Abstract Introduction: Checkpoint inhibition (CPI) has been shown to be an effective anti-tumor therapy, but CPI alone is frequently insufficient to control tumor growth, and costimulatory signals may also be required to produce clinically significant anti-tumor responses. PD1-PDL1 are established CPI targets and TMIGD2 is an inhibitory receptor expressed on T cells that is engaged by its cognate ligand HHLA2 on tumor cells, leading to inhibition of T cell responses. Novel biologics combining CD28 costimulation and CPI may yield promising tumor antigen-specific therapeutic candidates. Methods: Variants of CD86 with increased CD28 affinity were engineered using our directed evolution platform. PD1 and TMIGD2 variants were also engineered for increased affinity to PDL1 and HHLA2, respectively. Fusion proteins were generated including either PD1 or TMIGD2 domains, an effectorless Fc domain, and an engineered CD86 domain to generate proteins to provide target-dependent costimulation (TDC) and evaluated in multiple in vitro T cell stimulation assays. Costimulatory effects were confirmed by evaluating T cell proliferation, cytokine release, and tumor cell killing. Fusion proteins were also tested in vivo in a mouse tumor model using MC38 cells expressing human PDL1 or HHLA2, and in a humanized tumor model where an HPV+ squamous cell carcinoma cell line expressing either PDL1 or HHLA2 was implanted into NSG mice. Tumor-bearing NSG mice were given primary human T cells expressing an HPV peptide-specific TCR, with or without the corresponding fusion protein, and tumor growth was measured over time. Results: PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced T cell costimulation in multiple in vitro T cell response assays, and costimulation was dependent on target cell lines expressing PDL1 or HHLA2, respectively. There was no effect on T cell responses if the target cells did not express the relevant target protein. PD1 and TMIGD2 domains alone had little effect on T cell responses, indicating these TDC proteins were driving enhanced responses. PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced anti-tumor responses in vivo in a syngeneic MC38 implantation model only when MC38 cells expressed human PDL1 or HHLA2, respectively. Both proteins also enhanced antitumor responses in a humanized tumor implantation system using an HPV+ tumor cell line expressing the corresponding target antigen in the presence of human T cells expressing an HPV-specific TCR, but failed to control tumor growth when tumor cells lacked expression of the target protein. Conclusions: Tumor antigen-specific antitumor therapy can be achieved with fusion proteins that combine engineered CPI and CD28 costimulatory domains. Such novel biologics may provide promising approaches to enhancing the efficacy of CPI monotherapies and to address checkpoint inhibitor-resistant tumors. Citation Format: Steven D. Levin, Mark F. Maurer, Chelsea Gudgeon, Siddarth Chandrasekaran, Daniel Ardourel, Daniel Demonte, Joseph Kuijper, Martin Wolfson, Logan Garrett, Kayla N. Kleist, Sherri Mudri, Hieu Nguyen, Michelle Seaberg, Rachel Wang, Jing Yang, Katherine E. Lewis, Stacey R. Dillon, Mark Rixon, Stanford L. Peng. Engineered variant domain fusion proteins provide checkpoint inhibition and tumor antigen dependent CD28 costimulation resulting in potent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1740.

