- Email: [email protected]
527 Profiling of the immune tumour microenvironment generates a roadmap for checkpoint modulation in advanced melanoma
S112 node metastases compared to subcutaneous/cutaneous ones, therefore, cell densities in the two groups of metastases were evaluated separately. Nine patients were considered responders showing complete or partial response or stable disease for at least 6 months, including 3 patients deriving long-term benefit lasting more than 2 years. With the exception of PD-1, higher density of cells expressing the immune cell markers was found in lymph node metastases of the responders compared to nonresponders. In subcutaneous or cutaneous metastases, on the other hand, similar difference could be observed only in the case of the CD8 and CD45RO markers while non-responders exhibited higher number of PD-1+ lymphocytes. The density of CD8+ and CD45RO+ cells in nodal metastases also showed correlation with patient survival. Conclusions: Our results corroborate previous findings suggesting an association between an immunologically active tumor microenvironment and response to ipilimumab treatment, and propose new potential biomarkers which can be used for predicting treatment efficacy and disease outcome. However, to verify the usefulness of these immune biomarkers prospective studies on larger patient cohorts are warranted. The work was supported by the National Scientific Research Fund OTKA grant 105132. No conflict of interest. 525 POSTER SPOTLIGHT/POSTER Safety and efficacy of pembrolizumab (MK-3475) in patients (pts) with advanced biliary tract cancer: Interim results of KEYNOTE-028 Y.J. Bang1 , T. Doi2 , F. De Braud3 , S. Piha-Paul4 , A. Hollebecque5 , A.R. Abdul Razak6 , C.C. Lin7 , P.A. Ott8 , A.R. He9 , S.S. Yuan10 , M. Koshiji11 , B. Lam11 , R. Aggarwal12 . 1 Seoul National University Hospital, Internal Medicine, Seoul, Korea; 2 National Cancer Center Hospital East, Experimental Therapeutics, Chiba, Japan; 3 Fondazione IRCCS “Istituto Nazionale dei Tumori”, Oncology, Milano, Italy; 4 The University of Texas MD Anderson Cancer Center, Investigational Cancer Therapeutics, Houston, USA; 5 Gustave Roussy, Therapeutic Innovations, Villejuif, France; 6 Princess Margaret Cancer Centre, Medical Oncology, Toronto, Canada; 7 National Taiwan University Hospital, Oncology, Taipei, Taiwan; 8 Dana Farber Cancer Institute, Medical Oncology, Boston, USA; 9 Georgetown University, Medicine/Oncology, Washington DC, USA; 10 Merck & Co. Inc., BARDS, Kenilworth, USA; 11 Merck & Co. Inc., Clinical Oncology, Kenilworth, USA; 12 University of California San Francisco, Helen Diller Family Comprehensive Cancer Center, San Francisco, USA Background: Tumors frequently use the programmed cell death 1 (PD-1) pathway to evade the host antitumor immune response. Pembrolizumab is a highly selective, humanized monoclonal antibody against PD-1 that is designed to block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. We assessed the safety and antitumor activity of pembrolizumab in pts with PD-L1-positive advanced biliary tract cancer. Methods: KEYNOTE-028 (ClinicalTrials.gov, NCT02054806) is an ongoing, multicohort, phase 1b trial of pembrolizumab monotherapy for pts with PD-L1-positive advanced solid tumors. Pts were eligible for enrollment in the biliary cohort if they had PD-L1-positive adenocarcinoma of the gallbladder or biliary tree, excluding cancer of the ampulla of Vater. Additional eligibility criteria included progression on or inability to receive standard therapy, ECOG PS 0−1, and measurable disease per RECIST v1.1. PD-L1 positivity was defined as staining in 1% of cells in tumor nests or PD-L1-positive bands in stroma as assessed at a central laboratory by a prototype IHC assay. Pembrolizumab 10 mg/kg was given every 2 weeks for up to 2 years or until confirmed progression or unacceptable toxicity. End points were safety, tolerability, and objective response rate (ORR) per RECIST v1.1 by investigator review (assessed every 8 weeks for the first 6 months and every 12 weeks thereafter). Results: Of the 89 pts with biliary tract cancer who were screened for PD-L1 expression, 37 (42%) had PD-L1-positive tumors. Between Apr and Jul 