as to the synthetic polynucleotide, poly(I:C), which is known to mimic anti-viral responses by activating TLR (toll-like receptor) signaling. Intra-tumor administration of LG-02 and poly(I:C) in a xenograft M14 melanoma model provided therapeutic benefit leading to complete tumor regression in 90 % of the mice as compared to mice treated with vehicle or compounds alone. Molecular studies in melanoma cells demonstrated decreases not only in the anti-apoptotic cFLIP proteins but also in a number of IAPs including livin following LG-02 treatment. To date there are no withanolides reported to have this dual activity on reducing levels of different anti-apoptotic proteins.

Conclusions

Thus, we hypothesized that 17-BHWs represent a unique NP scaffold, structural modification of which would lead to potent non-toxic sensitizers of apoptosis by TLR signaling that utilizes a downstream pathway similar to that of TNF death receptor signaling. Further studies with 17-BHWs could lead to the identification of novel and common therapeutic targets involved in apoptosis signaling in response to both TNF death receptor family members as well as TLR ligands.

Acknowledgements

Funded by NCI Contract HHSN261200800001E.

P243 Intratumoral administration of the TLR7/8 agonist MEDI9197 inhibits tumor growth and modulates the tumor microenvironment

John P Vasilakos1, Tesha Alston1, Simon Dovedi2, James Elvecrog1, Iwen Grigsby1, Ronald Herbst3, Karen Johnson1, Craig Moeckly1, Stefanie Mullins2, Kristen Siebenaler1, Julius SternJohn1, Ashenafi Tilahun1, Mark A Tomai1, Katharina Vogel2, Robert W Wilkinson2

13M Company, St. Paul, MN, USA; 2Medimmune, Cambridge, England, UK; 3Medimmune, Gaithersburg, MD, USA
Correspondence: John P Vasilakos (jpvasilakos@mmm.com)

Background

Toll-like receptor (TLR) agonists, such as the TLR7 agonist imiquimod, have been evaluated topically and systemically for cancer. Topical administration has shown antitumor activity against various cancers, such as melanoma, squamous cell carcinoma, and cutaneous breast cancer. However, systemic administration of TLR agonists in cancer patients has resulted in limited efficacy, in part due to cytokine-induced systemic adverse effects, which limits the therapeutic window. Therefore, a lipophilic imidazoquinoline, MEDI9197, was designed to be retained within the tumor following injection with the primary objective of directing immune activation to the tumor.

Methods

The antitumor effects of intratumoral (IT) administered MEDI9197 were evaluated in 4 different mouse syngeneic subcutaneous implantation tumor models. Tumor and serum drug levels were quantified following IT administration. In addition, the tumor immune profile was assessed by qPCR, histology, and flow cytometry. Lastly, the antitumor effects of combination therapy using IT injected MEDI9197 in conjunction with CTLA-4 or PD-L1 antibodies were evaluated.

Results

MEDI9197 is a human TLR7/8 agonist. Following IT administration, pharmacokinetic analysis shows that the drug is retained in the tumor, and very low levels of the drug are detected in the serum. MEDI9197 mediates antitumor activity (tumor growth inhibition and enhanced survival) in B16F10 luc, B16-OVA, 4 T1, and CT-26 mouse tumor models. Administration of MEDI9197 by the IT route and by the SC route away from the tumor demonstrates that the antitumor effects of MEDI9197 require IT administration. IT dosed MEDI9197 modulates the local immune response characterized by an upregulation of genes involved in innate and adaptive immunity. IT dosed MEDI9197 induces tumor necrosis, leukocyte activation, and the formation of lymphoid aggregates evident by 7 days postdose. IT injected MEDI9197 increases the number of tumor infiltrating CD8+ T cells, while concomitantly decreasing the number of tumor infiltrating CD4+ T cells. Moreover, MEDI9197 induces prolonged activation of tumor T cells and NK cells. Additionally, combination of MEDI9197 with CTLA-4 and PD-L1 antibodies enhances the efficacy observed in syngeneic mouse tumor models.

Conclusions

The data presented shows IT administration of the TLR7/8 agonist MEDI9197 is retained in the tumor, modulates the tumor microenvironment in a manner consistent with an antitumor signature, and inhibits tumor growth in multiple mouse cancer models. Finally, the antitumor effects of MEDI9197 are further enhanced by combination therapy with checkpoint blockade therapies. MEDI9197 is currently being evaluated for safety and efficacy in human clinical trials (ClinicalTrials.gov Identifier: NCT02556463).

P244 Impact of intratumoral clonal heterogeneity on checkpoint inhibitor response

Eveline E Vietsch, Anton Wellstein

Georgetown University/Medical School/Lombardi Cancer Center, Washington, DC, USA
Correspondence: Anton Wellstein (wellstea@georgetown.edu)

Background

Cancer cells are subjected to evolutionary selection of clonal populations by changes in the microenvironment as well as their response to drug treatment. We wished to evaluate how this heterogeneity impacts efficacy of checkpoint inhibition.

Methods

To understand the contribution of clonal subpopulations to the malignant progression and to the response to drugs, we established a model of tumor heterogeneity from six syngeneic, clonal primary cancer cells isolated from a mutant Kras/P53 mouse pancreatic cancer (KPC). The clones were characterized molecularly and tumors reconstituted from mixes of the clonal cell lines.

Results

These clonal cells formed invasive and metastatic lesions when grafted into hosts. The original tumor and clonal cell lines harbored common mutations in 99 genes suggesting their common ancestry. Additional unique mutations in the clonal lines were used to identify and quantitate clones in heterogeneous cell pools. The clones showed different levels of MAP kinase signaling, unique morphologies, different growth rates in vitro and tumor growth rates in immune competent mice. Moreover, the sensitivity to ~200 anticancer drugs revealed an up to 25-fold varying in vitro sensitivity of the clones to signal transduction inhibitors and cytotoxic drugs. To our surprise, drug sensitivity of individual clones when included in a heterogeneous cell population was strikingly different from their drug sensitivity when growing on their own. In particular, the sensitivity of clones to MEK or PI3K inhibition was not predictive of their sensitivity when grown in a pool with the other clones. Furthermore, the sensitivity of clones to an anti-PD-1 checkpoint inhibitor was distinct across the clonal cells growing in the heterogeneous mixture. Some clones were resistant and others highly sensitive to the checkpoint inhibition. We will discuss pathways and drivers of resistance in the different subpopulations.