BackgroundPreclinical evidence supports combining checkpoint inhibition (CPI) with T cell costimulatory agonism to improve the breadth and durability of anti-tumor responses relative to CPI alone. Currently, there are a number of therapeutic approaches combining costimulatory receptor agonists (e.g. CD28, 4-1BB, OX40L, etc.) with tumor targeting agents and/or CPI. Identification of a pharmacodynamically-justified therapeutic dose can be challenging because traditional duration of target occupancy does not necessarily correlate with immunological activity in the case of costimulatory molecules, and an ‘always on’ dose risks immune exhaustion. ALPN-202, a variant CD80 vIgD-Fc fusion protein that mediates PD-L1-dependent CD28 costimulation and inhibits the PD-L1 and CTLA-4 checkpoints, is in development for the treatment of multiple advanced malignancies. To assess clinical CD28 agonism in the context of ALPN-202 treatment, we developed a novel, ex vivo whole blood target-dependent costimulation (TDC) assay.MethodsA TDC assay was developed using clinical samples from NEON-1 (NCT04186637), an ongoing dose escalation and expansion clinical trial of ALPN-202 for patients with advanced malignancies. The assay uses patient blood stimulated with paraformaldehyde-fixed, artificial antigen presenting cells (aAPC) expressing both cell-surface anti-CD3 and PD-L1. Pre-dose and end-of-infusion (EOI) blood was drawn from trial participants and co-cultured for 24 hours with the aAPCs in a pre-made assay plate. Plasma was collected and secreted IL-2 was quantified and used as a measure of PD-L1-dependent CD28 costimulation. Nonlinear regression was used to calculate area under the curve (AUC) for each condition, and sample AUC values were compared to a positive control (pre-dose blood stimulated with a fixed concentration of ALPN-202). Serum concentration of ALPN-202 and CD28 target saturation analyses were conducted concurrently to evaluate the exposure-response relationship.ResultsUsing the ex vivo TDC assay, ALPN-202 demonstrated PD-L1-dependent T cell costimulation at all dose levels tested to date in the NEON-1 clinical trial, consistent with preclinical assay development data. Similarly, CD28 target saturation levels on circulating T cells correlated with serum concentration of ALPN-202.ConclusionsWe have developed a novel ex vivo assay to assess induction of PD-L1-dependent CD28 costimulation in a therapeutic setting. This assay has been successfully employed to monitor controlled CD28 costimulation by the CD28 agonist therapeutic candidate ALPN-202, helping to establish a PK/PD relationship that is consistent with preclinical data. More broadly, this type of cell-based, ex vivo TDC assay could be adapted to assess costimulatory receptor engagement, particularly target-dependent costimulation, for other therapeutic agonists in clinical development.Ethics ApprovalThis study was approved by WCG IRB’s Human Subjects Review, approval number: 20211877.

Abstract Background: Despite significant clinical benefit of checkpoint inhibitors (CPI) in some settings, unfortunately, the majority of patients still fail to respond and/or develop resistance. This is likely due to a complex set of factors including, but not limited to, the presence of immunosuppressive myeloid cells and/or T cell exhaustion due to chronic TCR activation in the absence of sufficient costimulation. ALPN-202, a variant CD80 vIgD™-Fc fusion protein that mediates PD-L1-dependent CD28 costimulation and blocks PD-L1 and CTLA-4, was designed to overcome several of these suppressive mechanisms. The objective of these studies was to measure effects of ALPN-202 on the suppression of T cell activation by M2c macrophages and to determine in vivo effects of ALPN-202 on T cell exhaustion in an adoptively transferred human TCR transgenic tumor model. Methods: M2c macrophages differentiated from primary monocytes with M-CSF and IL-10 were cocultured for 72hr with T cells, anti-CD3 and a titration of ALPN-202, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies. Cytokine concentrations in the culture media were measured at 24 and 72 hrs, and T cells and macrophages characterized at 72 hrs by flow cytometry. To measure its anti-tumor activity and effect on T cell exhaustion in vivo, ALPN-202 was evaluated in a humanized model using anti-HPV E6 TCR-transduced human T cells transferred into immunodeficient NSG mice bearing HPV+ SCC152 squamous cell tumors stably expressing PD-L1. Tumor volume was measured twice weekly and on day 38 tumors were harvested, digested, and intratumoral T cells characterized for expression of exhaustion markers. Results: In the in vitro coculture assay, ALPN-202 increased T cell proliferation and production of IL-2, IFNγ, TNFα, GM-CSF, IL-6, and IL-21 significantly more potently than CPI alone. Additionally, the M2c macrophages in the presence of ALPN-202 displayed a dose-dependent elevation of MHC II, CD80, and CD86, indicative of a more pro-inflammatory, M1-like phenotype. In the SCC152 tumor model, ALPN-202 induced a more robust anti-tumor response than CPI while the intratumoral T cells expressed lower levels of exhaustion markers. Conclusions: As a dual checkpoint inhibitor and conditional CD28-costimulator, ALPN-202 induced robust and selective T cell costimulation in vitro which overcame M2c macrophage-mediated suppression more potently than CPI alone. Intriguingly, not only were T cells vigorously activated, but M2c macrophages transitioned to a more M1-like proinflammatory phenotype. In a humanized tumor model, ALPN-202 treatment resulted in potent anti-tumor activity with reduced signs of T cell exhaustion in the TME. The data suggest that by combining CD28 costimulation with CPI, ALPN-202 may provide a more robust and persistent anti-tumor T cell response compared to CPI alone. Citation Format: Mark F. Maurer, Siddarth Chandrasekaran, Katherine Lewis, Sherri Mudri, Kayla Kleist, Fariha Ahmed-Qadri, Chelsea Gudgeon, Steven D. Levin, Stacey R. Dillon, Kristine M. Swiderek, Stanford L. Peng. ALPN-202 combines checkpoint inhibition with conditional T cell costimulation to overcome T cell suppression by M2c macrophages and improve the durability of engineered T cell anti-tumor responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-085.