2014, 24 (65%) of these pts were enrolled. Pts were predominantly men (58%) and Asian (50%), and the median age was 64 years. All pts received 1 prior therapy, including 38% who received 3 prior therapies. Overall, 15 (63%) pts experienced a treatment-related AE, most commonly pyrexia (17%) and nausea (13%). Four (17%) pts experienced grade 3−4 treatment-related AEs (n = 1 each for grade 3 anemia, autoimmune hemolytic anemia, colitis, and dermatitis). There were no treatment-related deaths. ORR (confirmed and unconfirmed) was 17% (95% CI, 5%-39%). Four (17%) pts had partial response, 4 (17%) pts had stable disease, and 12 (52%) pts had progressive disease as their best response. Three (13%) pts had no postbaseline tumor assessment at the time of analysis. Five pts, including all responders, remain on treatment (duration of treatment, 40+ to 42+ weeks). Conclusion: Pembrolizumab was generally well tolerated and demonstrated promising antitumor activity, including durable responses, in a
Abstracts subset of pts with advanced biliary tract cancer that expressed PD-L1. These data suggest that pembrolizumab warrants further exploration as a treatment option for advanced biliary tract cancer. Conflict of interest: Advisory Board: YJB has served on an advisory board for Merck Sharp Dohme. FdB has served on advisory boards for Bristol-Myers Squibb, Roche, and Merck Sharp Dohme. CCL has served on advisory boards for Bristol-Myers Squibb and Merck Sharp Dohme. Corporate-sponsored Research: YJB has received corporatesponsored research funding from Merck Sharp Dohme. TD has received corporate-sponsored research funding from Merck Sharp Dohme KK. Other Substantive Relationships: CCL has received honoraria from Bristol-Myers Squibb and Merck Sharp Dohme. SSY is an employee of and owns stock in Merck & Co, Inc. MK is an employee of Merck & Co, Inc. and owns stock in Eli Lilly & Company. BL is an employee of Merck & Co, Inc. 526 POSTER Engineering MEDI6383; a novel, multivalent immune-modulating fusion protein that potently agonizes the TNFRSF receptor OX40 M. Damschroder1 , M. Oberst2 , C. Stracener1 , Q. Du1 , D. Spencer3 , R. Woods1 , C. Auge2 , K. Mulgrew2 , J. Hair4 , H. Wu1 , S. Hammond2 , W. Dall’Acqua1 . 1 MedImmune, Antibody Discovery & Protein Engineering, Research, Gaithersburg, USA; 2 MedImmune, Oncology, Research, Gaithersburg, USA; 3 MedImmune, Analytical Biotechnology Sciences and Strategy, Biopharmaceutical Development, Gaithersburg, USA; 4 MedImmune, Oncology, Research, Cambridge, United Kingdom OX40 is a member of the tumor necrosis factor receptor super family of co-stimulatory molecules that augment T-cell activation. OX40 ligand is a transmembrane protein that binds to OX40, forming a trimeric signaling complex that enhances activated T cell functions such as cytokine production, expansion, and survival. Immune modulating therapies are at the forefront of cancer therapy, and OX40 signaling is one means by which to enhance an antitumor T cell response. Although soluble recombinant OX40L assembles into trimers and binds to OX40, it only produces a weak co-stimulatory signal. However, oligomerizing the trimeric ligand strongly improves the activity. To generate a recombinant therapeutic with enhanced immune modulating properties we have engineered MEDI6383; a multivalent, human OX40 ligand fusion protein that agonizes OX40 receptor. Extensive characterization of MEDI6383 was performed to ensure the developability and activity of MEDI6383. Physicochemical characterization demonstrated the low thermal transition temperature of MEDI6383 is initiated by the unfolding of the OX40L portion of the fusion protein. Glycoprofiling by mass spectrometry reveals the oligosaccharide profile varies between lots but did not affect binding or potency. Cell viability at the time of harvest impacted the sialic acid levels of the ligand fusion protein, however had minimal effect on conformational stability as assessed by DSC, and had no impact on pharmacokinetics in mice. Surface Plasmon Resonance revealed MEDI6383 maintained pH dependent binding to FcRn although the fusion protein exhibited a short serum half-life in human FcRn transgenic mice. Preclinical pharmacological