Conclusions

We conclude that malignant progression and selection of checkpoint inhibitor sensitive cancer cell subpopulations is impacted by the crosstalk between clonal cell populations present in heterogeneous tumors and the host environment.

P245 Discovery and characterization of PF-06840003, a novel brain penetrant IDO1 inhibitor

Martin Wythes1, Stefano Crosignani2, Joseph Tumang1, Shilpa Alekar1, Patrick Bingham1, Sandra Cauwenberghs2, Jenny Chaplin1, Deepak Dalvie1, Sofie Denies2, Coraline De Maeseneire2, JunLi Feng1, Kim Frederix2, Samantha Greasley1, Jie Guo1, James Hardwick1, Stephen Kaiser1, Katti Jessen1, Erick Kindt1, Marie-Claire Letellier2, Wenlin Li1, Karen Maegley1, Reece Marillier2, Nichol Miller1, Brion Murray1, Romain Pirson2, Julie Preillon3, Virginie Rabolli2, Chad Ray1, Kevin Ryan1, Stephanie Scales1, Jay Srirangam1, Jim Solowiej1, Al Stewart1, Nicole Streiner1, Vince Torti1, Konstantinos Tsaparikos1, Xianxian Zheng1, Gregory Driessens2, Bruno Gomes2, Manfred Kraus1

1Pfizer, San Diego, CA, USA; 2iTeos, 6041 Gosselies, Brussels Hoofdstedelijk Gewest, Belgium; 3iTeos, Rue Auguste Piccard 48, Brussels Hoofdstedelijk Gewest, Belgium
B: Martin Wythes (martin.wythes@pfizer.com)

Background

Tumors use tryptophan-catabolizing enzymes such as indoleamine 2–3 dioxygenase (IDO1) to induce an immunosuppressive environment. IDO1 is induced in response to inflammatory stimuli and promotes immune tolerance, effector T cell anergy and enhanced Treg function. As such, IDO1 is a nexus for the induction of key immunosuppressive mechanisms and represents an important immunotherapeutic target in oncology.

Methods

We have identified and characterized a new, selective, orally bioavailable IDO1 inhibitor, PF-06840003.

Results

Key interactions of PF-06840003 with IDO1 will be presented, and rationalized using a novel X-ray crystal structure of PF-06840003 bound to human IDO1. In addition, binding studies with ferrous and ferric forms of human IDO1 have been performed. The results suggest that PF-06840003 is a tryptophan non-competitive, non-heme binding IDO1 inhibitor. Key in vitro and in vivo pharmacology data, including combination studies with checkpoint inhibitors, and ADME data of PF-06840003 will be discussed. PF-06840003 shows a very favorable ADME profile (solubility, human hepatocyte stability, low in vivo clearance in preclinical species, high permeability, and high fraction absorbed in preclinical species) leading to favorable predicted human pharmacokinetic properties, including a predicted t1/2 of 19 hours.

Conclusions

PF-06840003 is a selective IDO1 inhibitor with very favorable predicted human PK characteristics. Its prolonged projected human half-life should allow QD administration. CNS penetration suggests potential impact on brain metastases. Checkpoint antagonists against PD-L1 cause enhanced IDO1 expression and enhanced in vivo anti-tumor efficacy in combination with PF-06840003. These studies highlight the potential of PF-06840003 as a clinical candidate in Immuno-Oncology. A first in patient study for PF-06840003 in malignant gliomas is described at ClinicalTrials.gov (NCT02764151).

P246 Enhanced anti-tumor effect of combination therapy with NHS-muIL-12 and anti-PD-L1 antibody (avelumab) in a preclinical cancer model

Chunxiao Xu1, Yanping Zhang2, Giorgio Kradjian2, Guozhong Qin2, Jin Qi2, Xiaomei Xu2, Bo Marelli2, Huakui Yu2, Wilson Guzman2, Rober Tighe2, Rachel Salazar2, Kin-Ming Lo2, Jessie English2, Laszlo Radvanyi2, Yan Lan2

1EMD Serono, Belmont, MA, USA; 2EMD Serono, Billerica, MA, USA
Correspondence: Chunxiao Xu (chunxiao.xu@emdserono.com)

Background

Recent clinical studies have found that treatment with the immune checkpoint inhibitors anti-PD-1 or PD-L1 induce durable anti-tumor responses in some patients with advanced-stage cancers. However, many patients do not benefit from treatment because the induction, potency, and persistence of immune responses depend on a complex interplay between different immune cell populations. Thus, treatment with a combination of therapies that target distinct immune pathways may be a promising strategy to improve anti-tumor efficacy. NHS-IL-12 (MSB0010360N; M9241) is an investigational immunocytokine designed to target tumor necrotic regions as a method to deliver IL-12 into the tumor microenvironment. Avelumab* (MSB0010718C) is a fully human anti-PD-L1 IgG1 monoclonal antibody designed to selectively bind to PD-L1 and competitively inhibit it from binding to PD-1, which has shown antitumor activity in various malignancies in clinical trials.

Methods

In the pre-clinical studies described here, the anti-tumor efficacy of combination treatment with avelumab and the surrogate NHS-muIL-12 was investigated in an orthotopic EMT-6 breast cancer model’

Results

Treatment with NHS-muIL-12 and avelumab generated an enhanced anti-tumor effect relative to either monotherapy. Most mice treated with the combination therapy had complete tumor regression and generated tumor-specific immune memory, as demonstrated by their protection against rechallenge with EMT-6 tumor cells and the significant induction of effector and memory T cells. The combination treatment dose-dependently stimulated cytotoxic NK and CD8+ T cell proliferation. NHS-muIL-12 treatment induced CD8+ T cell infiltration into the tumor microenvironment consistent with the induction of chemoattractants. Also, avelumab monotherapy reversed T cell immunosuppression and restored the function of exhausted CD8+ T cells in the tumor microenvironment.