Abstract Introduction: PD-1 pathway antagonists have revealed the importance of checkpoint pathways in regulating antitumor immunity, but an existing immune response is generally required for clinical efficacy. Specific T cell costimulation through CD28 is central to this process, but the CD28 ligands CD80 and CD86 are often poorly expressed in the tumor microenvironment, accounting for a second important mechanism of immune evasion by tumors. In contrast, PD-L1 expression has been found extensively in multiple tumor cell types. Therapeutics that combine PD-L1/PD-1 antagonism coupled with PD-L1 dependent CD28 agonism may therefore provide a more potent, yet safe immunotherapeutic approach. Experimental Procedures: The variant Ig Domain (vIgD)TM platform has generated a diversity of human CD80 variants using yeast display affinity maturation and selections against all three CD80 counterstructures CD28, CTLA-4, and PD-L1. CD80 vIgDs were produced in a mammalian expression system as recombinant Fc fusion proteins (CD80 vIgD-Fc proteins) and their binding properties were quantified by flow cytometry. Functional activity was determined in vitro by assessing responses from human primary T cells or an IL-2-luciferase Jurkat T cell reporter line stimulated with PD-L1-expressing artificial antigen presenting cells (aAPCs). In vitro human T cell cytotoxicity assays with a human PD-L1-expressing tumor line were also performed. Antitumor activity was assessed in vivo with mice implanted with human PD-L1 transduced MC38 tumors. Data Summary: The human CD80 IgV fragment was found to be optimal for high affinity PD-L1 and CD28 binding. A large panel of CD80 vIgD-Fc proteins demonstrated a range of binding towards CD28, PD-L1, and/or CTLA-4, and CD80 vIgD-Fc proteins with high affinity for PD-L1 antagonized the PD-L1/PD-1 interaction. Some CD80 vIgD-Fc proteins agonized CD28 in a PD-L1 dependent fashion with increased luciferase activity in the Jurkat reporter assay as well as increased cytokine production by primary human T cells when stimulated with a PD-L1 expressing aAPC in vitro. The same candidates also showed specific killing of human PD-L1 expressing tumor cells in vitro compared to the parental tumor line lacking PD-L1 expression. Importantly, selected CD80 vIgD-Fc proteins caused significant tumor reduction in the MC38 in vivo tumor model. Conclusion: Engineered CD80 vIgD-Fc proteins that deliver a localized CD28 costimulatory signal to T cells while simultaneously antagonizing the inhibitory PD-L1/PD-1 pathway may provide a transformative mechanism of action to drive potent, tolerable antitumor immunity. Preclinical development of therapeutic candidates is under way. Citation Format: Ryan Swanson, Mark F. Maurer, Chris L. Navas, Chelsea J. Gudgeon, Joseph L. Kuijper, Martin Wolfson, Katherine E. Lewis, Stacey R. Dillon, Steve D. Levin, Michael G. Kornacker. CD80 vIgD-Fc proteins combine checkpoint antagonism and costimulatory signaling for potent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4550.

Background: Simultaneous blockade of LAG-3 and PD-1 may synergistically restore T-cell activation and enhance antitumor immunity. In a phase 1/2a study (NCT01968109), BMS-986016 (anti–LAG-3) + nivolumab (anti–PD-1) showed promising antitumor activity in the mel prior IO cohort (Ascierto et al. J Clin Oncol. 2017;35(suppl) [abstr 9520]). Here, we describe updated efficacy and safety in a larger mel prior IO cohort in all-comer and biomarker-enriched populations.

