- Email: [email protected]
CD95L signaling
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Poster abstracts
(rash, sweating), pain (headache and other pain), sore throat, rhinitis and endocrine (hot flashes). All were grade 1 or 2 in severity except for 1 patient with G3 fatigue (DL2) and 1 patient with G3 nausea, vomiting and dehydration (DL3), both of which occurred at the time of disease progression. PK were dose dependent and comparable to the predicted PK from other studies. NKG2A saturation was at 100% following two doses of monalizumab and pharmacodynamic changes were seen at all DLs. Response and pharmacodynamic data will be presented. Conclusion: Monalizumab is generally well-tolerated therapy with little toxicity. Based on PK and PD and toxicity profiles, the RP2D is 10 mg/kg given i.v. every 2 weeks. The study has been expanded to 4 cohorts including (1) platinum-resistant ovarian cancer, (2) platinum-sensitive ovarian cancer, (3) epithelial endometrial cancer, and (4) squamous cell carcinoma of the cervix. No conflict of interest. 297 Poster (Board P123) Pre-clinical efficacy in multiple syngeneic models with oral immune checkpoint antagonists targeting PD-L1 and TIM-3 P. Sasikumar1 , N. Sudarshan1 , R. Ramachandra1 , N. Gowda1 , D. Samiulla1 , P. Bilugudi1 , S. Adurthi1 , J. Mani1 , R. Nair1 , N. Gowda1 , M. Ramachandra1 . 1 Aurigene Discovery Technologies Ltd., Pre-clinical Biology, Bangalore, India Background: Antibody-mediated blockade of PD1 and PD-L1 has transformed the cancer therapy paradigm by eliciting durable antitumor responses and long-term remissions in a subset of patients with a broad spectrum of cancers. Interestingly, in an endeavor to enhance the response rate, a combination of antibodies targeting PD1 and CTLA-4 has resulted in significantly higher patient response rate. However, such combination has suffered from increased immune-related adverse events (irREs) due to the breaking of immune self-tolerance. Sustained target inhibition as a result of a long half-life (>15−20 days) and >70% target occupancy for months are likely contributing to irAEs observed. Towards addressing these shortcomings, we are developing small molecule agents targeting more than one immune checkpoint pathway to increase the response rate and dosing by oral route with relatively shorter pharmacokinetic exposure for better management of irREs. Material and Methods: Herein we report the pharmacological evaluation of the first-in-class small molecule antagonists capable of targeting both PD-L1 and TIM-3 immune checkpoint pathways. The design hypothesis for generating a dual antagonist is primarily based on the pockets of sequence similarity of PDL-1 and TIM-3 proteins. A focused library of compounds mimicking the interaction of checkpoint proteins was designed and synthesized. Screening and analysis of the resulting library led to the identification of hits capable of functional disruption of the PDL-1 and TIM-3 signaling pathways. Further optimization resulted in compounds displaying equipotent antagonism towards PD-L1 and TIM-3 with desirable physicochemical properties and exposure upon oral administration. Results: Potent functional activity comparable to that obtained with an antiPD1 or anti-TIM-3 antibody in rescuing lymphocyte proliferation and effector functions were observed with a lead compound, AUPM-327. AUPM-327 showed selectivity against other immune checkpoint proteins including CTLA-4, VISTA, LAG-3 and BTLA as well as in a broad panel of receptors and enzymes. In syngeneic preclinical models of melanoma, breast and colon cancers, AUPM-327 showed significant efficacy in inhibition of both primary tumor growth and metastasis upon once a day oral dosing with excellent tolerability. Anti-tumoral activity correlated well with drug exposure and activation of CD4+ and CD8+ T cells. Conclusions: The findings demonstrating the dual inhibition of PD-L1 and TIM-3 pathways resulting in activation of T cells and anti-tumor activities support further development of these orally bioavailable agents. Conflict of interest: Ownership: All authors are employees of Aurigene Discovery Technologies Limited. 298 Poster (Board P124) T cell activation and anti-tumor efficacy of anti-Lag3 mAbs are independent of Lag-3–MHC Class II blocking capacity R. De Waal Malefyt1 , S. Zhao2 , B. Haines3 , M. Chenard2 , J. Laskey3 , L. Cui3 , W. Blumenschein4 , T. Churakova5 , R. Shukla5 , E. Hsieh5 , M. Beaumont5 , L. Fayadat-Dilman5 , S. Cemerski2 . 1 MRL Palo Alto, Immunology & IMR, Palo Alto, USA; 2 MRL-Boston, Immunology & IMR, Boston, USA; 3 MRL-Boston, In Vivo Pharmacology, Boston, USA; 4 MRL Palo Alto, Gene expression and profiling, Palo Alto, USA; 5 MRL Palo Alto, Protein Sciences, Palo Alto, USA LAG-3 has been shown to act as an inhibitory molecule in the regulation of T cell activation, proliferation and homeostasis. Exhausted T cell