Conclusions

These preclinical findings indicate that the combination therapy with NHS-IL-12 and avelumab may provide a promising approach to treat patients with solid tumors. Asterisk (*) indicates a proposed nonproprietary name.

P247 Opposing effects of CTLA-4 and PD-1 blockade on follicular helper-like T cells with immunosuppressive functions

Roberta Zappasodi1, Sadna Budhu1, Matthew D Hellmann2, Michael Postow2, Yasin Senbabaoglu1, Billel Gasmi1, Hong Zhong1, Yanyun Li1, Cailian Liu1, Daniel Hirschhorhn-Cymerman1, Jedd D Wolchok2, Taha Merghoub1

1Ludwig Collaborative Laboratory, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 2Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Correspondence: Roberta Zappasodi (zappasor@mskcc.org)

Background

The mechanism underlying the improved anti-tumor activity of combined CTLA-4 and PD-1 blockade is not yet well understood. We reported that expansion of CD4+Foxp3 T cells expressing PD-1 (4PD-1hi) was associated with limited therapeutic improvement when a CTLA-4-blocking antibody was added to an anti-melanoma vaccine in B16-melanoma bearing mice. We went on to define functions and origin of 4PD-1hi to clarify the significance of their modulation by checkpoint blockade in mouse tumor models and cancer patients.

Methods

4PD-1hi frequency was monitored by flow cytometry. Their function was tested in standard in vitro suppression assays and 3D collagen-fibrin gel killing assays. RNAseq gene expression analyses were performed on a Proton sequencing system at the MSK Genomics Core Facility.

Results

Circulating and intra-tumor 4PD-1hi frequencies positively correlated and both increased as a function of tumor burden in anti-CTLA-4-treated and naïve B16-bearing mice, suggesting a pro-tumor role of 4PD-1hi. Accordingly, the ratio between effector T cell (Teff) and 4PD-1hi inversely correlated with tumor size. 4PD-1hi from spleens and tumors of naïve and B16-bearing Foxp3-GFP-transgenic mice treated or not with CTLA-4 blockade suppressed Teff functions. RNAseq gene expression analysis revealed an enrichment of follicular helper T cell-(Tfh)-associated genes in 4PD-1hi in comparison with regulatory T cells (Tregs) and CD4+Foxp3PD-1 T cells. We therefore immunized Foxp3-GFP transgenic mice with sheep red blood cells (sRBC) to boost development of Tfh and test their function in vitro. 4PD-1hi from sRBC-treated animals inhibited Teff more efficiently than those isolated from untreated mice. However, in contrast to Tregs and according to their Tfh-like phenotype, 4PD-1hi promoted activation and maturation of B cells in vitro. Moreover, concurrent PD-1 and CTLA-4 blockade, either alone or in combination with an anti-melanoma vaccine, prevented 4PD-1hi expansion and significantly improved anti-tumor responses in mice. In cancer patients, ipilimumab increased, whereas PD-1 blockade reduced, circulating 4PD-1hi. We took advantage of differential CD25 expression in 4PD-1hi and Tregs to isolate and compare these two cell subsets from healthy donors’ peripheral blood as well as patients’ tumors. Human 4PD-1hi inhibited Teff functions in vitro and expressed Tfh-associated markers, thus confirming our observations in mice.

Conclusions

Our study describes T cell suppression functions of Tfh-like cells expanded by CTLA-4 blockade. Importantly, we show that these cells exist in healthy individuals and expand in the presence of tumor. We provide evidence that PD-1 blockade counteracts anti-CTLA-4-mediated 4PD-1hi induction, thus underscoring one of the mechanisms potentially responsible for the improved therapeutic activity of combination checkpoint blockade.

Consent

Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

P248 WT1 peptide vaccine in Montanide or poly-ICLC triggers different immune responses in patients with myeloid leukemia

Yuanyuan Zha1, Gregory Malnassy2, Noreen Fulton2, Jae-Hyun Park2, Wendy Stock3, Yusuke Nakamura2, Thomas F Gajewski4, Hongtao Liu4

1University of Chicago, OSRF-HIM, Chicago, IL, USA; 2University of Chicago, Chicago, IL, USA; 3University of Chicago, Section of Hematology/Oncology, Chicago, IL, USA; 4University of Chicago Medical Center, Chicago, IL, USA
Correspondence: Yuanyuan Zha (yzha1@bsd.uchicago.edu)

Background

It has been well established that human T cells can recognize and destroy tumor cells. In solid tumors, it has been shown that peptide vaccine against tumor antigens can augment host anti-tumor immune response and achieve tumor control in some patients. WT1 is a defined leukemia-associated antigen, a transcription factor that over-expressed in AML, CML, ALL, and other tumors. WT1 is highly antigenic and is an attractive target for immunotherapy. However, the optimal strategy for vaccination to induce WT1-specific immune responses is not known.

Methods

In this pilot study, we randomized seven (4 males, 3 female ages 39 to 73) HLA-A02+ patients with myeloid leukemia in the minimal residual disease state to receive vaccination with WT1 126–134 peptide (RMFPNAPYL) in either Montanide or poly-ICLC (TLR3 agonist). Four patients were randomized to receive WT1 in Montanide and three were randomized to receive WT1 in poly-ICLC. The vaccine was administered every other week X 6 during the induction phase followed by monthly booster vaccinations X 6 months. Patients were monitored for disease and toxicity. Blood was collected to monitor WT1 transcript levels, antigen-specific CD8+ T cell responses, and TCR sequencing.