Elotuzumab, a humanized monoclonal antibody that binds human signaling lymphocytic activation molecule F7 (hSLAMF7) on myeloma cells, was developed to treat patients with multiple myeloma (MM). Elotuzumab has a dual mechanism of action that includes the direct activation of natural killer (NK) cells and the induction of NK cell-mediated antibody-dependent cellular cytotoxicity. This study aimed to characterize the effects of elotuzumab on NK cells in vitro and in patients with MM and to determine whether elotuzumab antitumor activity was improved by programmed death receptor-1 (PD-1) blockade. Elotuzumab promoted NK cell activation when added to a coculture of human NK cells and SLAMF7-expressing myeloma cells. An increased frequency of activated NK cells was observed in bone marrow aspirates from elotuzumab-treated patients. In mouse tumor models expressing hSLAMF7, maximal antitumor efficacy of a murine immunoglobulin G2a version of elotuzumab (elotuzumab-g2a) required both Fcγ receptor-expressing NK cells and CD8+ T cells and was significantly enhanced by coadministration of anti-PD-1 antibody. In these mouse models, elotuzumab-g2a and anti-PD-1 combination treatment promoted tumor-infiltrating NK and CD8+ T-cell activation, as well as increased intratumoral cytokine and chemokine release. These observations support the rationale for clinical investigation of elotuzumab/anti-PD-1 combination therapy in patients with MM.

Table of contents MELANOMA BRIDGE 2015 KEYNOTE SPEAKER PRESENTATIONS Molecular and immuno-advances K1 Immunologic and metabolic consequences of PI3K/AKT/mTOR activation in melanoma Vashisht G. Y. Nanda, Weiyi Peng, Patrick Hwu, Michael A. Davies K2 Non-mutational adaptive changes in melanoma cells exposed to BRAF and MEK inhibitors help the establishment of drug resistance Gennaro Ciliberto, Luigi Fattore, Debora Malpicci, Luigi Aurisicchio, Paolo Antonio Ascierto, Carlo M. Croce, Rita Mancini K3 Tumor-intrinsic beta-catenin signaling mediates tumor-immune avoidance Stefani Spranger, Thomas F. Gajewski K4 Intracellular tumor antigens as a source of targets of antibody-based immunotherapy of melanoma Yangyang Wang, Soldano Ferrone Combination therapies K5 Harnessing radiotherapy to improve responses to immunotherapy in cancer Claire Vanpouille-Box, Erik Wennerberg, Karsten A. Pilones, Silvia C. Formenti, Sandra Demaria K6 Creating a T cell-inflamed tumor microenvironment overcomes resistance to checkpoint blockade Haidong Tang, Yang Wang, Yang-Xin Fu K7 Biomarkers for treatment decisions? Reinhard Dummer K8 Combining oncolytic therapies in the era of checkpoint inhibitors Igor Puzanov K9 Immune checkpoint blockade for melanoma: should we combine or sequence ipilimumab and PD-1 antibody therapy? Michael A. Postow News in immunotherapy K10 An update on adjuvant and neoadjuvant therapy for melanom Ahmad Tarhini K11 Targeting multiple inhibitory receptors in melanoma Joe-Marc Chauvin, Ornella Pagliano, Julien Fourcade, Zhaojun Sun, Hong Wang, Cindy Sanders, John M. Kirkwood, Tseng-hui Timothy Chen, Mark Maurer, Alan J. Korman, Hassane M. Zarour K12 Improving adoptive immune therapy using genetically engineered T cells David F. Stroncek Tumor microenvironment and biomarkers K13 Myeloid cells and tumor exosomes: a crosstalk for assessing immunosuppression? Veronica Huber, Licia Rivoltini K14 Update on the SITC biomarker taskforce: progress and challenges Magdalena Thurin World-wide immunoscore task force: an update K15 The immunoscore in colorectal cancer highlights the importance of digital scoring systems in surgical pathology Tilman Rau, Alessandro Lugli K16 The immunoscore: toward an integrated immunomonitoring from the diagnosis to the follow up of cancer’s patients Franck Pagès Economic sustainability of melanoma