properties of MEDI6383 were assessed using in vitro and in vivo models. MEDI6383 bound specifically and with high affinity to human and monkey OX40 expressed on T cells, and activated the OX40 signaling pathway as measured by NFúB signaling in Jurkat T reporter cells expressing human OX40. Co-culture of these reporter cells with cells expressing Fcg receptors enhanced OX40 signaling, demonstrating that Fcg receptor mediated clustering of MEDI6383 augments the agonistic properties of the soluble fusion protein. Evaluation of a surrogate mouse fusion protein in FcgR KO mouse models demonstrated the expression of activating FcgRs (I, III and IV) are required for anti-tumor activity. MEDI6383 induced cell proliferation and cytokine release that enhanced T cell receptor mediated activation of primary human CD4+ T cells, and suppressed Treg cell activity in T cell co-cultures. Potent in vivo anti-tumor activity was dependent on the addition of alloreactive human T cells in a xenograft mouse model of human cancer. Taken together, these results demonstrate that we have successfully engineered a novel, developable format for immunomodulation. No conflict of interest. 527 POSTER Profiling of the immune tumour microenvironment generates a roadmap for checkpoint modulation in advanced melanoma A. Furness1 . 1 University College London, Cancer Immunology Unit, London, United Kingdom Modulation of co-inhibitory and co-stimulatory molecules on tumourinfiltrating lymphocytes (TILs) has emerged as a promising anti-cancer strategy. To date, ipilimumab and pembrolizumab, monoclonal antibodies
Abstracts targeting cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) respectively, have received FDA approval. CTLA-4 and PD-1 represent two of multiple immune checkpoint molecules expressed on activated TILs for which a pipeline of immune modulatory agents are in clinical development. Despite great promise, responses to such agents are limited to a fraction of treated patients, highlighting the need to decipher underlying mechanisms of response and resistance. We performed multi-parametric analysis of peripheral blood and TILs by multi-colour flow cytometry in 10 patients with advanced melanoma, characterising the expression of co-inhibitory and co-stimulatory immune checkpoint molecules of B7 and TNFR superfamilies. All studied molecules were significantly upregulated in the tumour relative to the periphery. Critically, marked variation in the density of expression was observed between CD3+ CD8+ (CD8), CD4+ FoxP3− effector (CD4eff) and CD4+ FoxP3+ regulatory T lymphocyte (Treg) subsets. This was consistent between patients, irrespective of current or prior therapies and mirrored that observed in various subcutaneous tumour models in mice. Recent pre-clinical data demonstrate that antibodies targeting GITR, OX-40 and CTLA-4 can also promote depletion of tumour-infiltrating regulatory T cells by antibody-dependent cell-mediated cytotoxicity (ADCC). In keeping with these observations, we identified significantly higher expression of these targets on Treg relative to CD4eff and CD8 T cells, supporting the rationale for evaluating the development of antibodies with capacity to promote intra-tumoural Treg depletion in addition to their recognised immune-modulatory activity. Interestingly, in contrast to these findings, PD-1 was consistently expressed at highest density on CD8 T cells, whilst 4−1BB was similarly expressed between Treg and CD8 T cell subsets. This study demonstrates, irrespective of whether targeting co-inhibitory or co-stimulatory immune checkpoint molecules, the importance of considering the lymphocyte subset upon which the target molecule is expressed most highly. It sheds light on the potential mechanisms underlying the synergy observed to date in clinical trials of combination immune checkpoint modulation and the toxicity secondary to immunerelated adverse events. Most importantly, it generates an immunological roadmap, guiding the development, selection and clinical application of antibodies optimised for agonistic, blocking or depleting activity according to the density and distribution of target molecule expression on individual T lymphocyte subsets. No conflict of interest. 