Results

After WT1 vaccination, three of four patients in the Montanide arm had deceased WT1 levels in circulation detected by qRT-PCR, and two of these demonstrated augmented WT1-specific CD8+ T cell responses detected by IFN-γ ELISPOT assay. All three patients had TCR clonal enrichment after WT1 vaccination suggested by TCR alpha and beta CDR3 sequencing. In contrast, in the two patients on the poly-ICLC arm, no increase in WT1-specific CD8+ T cell responses was detected by IFN-γ ELISPOT assay, and no clonal enrichment was detected by TCR alpha/beta sequencing. Interestingly, these two patients nonetheless demonstrated decreased WT1 transcript levels in circulation detected by qRT-PCR and remained in remission 3 years after the initiation of WT1 vaccination.

Conclusions

Our results show that vaccination with WT1 peptide emulsified in Montanide is a superior vaccine strategy based on increased WT1-specific CD8+ T cell responses with TCR clonal and specific TCR beta CDR3 enrichment and decreased WT1 transcripts as a measure of minimal residual disease. The fact that vaccination with WT1 peptide in poly-ICLC nonetheless was associated with decreased WT1 transcripts suggests that a distinct immune activation mechanism might be occurring, for example an effect on dendritic cells of poly-ICLC alone.

P249 Combination of Listeria-based human papillomavirus (HPV) E7 cancer vaccine (AXAL) with CD137 agonist antibody provides an effective immunotherapy for HPV-positive tumors in a mouse model

Xiaoming Ju, Rachelle Kosoff, Kimberly Ramos, Brandon Coder, Robert Petit, Michael Princiotta, Kyle Perry, Jun Zou

Advaxis Immunotherapies, Princeton, NJ, USA
Correspondence: Jun Zou (zou@advaxis.com)

Background

HPV can cause cervical, anal, vulvar, vaginal, penile, and oropharyngeal cancers. AXAL is a genetically engineered Listeria monocytogenes-based therapeutic cancer vaccine currently in clinical trials for cervical (phase III), anal (phase II), and head and neck (phase I/II) cancers, either as monotherapy or in combination with checkpoint inhibitor (PD-1 or PD-L1) antibodies. To identify potentially synergistic immunotherapies, we evaluated AXAL ± antibodies for T cell co-inhibitory or co-stimulatory receptors (checkpoint inhibitors: CTLA-4, PD-1, TIM-3, LAG-3; co-stimulators: CD137, OX40, GITR, and CD40) in a mouse HPV-positive tumor model.

Methods

C57BL/6 female mice and TC1 cells (C57BL/6 mouse lung epithelial cells co-transfected with HPV16 E6 and E7 and activated Ras) were obtained from Jackson Laboratories and ATCC, respectively. All antibodies were obtained from Bio X Cell. Mice were subcutaneously injected on the hind-leg flank with TC1 cells. AXAL ± the respective antibodies was injected intraperitoneally at 5 × 107 colony-forming units/mouse weekly for 3 total doses. For combinations with superior performance, the tumor microenvironment (TME) was further evaluated using flow cytometry to immunophenotype the tumor-infiltrating lymphocytes (TILs), spleen, and tumor-draining lymph node.

Results

Among 8 antibodies tested in combination with AXAL, CD137 and CTLA-4 antibodies were the most effective for tumor growth inhibition, tumor regression, and survival. Consistent with prior reports that CD137 is expressed on natural killer, dendritic, and T cells, and can potentiate antitumor responses by altering the cellular makeup of the TME [1], immunophenotyping revealed increased TILs and CD8/Treg ratio, and decreased levels of highly immunosuppressive CD103-positive Tregs after CD137 + AXAL treatment versus treatment with either agent alone. Additionally, increases were observed in PD-L1 expression on tumor cells and PD-1 expression on CD8-positive T cells. Mice with complete tumor regression after CD137 + AXAL treatment (n = 5) were subsequently rechallenged with TC1 cells. Two mice remained tumor free until study termination (an additional 6–7 weeks); the other 3 had delayed or slower tumor growth versus controls. CTLA-4 + AXAL treatment resulted in complete tumor regression in 3 mice evaluated. These mice remained tumor free even after rechallenge.

Conclusions

AXAL demonstrated strong anticancer activity in this preclinical model of HPV-positive cancer, especially in combination with CD137 and CTLA-4 antibodies. Moreover, these data suggest that addition of anti-PD-1 to anti-CD137 + AXAL could be a potent triple combination therapy.

References

1. Makkouk A, Chester C, Kohrt HE: Rationale for anti-CD137 cancer immunotherapy. Eur J Cancer 2016, 54:112–119.

Combinations: Immunotherapy/Standard of Care

P250 Tumor-resident T cells survive and mediate the antitumoral effects in a murine model of cancer therapy with localized ionizing radiation

Ainhoa Arina1, Christian Fernandez1, Wenxin Zheng1, Michael A Beckett1, Helena J Mauceri1, Yang-Xin Fu2, Ralph R Weichselbaum1

1The University of Chicago, Chicago, IL, USA; 2UT Southwestern, Dallas, TX, USA
Corespondence: aarina@bsd.uchicago.edu

Background

A role for T cells in the antitumor effects of radiation therapy is becoming increasingly clear [1]. Since lymphocytes are considered to be very sensitive to ionizing radiation (IR), and IR increases T cell infiltration of tumors, it is usually assumed that the newly infiltrating T cells mediate the therapeutic effects of IR. However, there is no clear data showing the contribution of tumor-resident vs. newly infiltrating T cells to the therapeutic effects of IR.

Methods

Longitudinal in vivo imaging of tumors using window chambers was performed as described [2]. “T cell reporter” mice were obtained by crossing CD4-Cre or Lck-cre mice with R26-stop-EYFP mice (Jackson).