treatments: regulatory, health technology assessment and market access issues K17 Nivolumab, the regulatory experience in immunotherapy Jorge Camarero, Arantxa Sancho K18 Evidence to optimize access for immunotherapies Claudio Jommi ORAL PRESENTATIONS Molecular and immuno-advances O1 Ipilimumab treatment results in CD4 T cell activation that is concomitant with a reduction in Tregs and MDSCs Yago Pico de Coaña, Maria Wolodarski, Yuya Yoshimoto, Giusy Gentilcore, Isabel Poschke, Giuseppe V. Masucci, Johan Hansson, Rolf Kiessling O2 Evaluation of prognostic and therapeutic potential of COX-2 and PD-L1 in primary and metastatic melanoma Giosuè Scognamiglio, Francesco Sabbatino, Federica Zito Marino, Anna Maria Anniciello, Monica Cantile, Margherita Cerrone, Stefania Scala, Crescenzo D’alterio, Angela Ianaro, Giuseppe Cirino, Paolo Antonio Ascierto, Giuseppina Liguori, Gerardo Botti O3 Vemurafenib in patients with BRAFV600 mutation–positive metastatic melanoma: final overall survival results of the BRIM-3 study Paul B. Chapman, Caroline Robert, James Larkin, John B. Haanen, Antoni Ribas, David Hogg, Omid Hamid, Paolo Antonio Ascierto, Alessandro Testori, Paul Lorigan, Reinhard Dummer, Jeffrey A. Sosman, Keith T. Flaherty, Huibin Yue, Shelley Coleman, Ivor Caro, Axel Hauschild, Grant A. McArthur O4 Updated survival, response and safety data in a phase 1 dose-finding study (CA209-004) of concurrent nivolumab (NIVO) and ipilimumab (IPI) in advanced melanoma Mario Sznol, Margaret K. Callahan, Harriet Kluger, Michael A. Postow, RuthAnn Gordan, Neil H. Segal, Naiyer A. Rizvi, Alexander Lesokhin, Michael B. Atkins, John M. Kirkwood, Matthew M. Burke, Amanda Ralabate, Angel Rivera, Stephanie A. Kronenberg, Blessing Agunwamba, Mary Ruisi, Christine Horak, Joel Jiang, Jedd Wolchok Combination therapies O5 Efficacy and correlative biomarker analysis of the coBRIM study comparing cobimetinib (COBI) + vemurafenib (VEM) vs placebo (PBO) + VEM in advanced BRAF-mutated melanoma patients (pts) Paolo A. Ascierto, Grant A. McArthur, James Larkin, Gabriella Liszkay, Michele Maio, Mario Mandalà, Lev Demidov, Daniil Stoyakovskiy, Luc Thomas, Luis de la Cruz-Merino, Victoria Atkinson, Caroline Dutriaux, Claus Garbe, Matthew Wongchenko, Ilsung Chang, Daniel O. Koralek, Isabelle Rooney, Yibing Yan, Antoni Ribas, Brigitte Dréno O6 Preliminary clinical safety, tolerability and activity results from a Phase Ib study of atezolizumab (anti-PDL1) combined with vemurafenib in BRAFV600-mutant metastatic melanoma Ryan Sullivan, Omid Hamid, Manish Patel, Stephen Hodi, Rodabe Amaria, Peter Boasberg, Jeffrey Wallin, Xian He, Edward Cha, Nicole Richie, Marcus Ballinger, Patrick Hwu O7 Preliminary safety and efficacy data from a phase 1/2 study of epacadostat (INCB024360) in combination with pembrolizumab in patients with advanced/metastatic melanoma Thomas F. Gajewski, Omid Hamid, David C. Smith, Todd M. Bauer, Jeffrey S. Wasser, Jason J. Luke, Ani S. Balmanoukian, David R. Kaufman, Yufan Zhao, Janet Maleski, Lance Leopold, Tara C. Gangadhar O8 Primary analysis of MASTERKEY-265 phase 1b study of talimogene laherparepvec (T-VEC) and pembrolizumab (pembro) for unresectable stage IIIB-IV melanoma Reinhard Dummer, Georgina V. Long, Antoni Ribas, Igor Puzanov, Olivier Michielin, Ari VanderWalde, Robert H.I. Andtbacka, Jonathan Cebon, Eugenio Fernandez, Josep Malvehy, Anthony J. Olszanski, Thomas F. Gajewski, John M. Kirkwood, Christine Gause, Lisa Chen, David R. Kaufman, Jeffrey Chou, F. Stephen Hodi News in immunotherapy O9 Two-year survival and safety update in patients (pts) with treatment-naïve advanced melanoma (MEL) receiving nivolumab (NIVO) or dacarbazine (DTIC) in CheckMate 066 Victoria Atkinson, Paolo A. Ascierto, Georgina V. Long, Benjamin Brady, Caroline Dutriaux, Michele