528 POSTER SPOTLIGHT/POSTER Intratumoural treatment with LTX-315, an oncolytic peptide immunotherapy, in patients with advanced metastatic disease induces CD8 effector cells and regression in some injected tumours J. Spicer1 , A. Awada2 , P. Brunsvig3 , A. Saunders4 , W.M. Olsen4 , B. Nicolaisen4 , O. Rekdal5 , M. Laruelle6 , F. Marjuadi7 , J. Vakili2 , P. Aftimos8 , P. Barthelemy2 , S. Deva9 , J.F. Baurain10 . 1 Guy’s and St. Thomas hospital, Clinical Oncology, London, United Kingdom; 2 Institut Jules Bordet, Universite Libre de Bruxelles, Clinical Oncology, Brussels, Belgium; 3 Oslo University Hospital, Clinical Oncology, Oslo, Norway; 4 Lytix Biopharma AS, Clinical, Oslo, Norway; 5 Lytix Biopharma AS, Preclinical, Oslo, Norway; 6 Cliniques Universitaires Saint-Luc, Oncologie, Brussels, Belgium; 7 University hospital Louvain, Oncology, Louvain, Belgium; 8 Institut Jules Bordet, Universite Libre de Bruxelles, Clinical Onclogy, Brussels, Belgium; 9 Guys & St. Thomas hospital, Clinical oncology, London, United Kingdom; 10 University Hospital Louvain, Clinical Oncology, Louvain, Belgium Background: LTX-315 has a unique mode of action releasing potent danger signals and tumour-associated antigens from tumour cells via disintegrating cytoplasmic organelle membranes. Preclinical studies in several rodent models demonstrate that LTX-315 induced complete regression, systemic anti-tumour responses and prevented recurrence following tumour rechallenge. In addition,LTX-315 has demonstrated strong synergy in preclinical studies with immune checkpoint inhibitors (ICI). A phase 1 open label,multi-centre dose escalation study is ongoing to evaluate the safety profile and determine the recommended phase 2 dose as measured by assessment of adverse events,laboratory values, immunological responses and tumour necrosis. Methods: In this study, patients with advanced metastatic disease, for conventional anti-tumour treatment is not appropriate, receive ultrasoundguided injections of LTX-315 on 3 consecutive days into a single, transdermally accessible tumour in week 1. During weeks 2−6, the injections are given once a week, and then every 2 weeks for a maximum of 20 weeks. Biopsies (3) are taken at baseline, at week 7 and at end of treatment. Changes in injected tumour lesions were assessed by ultrasound, while non-injected (target) lesions were assessed by CT scan as per irRC criteria.
S113 Results: Nine patients (1 chordoma, 1 sarcoma, 1 pancreas, 2 breast, 1 myo-epithelioma, 3 melanoma) have been enrolled to date. The median age is 58 years (range 32−80) and median number of prior treatments 2 (range 1−14). All but one LTX-315-related adverse events were CTC grade 1 (erythema at injection site, paraesthesia and transient hypotension). One patient had grade 2 LTX-315-related pain at injection site (resolution within minutes). No DLTs have occurred. Best response in 14 injected lesions in 7 evaluable patients included 2 complete responses (no detectable tumour), 4 partial responses (>50% reduction in tumour volume) and 1 stable disease. Infiltration of CD8+ effector T-cells occurred in 2 of 7 patients in which biopsied tumours were available (by IHC and Granzyme B). No responses in non-injected tumours have been observed in 7 evaluable patients. Four of the 7 patients had stable disease (at any time on study assessed by CT and irRC) in non-injected tumours (including proximate to and distant from injected lesions). Conclusions: This phase 1 study demonstrates that intratumoural LTX315 is safe and can induce tumour specific T-cell responses in heavily pretreated patients. Partial and complete responses in injected tumours was observed. LTX-315 is an attractive combination partner for clinical studies with anti-PD1 and anti-CTLA4. No conflict of interest. 529 POSTER LAP TGF-beta subset of CD4+CD25+CD127− Treg cells is increased and overexpresses LAP TGF-beta in lung adenocarcinoma patients L. Islas-Vazquez1 , H. Prado-Garcia1 , D. Aguilar-Cazares1 , M. MenesesFlores1 , M. Galicia-Velasco1 , S. Romero-Garcia1 , C. Camacho-Mendoza1 , J.S. Lopez-Gonzalez1 . 