Results

Panc02SIYCerulean cancer cells were injected s.c. into EYFP+ T cell reporter mice bearing dorsal window chambers. When tumors were established (around day 21), mice received 800 cGy of whole-body irradiation (WBI) while their tumor was shielded. This procedure depleted most peripheral T cells while preserving tumor-resident EYFP+ T cells. Following bone-marrow reconstitution with DsRed+Rag−/− cells, EGFP+ 2C CD8+ T cells specific for the SIY antigen were adoptively transferred, to distinguish newly infiltrating T cells. 3–4 days after 2C transfer, some mice received local IR as follows. 2 experiments using (i) 5 doses of 1.8 Gy each, 24 hours apart (fractionated IR model), and (ii) a single dose of 20 Gy (SBRT model) showed that a significant proportion of tumor-resident EYFP+ T cells were still detected in the tumor and kept their motility even after 20 Gy of IR, for up to 2 weeks. 2C-EGFP+ T cells infiltrated IR-treated tumors with some delay, but eventually reached high numbers in IR-treated and untreated tumors. Treating MC38 tumor-bearing mice with increasing doses of WBI (1–10 Gy) showed that tumor-resident T cells were more resistant to IR than circulating T cells. Experiments treating MC38-bearing animals with 20 Gy local IR and systemic sphingosine 1-phosphate receptor agonist FTY720, suggest that tumor-resident T cells might suffice for the antitumoral effects of single high-dose IR.

Conclusions

Tumor-resident T cells show preferential survival to IR compared to circulating T cells and can contribute to the therapeutic effects of radiotherapy.

References

1. Burnette B, Fu YX, Weichselbaum RR: The confluence of radiotherapy and immunotherapy. Front Oncol 2012, 2:143.

2. Schietinger A, Arina A, et al.: Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer. Oncoimmunology 2013, 2:e26677.

P251 Multi-kinase inhibitors for the treatment of mRCC: implications for combined therapy with AGS-003, an autologous dendritic cell immunotherapy

Mark DeBenedette, Whitney Lewis, Alicia Gamble, Charles Nicolette

Argos Therapeutics, Durham, NC, USA
Correspondence: Mark DeBenedette (mdebenedette@argostherapeutics.com)

Background

AGS-003, is an autologous dendritic cell (DC) immunotherapy consisting of matured DCs co-electroporated with amplified autologous tumor RNA and CD40L RNA. AGS-003, is being evaluated in the pivotal ADAPT phase III clinical trial for the treatment of metastatic renal cell carcinoma (mRCC) in combination with standard-of-care, based on a phase II clinical trial suggesting that response combination of sunitinib + AGS-003 was greater than sunitinib alone. The standard-of-care tyrosine kinase inhibitors including sunitinib, sorafinib, axitinib, pazopanib, cabozantinib and tivozanib, and the mTOR inhibitors, everolimus and temsirolimus, are anti-angiogenic therapeutics targeting signaling pathways implicated in the progression of RCC. However, these same signaling pathways are essential for the activation of antigen-specific T cell responses. Combining kinase inhibitor therapy with an active immunotherapeutic, such as AGS-003, may be ineffective, if kinase inhibitor therapy impedes the induction of CTL responses in vivo, which is the proposed mechanism of action (MOA) of AGS-003. Therefore, it was of interest to test these combinations in vitro with DCs representative of AGS-003 to observe the effects of combination therapy on antigen-specific CTL proliferation and CTL functional responses.

Methods

DCs derived from normal donor monocytes were co-electroporated with MART-1 RNA and CD40L RNA to represent AGS-003 DC products. In vitro co-cultures were set up with autologous CD8+ T cells and MART-1/CD40L-DCs in the presence of various concentrations of the kinase inhibitors. Kinase inhibitor concentrations were chosen to represent steady-state concentrations reported in patients receiving active therapy. Subsequent expansion of MART-1 specific CTLs and multi-functional responses were mapped using multi-color flow cytometry.

Results

Our in vitro analysis demonstrated that sunitinib, axitinib, cabozantinib, tivozanib, everolimus and temsirolimus did not impact the priming nor proliferation of MART-1-specific CTL responses. Furthermore, these kinase inhibitors did not impact multi-functionality of CD28+/CD45RA effector/memory CTL. However, sorafenib, when present in the CTL/DC co-cultures, did significantly impair anti-MART-1-specific CTL expansion and CTL multi-functionality.

Conclusions

Autologous DCs co-electroporated with MART-1 RNA/CD40L RNA exhibit a similar MOA in vitro to AGS-003 administered in vivo, whereby both DC preparations induce antigen-specific multi-functional CTLs. Understanding the MOA of AGS-003 in vitro, allows for the testing of a broad range of potential combination therapies to provide feasibility data to support clinical trials of combination therapy for mRCC. Data provided show that most, but not all, kinase inhibitors are compatible with the MOA of AGS-003, the induction of effector/memory CTL responses, and that each therapeutic agent warrants testing.

Trial Registration

ClinicalTrials.gov identifier NCT01582672.

P252 Multiple tumor antigen-activated T cell therapy elicits Individual and dynamic T cell responses in patients with hepatocellular carcinoma

Yanyan Han1, Yeting Wu2, Chou Yang2, Jing Huang2, Dongyun Wu3, Jin Li3, Xiaoling Liang1, Xiangjun Zhou3, Jinlin Hou2

1R&D Department, HRYZ Biotech Co., Shenzhen, Guangdong, People’s Republic of China; 2Department of Infectious Diseases and Hepatology Unit, Nanfang Hospital, Guangzhou, Guangdong, People’s Republic of China; 3HRYZ Biotech Co., Shenzhen, Guangdong, People’s Republic of China
Correspondence: Yanyan Han (hanyy@thyx.com)

Background

We have previously reported that the immunotherapy with multiple tumor antigens activated autologous T cells (MASCT) was a safe treatment, which may improve the immunologic function and clinical outcome of the patients with hepatocellular carcinoma (HCC). In this study, we investigated the dynamics of MASCT-induced immune responses and demonstrated the mechanism and advantages of using multiple tumor antigens.

Methods

13 patients with stage B stage (BCLC) were treated with MASCT for three courses after tumor resection. During each course, the patients received two subcutaneous injections of mature dendritic cells (mDCs) pulsed with a peptide pool of multiple tumor antigens, and three i.v. injections of autologous T cells activated by mDCs described above. Each course lasted 14–15 weeks.