Maio, Laurent Mortier, Jessica C. Hassel, Piotr Rutkowski, Catriona McNeil, Ewa Kalinka-Warzocha, Celeste Lebbé, Lars Ny, Matias Chacon, Paola Queirolo, Carmen Loquai, Parneet Cheema, Alfonso Berrocal, Karmele Mujika Eizmendi, Luis De La Cruz-Merino, Gil Bar-Sela, Christine Horak, Joel Jiang, Helene Hardy, Caroline Robert O10 Efficacy and safety of nivolumab (NIVO) in patients (pts) with advanced melanoma (MEL) who were treated beyond progression in CheckMate 066/067 Georgina V. Long, Jeffrey S. Weber, James Larkin, Victoria Atkinson, Jean-Jacques Grob, Reinhard Dummer, Caroline Robert, Ivan Marquez-Rodas, Catriona McNeil, Henrik Schmidt, Karen Briscoe, Jean-François Baurain, F. Stephen Hodi, Jedd D. Wolchok Tumor microenvironment and biomarkers O11 New biomarkers for response/resistance to BRAF inhibitor therapy in metastatic melanoma Rosamaria Pinto, Simona De Summa, Vito Michele Garrisi, Sabino Strippoli, Amalia Azzariti, Gabriella Guida, Michele Guida, Stefania Tommasi O12 Chemokine receptor patterns in lymphocytes mirror metastatic spreading in melanoma and response to ipilimumab Nicolas Jacquelot, David Enot, Caroline Flament, Jonathan M. Pitt, Nadège Vimond, Carolin Blattner, Takahiro Yamazaki, Maria-Paula Roberti, Marie Vetizou, Romain Daillere, Vichnou Poirier-Colame, Michaëla Semeraro, Anne Caignard, Craig L Slingluff Jr, Federica Sallusto, Sylvie Rusakiewicz, Benjamin Weide, Aurélien Marabelle, Holbrook Kohrt, Stéphane Dalle, Andréa Cavalcanti, Guido Kroemer, Anna Maria Di Giacomo, Michaele Maio, Phillip Wong, Jianda Yuan, Jedd Wolchok, Viktor Umansky, Alexander Eggermont, Laurence Zitvogel O13 Serum levels of PD1- and CD28-positive exosomes before Ipilimumab correlate with therapeutic response in metastatic melanoma patients Passarelli Anna, Tucci Marco, Stucci Stefania, Mannavola Francesco, Capone Mariaelena, Madonna Gabriele, Ascierto Paolo Antonio, Silvestris Franco O14 Immunological prognostic factors in stage III melanomas María Paula Roberti, Nicolas Jacquelot, David P Enot, Sylvie Rusakiewicz, Michaela Semeraro, Sarah Jégou, Camila Flores, Lieping Chen, Byoung S. Kwon, Ana Carrizossa Anderson, Caroline Robert, Christophe Borg, Benjamin Weide, François Aubin, Stéphane Dalle, Michele Maio, Jedd D. Wolchok, Holbrook Kohrt, Maha Ayyoub, Guido Kroemer, Aurélien Marabelle, Andréa Cavalcanti, Alexander Eggermont, Laurence Zitvogel POSTER PRESENTATIONS Molecular and immuno-advances P1 Human melanoma cells resistant to B-RAF and MEK inhibition exhibit mesenchymal-like features Anna Lisa De Presbiteris, Fabiola Gilda Cordaro, Rosa Camerlingo, Federica Fratangelo, Nicola Mozzillo, Giuseppe Pirozzi, Eduardo J. Patriarca, Paolo A. Ascierto, Emilia Caputo P2 Anti-proliferative and pro-apoptotic effect of ABT888 on melanoma cell lines and its potential role in the treatment of melanoma resistant to B-RAF inhibitors Federica Fratangelo, Rosa Camerlingo, Emilia Caputo, Maria Letizia Motti, Rosaria Falcone, Roberta Miceli, Mariaelena Capone, Gabriele Madonna, Domenico Mallardo, Maria Vincenza Carriero, Giuseppe Pirozzi and Paolo Antonio Ascierto P3 Involvement of the L-cysteine/CSE/H2S pathway in human melanoma progression Elisabetta Panza, Paola De Cicco, Chiara Armogida, Giuseppe Ercolano, Rosa Camerlingo, Giuseppe Pirozzi, Giosuè Scognamiglio, Gerardo Botti, Giuseppe Cirino, Angela Ianaro P4 Cancer stem cell antigen revealing pattern of antibody variable region genes were defined by immunoglobulin repertoire analysis in patients with malignant melanoma Beatrix Kotlan, Gabriella Liszkay, Miri Blank, Timea Balatoni, Judit Olasz, Emil Farkas, Andras Szollar, Akos Savolt, Maria Godeny, Orsolya Csuka, Szabolcs Horvath, Klara Eles, Yehuda Shoenfeld and Miklos Kasler P5 Upregulation of Neuregulin-1 expression is a hallmark of