1 Instituto Nacional de Enfermedades Respiratorias Ismael Cosio Villegas, Chronic-degenerative diseases, Mexico City, Mexico Background: Lung cancer is the leading cause of cancer death worldwide. Adenocarcinoma, the most commonly diagnosed histologic type of lung cancer, is associated with smoking. Cigarette smoke promotes inflammation of the airways, this inflammatory environment perhaps mediated by Th17 cells may contribute to tumor development. In other aspect, tumors may also induce immunosuppressive regulatory T-cells (Tregs) to dampen immune reactivity, supporting tumor growth and progression. To clarify whether smoking-associated chronic inflammation or tumor-induced suppression contribute to lung adenocarcinoma progression, in this study, several cytokines and percentages of Th17 and Treg cells were detected in lung adenocarcinoma patients and compared with non-smoking and smoking control subjects. Material and Methods: A total of 28 patients with lung adenocarcinoma with clinical stage IV and heavy smokers were studied. As control groups, 13 non-smoking and 15 heavy smoking healthy volunteers were included. From blood samples, plasma and peripheral blood mononuclear cells (PBMCs) were collected. In plasma, Th1, Th2 and Th17 cytokines were quantified using a CBA assay. TGF-beta was quantified using the Quantikine Human TGF-beta1 ELISA assay. In stimulated PBMCs, percentages of IL-17+CD4+ T-cells were detected. In CD4+ T-cells isolated by negative selection, Th17 and Treg cells were phenotyped using multiparametric flow cytometry. Percentages of Th17 expressing ROR-gamma and percentages of CD4+CD25+CD127− Tregs were determined. Also, expression levels of CTLA-4, IL-10 and LatencyAssociated Peptide (LAP) TGF-beta in Tregs were detected. Results: High proportions of IL-17+CD4+ T-cells were found in smoking control subjects and lung adenocarcinoma patients compared to nonsmoking control subjects. In lung adenocarcinoma patients, increases of IL-2, IL-4, IL-6, and IL-10 plasma concentrations were found. In addition, high proportions of LAP TGF-beta subset of CD4+CD25+CD127− Treg cells, which overexpressed LAP TGF-beta were found. Conclusions: In lung adenocarcinoma patients, the tumor altered the Th17/Treg balance favoring the increase of Tregs expressing LAP TGFbeta. This subset may mediate the immunosuppression reported in cancer patients. This knowledge may lead to the development of immunotherapies that inhibit the suppressor activity mediated by the LAP TGF-beta subset of CD4+CD25+CD127− Treg cells to promote reactivity of immune cells against lung adenocarcinoma cells. L. Islas-Vazquez, is a student from the Posgrado en Ciencias Biologicas, Universidad Nacional Autonoma de Mexico (UNAM), and recipient of a fellowship from CONACyT (307085). This study was conducted as part of his doctoral thesis. No conflict of interest.
Abstracts subset of pts with advanced biliary tract cancer that expressed PD-L1. These data suggest that pembrolizumab warrants further exploration as a treatment option for advanced biliary tract cancer. Conflict of interest: Advisory Board: YJB has served on an advisory board for Merck Sharp Dohme. FdB has served on advisory boards for Bristol-Myers Squibb, Roche, and Merck Sharp Dohme. CCL has served on advisory boards for Bristol-Myers Squibb and Merck Sharp Dohme. Corporate-sponsored Research: YJB has received corporatesponsored research funding from Merck Sharp Dohme. TD has received corporate-sponsored research funding from Merck Sharp Dohme KK. Other Substantive Relationships: CCL has received honoraria from Bristol-Myers Squibb and Merck Sharp Dohme. SSY is an employee of and owns stock in Merck & Co, Inc. MK is an employee of Merck & Co, Inc. and owns stock in Eli Lilly & Company. BL is an employee of Merck & Co, Inc. 526 POSTER Engineering MEDI6383; a novel, multivalent immune-modulating fusion protein that potently agonizes the TNFRSF receptor OX40 M. Damschroder1 , M. Oberst2 , C. Stracener1 , Q. Du1 , D. Spencer3 , R. Woods1 , C. Auge2 , K. Mulgrew2 , J. Hair4 , H. Wu1 , S. Hammond2 , W. Dall’Acqua1 . 