Results

After repeated treatment of MASCT, the frequency of regulatory T cells in the patients’ PBMCs was significantly decreased, while antigen peptide pool-triggered T cell proliferation and IFNγ-production were significantly enhanced in the patients’ PBMCs. Moreover, the specific immune responses of T cells against each kind of tumor antigen peptide in the pool were also measured by IFNγ ELISPOT assay. These specific immune responses could be detected in 11 out of 13 patients’ PBMCs but with individual and dynamic patterns during the treatments of MASCT. After 1 course of treatment, the best patient has specific immune responses against 9 tumor antigens out of 14 in the pool, and the worst patient has responses against 2 tumor antigens. These numbers have increased to 11 and 3 after the second course. The most immunogenic tumor antigens are survivin (7/13), cyclin D1 (CCND1, 6/13), carcinoembryonic antigen (CEA, 5/13), and HBV DNA polymerase (5/13). There were 7 patients left without progression 1 year after the immunotherapy initiation. And, the specific immune responses detected in these patients’ PBMCs were significantly stronger than that in the patients with progression.

Conclusions

Our study demonstrates that individual and dynamic tumor antigen-specific T cell responses can be induced in HCC patients after repeated treatments of MASCT, providing evidence to show the advantage of using multiple tumor antigens in immunotherapy instead of single antigen. In addition, these specific immune responses may correlate with the clinical outcomes.

P253 Live, attenuated, double-deleted Listeria monocytogenes expressing mesothelin (CRS-207) with immuno-modulatory doses of cyclophosphamide, combined with chemotherapy as treatment for malignant pleural mesothelioma (MPM)

Raffit Hassan1, Thierry Jahan2, Scott J Antonia3, Hedy L Kindler4, Evan W Alley5, Somayeh Honarmand6, Weiqun Liu6, Meredith L Leong6, Chan C Whiting6, Nitya Nair6, Amanda Enstrom6, Edward E Lemmens6, Takahiro Tsujikawa7, Sushil Kumar7, Lisa M Coussens7, Aimee L Murphy6, Dirk G Brockstedt6

1Thoracic and GI Oncology Branch, National Cancer Institute, Bethesda, MD, USA; 2Department of Medicine, Division of Hematology Oncology, UCSF, San Francisco, CA, USA; 3H. Lee Moffitt Cancer Center, Tampa, FL, USA; 4Gastrointestinal Oncology and Mesothelioma Programs, Section of Hematology/Oncology, University of Chicago, Chicago, IL, USA; 5Penn Prebyterian Medical Center, University of Pennsylvania, Philadelphia, PA, USA; 6Aduro Biotech, Inc., Berkeley, CA, USA; 7Oregon Health & Science University, Portland, OR, USA
Correspondence: Somayeh Honarmand (shonarmand@aduro.com)

Background

CRS-207 is live, attenuated, double-deleted Listeria monocytogenes (LADD) engineered to express mesothelin, a tumor-associated antigen over-expressed in several cancers, including MPM, an aggressive treatment-refractory disease with poor prognosis. CRS-207 activates innate and adaptive immunity and may act synergistically with chemotherapy to increase the susceptibility of the tumor microenvironment to immune-mediated killing. CRS-207 in combination with standard of care (SOC) pemetrexed/cisplatin demonstrated clinical activity in a phase Ib study. Low-dose cyclophosphamide (Cy) has been shown to decrease regulatory T cells and enhance vaccine-induced responses. Preclinical data demonstrate CRS-207 with low-dose Cy improves survival in a murine lung metastasis model.

Methods

60 patients were enrolled into 2 cohorts in this phase Ib study. Eligibility required unresectable, untreated MPM, ECOG 0 or 1, and adequate organ function. Patients in Cohort 1 received 2 CRS-207 2 weeks apart, 6 cycles pemetrexed/cisplatin 3 weeks apart, followed by 2 CRS-207 3 weeks apart. Clinically stable patients continued CRS-207 every 8 weeks. Patients in Cohort 2 received Cy (200 mg/m2) 1 day prior to each CRS-207. Safety, immunogenicity, tumor responses, survival and tumor markers were assessed.

Results

22 patients were enrolled into the Cy/CRS-207 cohort of this study; 77 % male, median age 70. The most common Cy/CRS-207 related adverse events (AEs) were grades 1/2 fever, chills, hypotension and nausea/vomiting, with no treatment-related serious AEs or deaths. As of August 2016, of 22 evaluable patients receiving Cy/CRS-207 + chemotherapy, 86 % (19/22) had disease control with 11 (50 %) whose best overall response was partial response (PR) and 8 (36 %) had stable disease. Tumor shrinkage was observed in 8/22 (36 %) patients including 3 PR after 2 doses of Cy/CRS-207 prior to chemotherapy initiation. Comprehensive immune profiling including multidimensional immunohistochemistry (IHC) analyses will be presented.

Conclusions

Addition of immune-modulating doses of Cy to a regimen of CRS-207 and SOC chemotherapy appears to be well-tolerated with no increase in toxicity compared to those receiving CRS-207 alone with chemotherapy. Preliminary results show signs of tumor activity following 2 doses of Cy/CRS-207 prior to chemotherapy (36 % tumor shrinkage) and the combination with chemotherapy resulted in 86 % disease control and 50 % response rate compared to published response rates of 20-41 % with chemotherapy alone. Immune analyses and further follow-up are warranted to evaluate the Cy/CRS-207 + chemotherapy regimen as a treatment for MPM.

Trial Registration

ClinicalTrials.gov identifier NCT01675765.