adaptive response to BRAF/MEK inhibitors in melanoma Debora Malpicci, Luigi Fattore, Susan Costantini, Francesca Capone, Paolo Antonio Ascierto, Rita Mancini, Gennaro Ciliberto P6 HuR positively regulates migration of HTB63 melanoma cells Farnaz Moradi, Pontus Berglund, Karin Leandersson, Rickard Linnskog, Tommy Andersson, Chandra Prakash Prasad P7 Prolyl 4- (C-P4H) hydroxylases have opposing effects in malignant melanoma: implication in prognosis and therapy Cristiana Lo Nigro, Laura Lattanzio, Hexiao Wang, Charlotte Proby, Nelofer Syed, Marcella Occelli, Carolina Cauchi, Marco Merlano, Catherine Harwood, Alastair Thompson, Tim Crook P8 Urokinase receptor antagonists: novel agents for the treatment of melanoma Maria Letizia Motti, Katia Bifulco, Vincenzo Ingangi, Michele Minopoli, Concetta Ragone, Federica Fratangelo, Antonello Pessi, Gennaro Ciliberto, Paolo Antonio Ascierto, Maria Vincenza Carriero P9 Exosomes released by melanoma cell lines enhance chemotaxis of primary tumor cells Francesco Mannavola, Stella D’Oronzo, Claudia Felici, Marco Tucci, Antonio Doronzo, Franco Silvestris P10 New insights in mitochondrial metabolic reprogramming in melanoma Anna Ferretta, Gabriella Guida, Stefania Guida, Imma Maida, Tiziana Cocco, Sabino Strippoli, Stefania Tommasi, Amalia Azzariti, Michele Guida P11 Lenalidomide restrains the proliferation in melanoma cells through a negative regulation of their cell cycle Stella D’Oronzo, Anna Passarelli, Claudia Felici, Marco Tucci, Davide Quaresmini, Franco Silvestris Combination therapies P12 Chemoimmunotherapy elicits polyfunctional anti-tumor CD8 + T cells depending on the activation of an AKT pathway sustained by ICOS Ornella Franzese, Belinda Palermo, Cosmo Di Donna, Isabella Sperduti, MariaLaura Foddai, Helena Stabile, Angela Gismondi, Angela Santoni, Paola Nisticò P13 Favourable toxicity profile of combined BRAF and MEK inhibitors in metastatic melanoma patients Andrea P. Sponghini, Francesca Platini, Elena Marra, David Rondonotti, Oscar Alabiso, Maria T. Fierro, Paola Savoia, Florian Stratica, Pietro Quaglino P14 Electrothermal bipolar vessel sealing system dissection reduces seroma output or time to drain removal following axillary and ilio-inguinal node dissection in melanoma patients: a pilot study Di Monta Gianluca, Caracò Corrado, Di Marzo Massimiliano, Marone Ugo, Di Cecilia Maria Luisa, Mozzillo Nicola News in immunotherapy P15 Clinical and immunological response to ipilimumab in a metastatic melanoma patient with HIV infection Francesco Sabbatino, Celeste Fusciello1, Antonio Marra, Rosario Guarrasi, Carlo Baldi, Rosa Russo, Di Giulio Giovanni, Vincenzo Faiola, Pio Zeppa, Stefano Pepe P16 Immunotherapy and hypophysitis: a case report Elisabetta Gambale, Consiglia Carella, Alessandra Di Paolo, Michele De Tursi Tumor microenvironment and biomarkers P17 New immuno- histochemical markers for the differential diagnosis of atypical melanocytic lesions with uncertain malignant potential Laura Marra, Giosuè Scognamiglio, Monica Cantile, Margherita Cerrone, Fara De Murtas, Valeria Sorrentino, Anna Maria Anniciello, Gerardo Botti P18 Utility of simultaneous measurement of three serum tumor markers in melanoma patients Angela Sandru, Silviu Voinea, Eugenia Panaitescu, Madalina Bolovan, Adina Stanciu, Sabin Cinca P19 The significance of various cut-off levels of melanoma inhibitory activity in evaluation of cutaneous melanoma patients Angela Sandru, Silviu Voinea, Eugenia Panaitescu, Madalina Bolovan, Adina Stanciu, Sabin Cinca P20 The long noncoding RNA HOTAIR is associated to metastatic progression of melanoma and it can be identified in the blood of patients with advanced disease Chiara Botti, Giosuè Scognamiglio, Laura Marra, Gabriella Aquino, Rosaria Falcone, Annamaria Anniciello, Paolo Antonio