1 MedImmune, Antibody Discovery & Protein Engineering, Research, Gaithersburg, USA; 2 MedImmune, Oncology, Research, Gaithersburg, USA; 3 MedImmune, Analytical Biotechnology Sciences and Strategy, Biopharmaceutical Development, Gaithersburg, USA; 4 MedImmune, Oncology, Research, Cambridge, United Kingdom OX40 is a member of the tumor necrosis factor receptor super family of co-stimulatory molecules that augment T-cell activation. OX40 ligand is a transmembrane protein that binds to OX40, forming a trimeric signaling complex that enhances activated T cell functions such as cytokine production, expansion, and survival. Immune modulating therapies are at the forefront of cancer therapy, and OX40 signaling is one means by which to enhance an antitumor T cell response. Although soluble recombinant OX40L assembles into trimers and binds to OX40, it only produces a weak co-stimulatory signal. However, oligomerizing the trimeric ligand strongly improves the activity. To generate a recombinant therapeutic with enhanced immune modulating properties we have engineered MEDI6383; a multivalent, human OX40 ligand fusion protein that agonizes OX40 receptor. Extensive characterization of MEDI6383 was performed to ensure the developability and activity of MEDI6383. Physicochemical characterization demonstrated the low thermal transition temperature of MEDI6383 is initiated by the unfolding of the OX40L portion of the fusion protein. Glycoprofiling by mass spectrometry reveals the oligosaccharide profile varies between lots but did not affect binding or potency. Cell viability at the time of harvest impacted the sialic acid levels of the ligand fusion protein, however had minimal effect on conformational stability as assessed by DSC, and had no impact on pharmacokinetics in mice. Surface Plasmon Resonance revealed MEDI6383 maintained pH dependent binding to FcRn although the fusion protein exhibited a short serum half-life in human FcRn transgenic mice. Preclinical pharmacological properties of MEDI6383 were assessed using in vitro and in vivo models. MEDI6383 bound specifically and with high affinity to human and monkey OX40 expressed on T cells, and activated the OX40 signaling pathway as measured by NFúB signaling in Jurkat T reporter cells expressing human OX40. Co-culture of these reporter cells with cells expressing Fcg receptors enhanced OX40 signaling, demonstrating that Fcg receptor mediated clustering of MEDI6383 augments the agonistic properties of the soluble fusion protein. Evaluation of a surrogate mouse fusion protein in FcgR KO mouse models demonstrated the expression of activating FcgRs (I, III and IV) are required for anti-tumor activity. MEDI6383 induced cell proliferation and cytokine release that enhanced T cell receptor mediated activation of primary human CD4+ T cells, and suppressed Treg cell activity in T cell co-cultures. Potent in vivo anti-tumor activity was dependent on the addition of alloreactive human T cells in a xenograft mouse model of human cancer. Taken together, these results demonstrate that we have successfully engineered a novel, developable format for immunomodulation. No conflict of interest. 527 POSTER Profiling of the immune tumour microenvironment generates a roadmap for checkpoint modulation in advanced melanoma A. Furness1 . 1 University College London, Cancer Immunology Unit, London, United Kingdom Modulation of co-inhibitory and co-stimulatory molecules on tumourinfiltrating lymphocytes (TILs) has emerged as a promising anti-cancer strategy. To date, ipilimumab and pembrolizumab, monoclonal antibodies
Abstracts targeting cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) respectively, have received FDA approval. CTLA-4 and PD-1 represent two of multiple immune checkpoint molecules expressed on activated TILs for which a pipeline of immune modulatory agents are in clinical development. Despite great promise, responses to such agents are limited to a fraction of treated patients, highlighting the need to decipher underlying mechanisms of response and resistance. We performed multi-parametric analysis of peripheral blood and TILs by multi-colour flow cytometry in 10 patients with advanced melanoma, characterising the expression of co-inhibitory and co-stimulatory immune checkpoint molecules of B7 and TNFR superfamilies. All studied molecules were significantly upregulated in the tumour relative to the