P254 Phase Ib trial of RNActive® cancer vaccine BI1361849 (CV9202) and local radiotherapy in stage IV non-small cell lung cancer (NSCLC) patients with disease control after 1st-line therapy: updated clinical results and immune responses

Sven D Koch1, Martin Sebastian2, Christian Weiss3, Martin Früh4, Miklos Pless5, Richard Cathomas6, Wolfgang Hilbe7, Georg Pall8, Thomas Wehler9, Jürgen Alt9, Helge Bischoff10, Michael Geissler11, Frank Griesinger12, Jens Kollmeier13, Alexandros Papachristofilou14, Fatma Doener1, Mariola Fotin-Mleczek1, Madeleine Hipp1, Henoch S Hong1, Karl-Josef Kallen1, Ute Klinkhardt15, Claudia Stosnach15, Birgit Scheel1, Andreas Schroeder15, Tobias Seibel15, Ulrike Gnad-Vogt15, Alfred Zippelius14

1CureVac AG, Tubingen, Baden-Wurttemberg, Germany; 2University Hospital Frankfurt, Medical Clinic II, Goethe University, Frankfurt, Hessen, Germany; 3Klinikum Darmstadt GmbH, Darmstadt, Hessen, Germany; 4Kantonsspital St. Gallen, St. Gallen, Switzerland; 5Kantonsspital Winterthur, Winterthur, Zurich, Switzerland; 6Kantonsspital Graubünden, Chur, Graubunden, Switzerland; 7Wilhelminenspital Wien, Wien, Austria; 8Fachkliniken Wangen, Wangen (Allgäu), Baden-Wurttemberg, Germany; 9J. Gutenberg University Hospital Mainz, Mainz, Rheinland-Pfalz, Germany; 10Thoraxklinik Heidelberg gGmbH, Heidelberg, Baden-Wurttemberg, Germany; 11Klinikum Esslingen GmbH, Esslingen, Baden-Wurttemberg, Germany; 12Pius Hospital Oldenburg, Oldenburg, Niedersachsen, Germany; 13Heckeshorn Lung Clinic, Berlin, Germany; 14University Hospital Basel, Basel-Stadt, Switzerland; 15CureVac AG, Frankfurt am Main, Hessen, Germany
Correspondence: Sven D Koch (sven.koch@curevac.com)

Background

Preclinical studies demonstrated that local radiotherapy (RT) acts synergistically with RNActive® mRNA vaccines to enhance anti-tumor effects and increase tumor-infiltrating lymphocytes. BI1361849 is a therapeutic vaccine comprising optimized mRNA constituents encoding six NSCLC-associated antigens. Interim data of a phase Ib study, employing local RT to increase the immune mediated tumor control by BI1361849, have been previously published [1]. Here we report results of immune response analyses as well as updated safety and efficacy data.

Methods

26 patients (pts) with stage IV NSCLC were enrolled in three cohorts based on histological and molecular NSCLC subtypes (squamous and non-squamous cell with/without activating EGFR mutations). Pts received two vaccinations with BI1361849 before local RT to a single tumor lesion was administered in four consecutive daily fractions of 5 GY. Vaccination was continued until start of subsequent anti-cancer therapy. Maintenance pemetrexed (mP) and EGFR-TKIs were allowed where indicated. Cellular and humoral immune responses were measured ex vivo by multifunctional intracellular cytokine staining, IFN-g ELISpot, and ELISA in pre- and post-treatment blood samples. The induction of humoral immune responses against 27 lung cancer antigens not encoded by the vaccine was measured by antibody array.

Results

26 pts were enrolled. 15 pts received mP, two received EGFR TKIs. Most frequent AEs were mild to moderate injection-site reactions and flu-like symptoms. No BI1361849-related SAEs were reported. Based on preliminary data following up to 110 weeks of exposure, one confirmed PR was observed in a pt on mP, 13 pts (52 %) experienced SD (8 pts on mP, 2 pts on EGFR-TKI and 3 pts without concomitant maintenance treatment, associated with 15 % tumor shrinkage outside the radiation field in one of them). 25 pts were available for immune response analysis. Preliminary data indicate that BI1361849 was capable of eliciting antigen-specific immune responses in of the majority of the patients including cellular and humoral immune responses. Moreover all encoded antigens were immunogenic and responses against multiple antigens were observed. Treatment induced immune responses against other lung cancer antigens were detected in several patients.

Conclusions

BI1361849 can be safely combined with local RT and mP treatment. Shrinkage of non-irradiated lesions and prolonged disease stablization was observed in a subset of pts, mainly in combination with mP. Data indicate immunogenicity of BI1361849. Analyses of cellular and humoral immune responses will be updated, as well as updated clinical data.

References

1. J Clin Oncol 2016, 34(supl):Abstr e20627.

P255 Overcoming resistance to tyrosine kinase inhibitor by natural killer (NK) cells in non-small cell lung cancer (NSCLC) cells

Ha-Ram Park1, Yong-Oon Ahn1, Tae Min Kim2, Soyeon Kim1, Seulki Kim1, Yu Soo Lee1, Bhumsuk Keam2, Dong-Wan Kim2, Dae Seog Heo2

1SNU Cancer Research Institute, Seoul, Republic of Korea; 2Seoul National University Hospital, SNU Cancer Research Institute, Seoul, Republic of Korea
Correspondence: Ha-Ram Park (halam92@naver.com)

Background

Receptor tyrosine kinase signals are altered in NSCLC and tyrosine kinase inhibitors (TKIs) have been used to treat NSCLC harboring driver mutations (e.g. ALK fusion and EGFR). Although TKIs are sensitive to NSCLC with driver mutations, acquired resistance to TKIs is inevitable by various mechanisms including gatekeeper mutation and alternative pathway activation. Considering immunotherapy is one of the main strategies that override drug resistance and cancer stemness, we evaluated an immunologic strategy to overcome acquired resistance to TKIs using NK cells in NSCLC.

Methods

TKI-resistant NSCLC cell lines (H3122CR1, H3122LR1, H3122CR1LR1, EBC-R1, EBC-R2, PC-9GR, and PC-9ER) were established from NCI-H3122 (EML4-ALK fusion), EBC-1 (MET amplification), and PC-9 (EGFR exon 19 deletion) after continuous exposure to crizotinib, ceritinib, capmatinib, gefitinib, and erlotinib. NK cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) using anti-EGFR monoclonal antibody (mAb) cetuximab were measured using ‘off-the-shelf’ NK92-CD16 cell line as effectors and detected by 51chromium-release assay. Expression of the ligands for NK cell receptors and total EGFR were analyzed by flow cytometry.