Ascierto, Gerardo Botti, Monica Cantile Other P21 The effect of Sentinel Lymph Node Biopsy in melanoma mortality: timing of dissection Cristina Fortes, Simona Mastroeni, Alessio Caggiati, Francesca Passarelli, Alba Zappalà, Maria Capuano, Riccardo Bono, Maurizio Nudo, Claudia Marino, Paola Michelozzi P22 Epidemiological survey on related psychopathology in melanoma Valeria De Biasio, Vincenzo C. Battarra IMMUNOTHERAPY BRIDGE KEYNOTE SPEAKER PRESENTATIONS Immunotherapy beyond melanoma K19 Predictor of response to radiation and immunotherapy Silvia Formenti K20 Response and resistance to PD-1 pathway blockade: clues from the tumor microenvironment Maria Libera Ascierto, Tracee L. McMiller, Alan E. Berger, Ludmila Danilova, Robert A. Anders, George J. Netto, Haiying Xu, Theresa S. Pritchard, Jinshui Fan, Chris Cheadle, Leslie Cope, Charles G. Drake, Drew M. Pardoll, Janis M. Taube and Suzanne L. Topalian K21 Combination immunotherapy with autologous stem cell transplantation, protein immunization, and PBMC reinfusion in myeloma patients Sacha Gnjatic, Sarah Nataraj, Naoko Imai, Adeeb Rahman, Achim A. Jungbluth, Linda Pan, Ralph Venhaus, Andrew Park, Frédéric F. Lehmann, Nikoletta Lendvai, Adam D. Cohen, and Hearn J. Cho K22 Anti-cancer immunity despite T cell “exhaustion” Speiser Daniel Immunotherapy in oncology (I-O): data from clinical trial K23 The Checkpoint Inhibitors for the Treatment of Metastatic Non-small Cell Lung Cancer (NSCLC) Vera Hirsh

HIV infection induces phenotypic and functional changes to CD8(+) T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8(+) T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT(+) and TIGIT(+) PD-1(+)CD8(+) T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8(+) T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8(+) T cell effector responses. The frequency of TIGIT(+) CD4(+) T cells correlated with the CD4(+) T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA T-cell Immunoglobulin and ITIM Domain (TIGIT) is an inhibitory receptor expressed by activated T cells, regulatory T cells and NK cells. Here, we show that tumor antigen (TA)-specific CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) upregulate TIGIT and most often co-express TIGIT and PD-1 in melanoma patients. CD8+ TILs downregulate CD226, supporting an imbalance of TIGIT/CD226 expression in metastatic melanoma. TIGIT is a marker of early T cell activation, which is further upregulated by T cells upon PD-1 blockade and by dysfunctional PD-1+Tim-3+ TA-specific CD8+ T cells. PD-1+TIGIT+, PD-1−TIGIT+ and PD-1+ TIGIT− CD8+ TILs exhibit similar functional capacities ex vivo, suggesting that TIGIT upregulation alone or together with PD-1 is not a marker of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT blockade adds to PD-1 blockade to increase the proliferation, cytokine production and degranulation of TA-specific CD8+ T cells and CD8+ TILs. Collectively, our findings show that TIGIT together with PD-1 regulate the expansion and function of TA-specific CD8+ T cells and CD8+TILs in melanoma patients. They also support the use of dual TIGIT and PD-1 blockade to stimulate potent antitumor CD8+ T cells responses in patients with advanced melanoma. Citation Format: Joe-Marc Chauvin, Ornella Pagliano, Julien Fourcade, Zhaojun Sun, Cindy Sanders, John M. Kirkwood, Tseng-hui T. Chen, Mark Maurer, Alan Korman, Hassane Zarour. Targeting TIGIT and PD-1 to increase the expansion and function of tumor antigen-specific CD8+ T cells in melanoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 459. doi:10.1158/1538-7445.AM2015-459

T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen-specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1⁻TIGIT⁺, and PD-1⁺TIGIT⁻ CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.