periphery. Critically, marked variation in the density of expression was observed between CD3+ CD8+ (CD8), CD4+ FoxP3− effector (CD4eff) and CD4+ FoxP3+ regulatory T lymphocyte (Treg) subsets. This was consistent between patients, irrespective of current or prior therapies and mirrored that observed in various subcutaneous tumour models in mice. Recent pre-clinical data demonstrate that antibodies targeting GITR, OX-40 and CTLA-4 can also promote depletion of tumour-infiltrating regulatory T cells by antibody-dependent cell-mediated cytotoxicity (ADCC). In keeping with these observations, we identified significantly higher expression of these targets on Treg relative to CD4eff and CD8 T cells, supporting the rationale for evaluating the development of antibodies with capacity to promote intra-tumoural Treg depletion in addition to their recognised immune-modulatory activity. Interestingly, in contrast to these findings, PD-1 was consistently expressed at highest density on CD8 T cells, whilst 4−1BB was similarly expressed between Treg and CD8 T cell subsets. This study demonstrates, irrespective of whether targeting co-inhibitory or co-stimulatory immune checkpoint molecules, the importance of considering the lymphocyte subset upon which the target molecule is expressed most highly. It sheds light on the potential mechanisms underlying the synergy observed to date in clinical trials of combination immune checkpoint modulation and the toxicity secondary to immunerelated adverse events. Most importantly, it generates an immunological roadmap, guiding the development, selection and clinical application of antibodies optimised for agonistic, blocking or depleting activity according to the density and distribution of target molecule expression on individual T lymphocyte subsets. No conflict of interest. 528 POSTER SPOTLIGHT/POSTER Intratumoural treatment with LTX-315, an oncolytic peptide immunotherapy, in patients with advanced metastatic disease induces CD8 effector cells and regression in some injected tumours J. Spicer1 , A. Awada2 , P. Brunsvig3 , A. Saunders4 , W.M. Olsen4 , B. Nicolaisen4 , O. Rekdal5 , M. Laruelle6 , F. Marjuadi7 , J. Vakili2 , P. Aftimos8 , P. Barthelemy2 , S. Deva9 , J.F. Baurain10 . 1 Guy’s and St. Thomas hospital, Clinical Oncology, London, United Kingdom; 2 Institut Jules Bordet, Universite Libre de Bruxelles, Clinical Oncology, Brussels, Belgium; 3 Oslo University Hospital, Clinical Oncology, Oslo, Norway; 4 Lytix Biopharma AS, Clinical, Oslo, Norway; 5 Lytix Biopharma AS, Preclinical, Oslo, Norway; 6 Cliniques Universitaires Saint-Luc, Oncologie, Brussels, Belgium; 7 University hospital Louvain, Oncology, Louvain, Belgium; 8 Institut Jules Bordet, Universite Libre de Bruxelles, Clinical Onclogy, Brussels, Belgium; 9 Guys & St. Thomas hospital, Clinical oncology, London, United Kingdom; 10 University Hospital Louvain, Clinical Oncology, Louvain, Belgium Background: LTX-315 has a unique mode of action releasing potent danger signals and tumour-associated antigens from tumour cells via disintegrating cytoplasmic organelle membranes. Preclinical studies in several rodent models demonstrate that LTX-315 induced complete regression, systemic anti-tumour responses and prevented recurrence following tumour rechallenge. In addition,LTX-315 has demonstrated strong synergy in preclinical studies with immune checkpoint inhibitors (ICI). A phase 1 open label,multi-centre dose escalation study is ongoing to evaluate the safety profile and determine the recommended phase 2 dose as measured by assessment of adverse events,laboratory values, immunological responses and tumour necrosis. Methods: In this study, patients with advanced metastatic disease, for conventional anti-tumour treatment is not appropriate, receive ultrasoundguided injections of LTX-315 on 3 consecutive days into a single, transdermally accessible tumour in week 1. During weeks 2−6, the injections are given once a week, and then every 2 weeks for a maximum of 20 weeks. Biopsies (3) are taken at baseline, at week 7 and at end of treatment. Changes in injected tumour lesions were assessed by ultrasound, while non-injected (target) lesions were assessed by CT scan as per irRC criteria.