Poster Session – Immunotherapy, Wednesday 29 November 2016 populations that evolve in the tumor microenvironment or during chronic viral infections show coordinated expression of LAG3 and PD-1. LAG-3 has been identified as a protein structurally related to CD4 and which also binds to MHC-II. Anti-LAG-3 antibodies have shown preclinical efficacy in several disease models in particular when combined with anti-PD-1 antibodies. It has been demonstrated that LAG-3 blockade is efficacious in both CD4+ and CD8+ T cells despite the lack of significant MHCII levels on CD8+ T cell. We describe studies that evaluated if anti-LAG-3 efficacy is dependent on the ability of the antibody to inhibit the binding of LAG-3 to MHCII. We have compared, the anti-mLAG-3 antibody (C9B7W) which does not block LAG-3–MHCII interaction and an in-house generated anti-LAG-3 antibody (28G10) that strongly inhibits the interaction between LAG-3 and its ligand (MHCII) in a series of in vitro assays. Biophysical characterization confirmed the differences of these antibodies to disrupt LAG-3–MHCII binding and epitope mapping indicated that this may be associated with the different regions of LAG-3 that these antibodies bind to. Furthermore, in in vitro functional assays performed using TCR transgenic CD4+ T cells, no differences were seen between the two anti-LAG-3 antibodies to enhance antigen-specific T cell responses. In addition, their ability to synergize with an anti-PD-1 antibody was comparable. To understand if the overall enhancement in CD4+ T cell activation obtained with C9B7W and 28G10 was achieved through different mechanisms of action, we evaluated gene induction following anti-LAG-3 antibody treatment and did not identify any significant differences between the gene signatures induced by these antibodies. Consistent with this observation, we did not see an additive effect of C9B7W and 28G10 when used together in vitro. Neither antibody demonstrated a significant effect on the activity of TCR transgenic CD8+ T cells in in vitro functional assays. Studies in mouse syngeneic tumor models also showed comparable antitumor efficacy of the LAG-3–MHCII blocking and non-blocking when dosed in combination with anti-PD-1. In conclusion, we demonstrate that efficacy of LAG-3-targeting antibodies is not associated with their ability to disrupt LAG-3–MHCII interactions. Conflict of interest: Corporate-sponsored Research: All authors are employees of Merck, Sharpe and Dome. 299 Poster (Board P125) APG101 protects immune cells from activation-induced cell death by blocking pro-apoptotic CD95/CD95L signaling C. Merz1 , J. Sykora1 , D.M. Richards2 , H. Fricke3 , C. Gieffers4 . 1 Apogenix AG, Cellular Analytics, Heidelberg, Germany; 2 Apogenix AG, Immunology, Heidelberg, Germany; 3 Apogenix AG, CMO, Heidelberg, Germany; 4 Apogenix AG, Protein Chemistry/Analytics, Heidelberg, Germany Background: Inhibitory receptors on immune cells like PD-1 or CTLA-4 − now termed immune checkpoints − are used by cancers expressing cognate checkpoint ligands in order to escape immune surveillance. The classical death receptor CD95 is a regulator of immune homeostasis and is consequently upregulated following activation of immune cells. Initiation of apoptosis in activated immune cells upon contact with CD95 ligand (CD95L) makes CD95 another putative immune checkpoint. Like other checkpoint ligands, the CD95 ligand is frequently overexpressed in cancers and tumor-associated blood vessels, which possibly facilitates killing or inactivation of immune cells (i.e., tumor counter-attack). Here we examined the mode-of-action of APG101, a drug designed to neutralize CD95L, in protection of immune cells from activation induced cell death and subsequent effects on tumor cell killing. Methods: Monocytes isolated from healthy-donor blood samples were differentiated in vitro into either M1- or M2-type macrophages, which was confirmed by multicolor-flow cytometry for subtype markers. Subsequently, we analyzed the respective M1 and M2-type macrophages regarding apoptosis induction upon CD95L exposure. Analogous experiments were performed using purified T-cells. Analytical FACS was used to monitor apoptosis by appearance of cleaved PARP. Real-time cell analysis was performed on direct co-cultures of activated T-Lymphocytes and monocyte-depleted PBMCs with tumor cell lines to monitor tumor cell killing by immune cells. Results: Differentiated M1- and M2-type macrophages express specific marker antigens including CD95. Exposure of macrophages to soluble recombinant CD95L induces apoptosis in both populations. Higher sensitivity of M1- compared to M2-macrophages is observed, coinciding with higher expression of CD95 on M1-macrophages. Addition of APG101 dose-dependently protects macrophages from CD95L-induced cell death. Similar results are obtained after treatment of T-cells with CD95L and APG101 in apoptosis-assays. In direct co-culture, purified T-Lymphocytes and monocyte-depleted PBMCs are able to induce apoptosis in several tumor cell lines. Exogenously added APG101 does not interfere with chemical-induced activation or stimulation of immune cells by anti-CD3 and -CD28 antibodies and, importantly, tumor cell killing is not impaired. Conclusion: APG101 is a potent inhibitor of pro-apoptotic CD95/CD95L signaling and protects activated immune cells from AICD by undergoing