Results

Most of TKI-resistant NSCLC cell lines were more susceptible to NK92-CD16 cells compared with their parental cell lines. The percentage of cytotoxicity was determined to be 0.2 % in H3122 and 13.4 %, 30.2 % and 39.1 % in TKI-resistant H3122 group with an effector:target ratio of 30:1. (PC-9: 18.2 % vs. 38.8 % vs. 24.8 %). The expression of ICAM-1, which is a ligand for LFA-1 in NK cells, is higher in TKI-resistant NSCLC cells than in parental cells. When we blocked ICAM1-CD11a interaction during a cytotoxic assay, the cytotoxicity was decreased about 10 %. Cetuximab-mediated ADCC was higher in resistant cells due to the increased expression level of total EGFR in resistant cells.

Conclusions

TKI-resistant NSCLC cells are more sensitive to NK92 cell-mediated cytotoxicity that is partially dependent on up-regulation of ICAM-1 via an immunological synapse. In addition, cetuximab, an EGFR-targeting mAb, significantly increases NK cell cytotoxicity in TKI-resistant NSCLC cells. Taken together, NK-cell based immunotherapy with cetuximab might be feasible to treat NSCLC patients with acquired resistance to TKIs.

P256 Intralesional injection with Rose Bengal and systemic chemotherapy induces anti-tumor immunity in a murine model of pancreatic cancer

Shari Pilon-Thomas, Amy Weber, Jennifer Morse, Krithika Kodumudi, Hao Liu, John Mullinax, Amod A Sarnaik

H. Lee Moffitt Cancer Center, Tampa, FL, USA
Correspondence: Shari Pilon-Thomas (shari.pilon-thomas@moffitt.org)

Background

Rose Bengal is a xanthene dye that has been utilized for liver function studies and is currently used topically in ophthalmology. Intralesional (IL) Rose Bengal (PV-10) has been shown in murine models and melanoma clinical trials to induce regression of treated melanoma lesions and uninjected bystander lesions. This study was undertaken to measure whether IL PV-10 can induce systemic anti-tumor effects alone or in combination with gemcitabine (Gem) therapy in a murine model of pancreatic cancer.

Methods

C57BL/6 mice received Panc02 pancreatic tumor cells subcutaneously (SC) on one flank to establish a single tumor. On day 7, tumor was treated with IL PV-10. Control mice received IL phosphate buffered saline (PBS). Tumor growth was measured. Splenic T cells were collected and co-cultured with Panc02 or irrelevant B16 cells. Supernatants were collected to measure Panc02-specific T cell responses by IFN-gamma ELISA. To measure the effect of IL PV-10 on the growth of an untreated, bystander tumor, mice received Panc02 cells in bilateral flanks. The resulting right tumor was injected IL with PV-10 or PBS. Tumor sizes were measured for both the right (treated) and left (untreated/bystander) tumors. To determine the efficacy of combination therapy with IL PV-10 and systemic Gem, mice bearing a single or bilateral Panc02 tumors were treated with PV-10 alone or in combination with Gem. Mice received 60 mg/kg Gem intraperitoneally (IP) twice per week.

Results

C57BL/6 mice bearing Panc02 tumors treated with IL PV-10 had significantly smaller tumors than mice treated with PBS (p < 0.001). A significant increase in the IFN-gamma production in response to Panc02 was measured in the splenocytes of mice treated with PV-10 as compared to mice treated with PBS (p < 0.05). Mice with bilateral tumors had a significant regression of tumors injected IL with PV-10 and there was a reduction in the untreated (bystander) flank Panc02 tumor (p < 0.01). Gem therapy in combination with IL PV-10 injection led to enhanced tumor regression (p < 0.05) compared to IL PV-10 or Gem alone in both a single tumor model and a bilateral tumor model.

Conclusions

Regression of untreated pancreatic tumors by IL injection of PV-10 in concomitant tumor supports the induction of a systemic anti-tumor response. Addition of Gem chemotherapy enhances the effects of IL PV-10 therapy. Given that patients with metastatic pancreatic cancer have a dismal prognosis, combination therapy of IL PV-10 combined with Gem may benefit patients with metastatic pancreatic cancer.

P257 Multi-institution evaluation of outcomes following radiation and PD-1 inhibition

Luke Pike1, Andrew Bang2, Patrick A. Ott3, Tracy Balboni1, Allison Taylor1, Alexander Spektor1, Tyler Wilhite1, Monica Krishnan1, Daniel Cagney1, Brian Alexander1, Ayal Aizer1, Elizabeth Buchbinder1, Mark Awad1, Leena Ghandi1, F Stephen Hodi3, Jonathan Schoenfeld1

1Brigham and Women's/Dana-Farber Cancer Center, Harvard University, Boston, MA, USA; 2Harvard Radiation Oncology Program, Boston, MA, USA; 3Dana-Farber Cancer Institute, Harvard University, Boston, MA, USA
Correspondence: Jonathan Schoenfeld (jdschoenfeld@partners.org)

Background

Preclinical models suggest radiation may synergize with immunotherapy; for instance, increased responses and prolonged survival have been observed in mice treated with radiation and either PD-1 inhibition or combined CTLA-4/PD-1 blockade. We previously observed that radiation was associated with favorable responses in melanoma patients treated with ipilimumab [1]. However, clinical data are lacking in regards to combining radiation with PD-1 inhibitors with or without CTLA-4 blockade.

Methods

We conducted an IRB-approved retrospective multi-institution analysis of patients with metastatic melanoma, non-small cell lung cancer (NSCLC), and renal cell carcinoma (RCC) treated at 6 centers with palliative radiation and PD-1 inhibitors, either before, after, or concurrent with radiation.