S113 Results: Nine patients (1 chordoma, 1 sarcoma, 1 pancreas, 2 breast, 1 myo-epithelioma, 3 melanoma) have been enrolled to date. The median age is 58 years (range 32−80) and median number of prior treatments 2 (range 1−14). All but one LTX-315-related adverse events were CTC grade 1 (erythema at injection site, paraesthesia and transient hypotension). One patient had grade 2 LTX-315-related pain at injection site (resolution within minutes). No DLTs have occurred. Best response in 14 injected lesions in 7 evaluable patients included 2 complete responses (no detectable tumour), 4 partial responses (>50% reduction in tumour volume) and 1 stable disease. Infiltration of CD8+ effector T-cells occurred in 2 of 7 patients in which biopsied tumours were available (by IHC and Granzyme B). No responses in non-injected tumours have been observed in 7 evaluable patients. Four of the 7 patients had stable disease (at any time on study assessed by CT and irRC) in non-injected tumours (including proximate to and distant from injected lesions). Conclusions: This phase 1 study demonstrates that intratumoural LTX315 is safe and can induce tumour specific T-cell responses in heavily pretreated patients. Partial and complete responses in injected tumours was observed. LTX-315 is an attractive combination partner for clinical studies with anti-PD1 and anti-CTLA4. No conflict of interest. 529 POSTER LAP TGF-beta subset of CD4+CD25+CD127− Treg cells is increased and overexpresses LAP TGF-beta in lung adenocarcinoma patients L. Islas-Vazquez1 , H. Prado-Garcia1 , D. Aguilar-Cazares1 , M. MenesesFlores1 , M. Galicia-Velasco1 , S. Romero-Garcia1 , C. Camacho-Mendoza1 , J.S. Lopez-Gonzalez1 . 1 Instituto Nacional de Enfermedades Respiratorias Ismael Cosio Villegas, Chronic-degenerative diseases, Mexico City, Mexico Background: Lung cancer is the leading cause of cancer death worldwide. Adenocarcinoma, the most commonly diagnosed histologic type of lung cancer, is associated with smoking. Cigarette smoke promotes inflammation of the airways, this inflammatory environment perhaps mediated by Th17 cells may contribute to tumor development. In other aspect, tumors may also induce immunosuppressive regulatory T-cells (Tregs) to dampen immune reactivity, supporting tumor growth and progression. To clarify whether smoking-associated chronic inflammation or tumor-induced suppression contribute to lung adenocarcinoma progression, in this study, several cytokines and percentages of Th17 and Treg cells were detected in lung adenocarcinoma patients and compared with non-smoking and smoking control subjects. Material and Methods: A total of 28 patients with lung adenocarcinoma with clinical stage IV and heavy smokers were studied. As control groups, 13 non-smoking and 15 heavy smoking healthy volunteers were included. From blood samples, plasma and peripheral blood mononuclear cells (PBMCs) were collected. In plasma, Th1, Th2 and Th17 cytokines were quantified using a CBA assay. TGF-beta was quantified using the Quantikine Human TGF-beta1 ELISA assay. In stimulated PBMCs, percentages of IL-17+CD4+ T-cells were detected. In CD4+ T-cells isolated by negative selection, Th17 and Treg cells were phenotyped using multiparametric flow cytometry. Percentages of Th17 expressing ROR-gamma and percentages of CD4+CD25+CD127− Tregs were determined. Also, expression levels of CTLA-4, IL-10 and LatencyAssociated Peptide (LAP) TGF-beta in Tregs were detected. Results: High proportions of IL-17+CD4+ T-cells were found in smoking control subjects and lung adenocarcinoma patients compared to nonsmoking control subjects. In lung adenocarcinoma patients, increases of IL-2, IL-4, IL-6, and IL-10 plasma concentrations were found. In addition, high proportions of LAP TGF-beta subset of CD4+CD25+CD127− Treg cells, which overexpressed LAP TGF-beta were found. Conclusions: In lung adenocarcinoma patients, the tumor altered the Th17/Treg balance favoring the increase of Tregs expressing LAP TGFbeta. This subset may mediate the immunosuppression reported in cancer patients. This knowledge may lead to the development of immunotherapies that inhibit the suppressor activity mediated by the LAP TGF-beta subset of CD4+CD25+CD127− Treg cells to promote reactivity of immune cells against lung adenocarcinoma cells. L. Islas-Vazquez, is a student from the Posgrado en Ciencias Biologicas, Universidad Nacional Autonoma de Mexico (UNAM), and recipient of a fellowship from CONACyT (307085). This study was conducted as part of his doctoral thesis. No conflict of interest.