Poster Session – Immunotherapy, Wednesday 29 November 2016 apoptosis through exposure to CD95 ligand. Importantly, APG101 does not interfere with macrophage differentiation or activation of the cytolytic functions of T-Lymphocytes and PBMCs in vitro. Conflict of interest: Other Substantive Relationships: All authors are employees and/or stock holders of Apogenix AG. 300 Poster (Board P126) Immune cell activation by novel hexavalent CD40 agonist APG1233 compared to trimeric formats or agonistic anti-CD40 antibodies D.M. Richards1 , C. Merz2 , J. Sykora2 , M. Thiemann3 , T. Beyer2 , S. Kuehn3 , H. Fricke4 , C. Gieffers3 , O. Hill5 . 1 Apogenix AG, Immunology, Heidelberg, Germany; 2 Apogenix AG, Cellular Analytics, Heidelberg, Germany; 3 Apogenix AG, Protein Chemistry/Analytics, Heidelberg, Germany; 4 Apogenix AG, CMO, Heidelberg, Germany; 5 Apogenix AG, Molecular Biology, Heidelberg, Germany Introduction: The co-stimulatory receptor CD40 is strongly expressed on B cells, monocytes and antigen-presenting cells (APC). By promoting their maturation, activation and survival, CD40 signaling greatly contributes to anti-tumor responses of the immune system. The HERA-Technology developed by Apogenix is a powerful engineering platform for the production of modular fusion proteins targeting the TNF-receptor superfamily. Here we compared the efficacy of different CD40 agonist formats, including the novel hexavalent scCD40L-RBD-Fc (APG1233), and the functional consequences of differential receptor clustering. Materials and Methods: Immune cells were isolated from healthydonor blood samples and profiled by multicolor flow cytometry (MC-FC). Subsequently, immune cells were cultured in growth media containing various forms of CD40 agonists. Upregulation of activation markers on B cells and monocytes (e.g. CD69, CD86, HLA-DR) and T cellinduced killing of tumor cells in direct co-culture was assessed by MC-FC and employing a real-time cell analysis system (xCELLigence), respectively. Secretion of cytokines in response to CD40 ligation and the pharmacokinetic properties of the fully human APG1233 and the chimeric murine/human APG1274 were determined by ELISA. Results: In vivo stability of APG1233 was demonstrated in a single dose mouse PK study revealing a terminal half-life of 84 hours. The chimeric surrogate molecule APG1274 which binds murine CD40 is eliminated much quicker (t1/2 of 4 hours) demonstrating the specificity of both compounds. In vitro only the hexavalent APG1233 efficiently stimulated B cells, monocytes and PBMCs. In contrast, neither trimeric CD40L nor an agonistic antibody against CD40 were able to upregulate expression of activation markers. Similarly, the secretion of proinflammatory cytokines such as IL-12, CD95L and IFNg by PBMCs was only stimulated after exposure to APG1233 and not in the presence of other CD40 agonists. In functional co-culture assays, after exposure to APG1233, in vitro generated M2-macrophages underwent conversion and acquired M1-type surface markers which strongly enhanced proliferation of na¨ıve CD4+ T cells. Consistent with these data, only APG1233 efficiently increased direct cytotoxic activity of immune cells against tumor cells measured by a realtime cell analysis assay. Conclusion: The CD40 agonist APG1233 is a member of a novel class of hexavalent TNFRSF agonists which binds its target with high specificity, exhibits excellent in vivo stability and superior biological activity over other agonistic formats (e.g. agonistic antibodies to CD40). Conflict of interest: Other Substantive Relationships: All authors are employees and/or stock holders of Apogenix AG. 301 Poster (Board P127) Costimulatory T-cell engagement by PRS-343, a CD137 (4-1BB)/HER2 bispecific, leads to tumor growth inhibition and TIL expansion in a humanized mouse model M.J. Hinner1 , R.S. Bel Aiba1 , C. Schlosser1 , T. Jaquin1 , A. Allersdorfer1 , ¨ 2, S. Berger1 , A. Wiedenmann1 , G. Matschiner1 , J. Schuler U. Moebius1 , C. Rothe1 , S.A. Olwill1 . 1 Pieris Pharmaceuticals GmbH, Immuno-Oncology, Freising, Germany; 2 Oncotest GmbH, In vivo operations, Freiburg, Germany Background: CD137 (4-1BB) is a key costimulatory immunoreceptor and a highly promising therapeutic target in cancer. To overcome limitations of current mAb-based approaches which monospecifically target CD137, we generated PRS-343, a CD137/HER2 bispecific designed to promote CD137 clustering by bridging CD137-positive T cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T cells. Methods: We have previously described the generation of PRS-343 as a fusion of a CD137-specific Anticalin® protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone. PRS-343 was found to efficiently activate T cells ex vivo in the presence of HER2-positive cells. Here, in vivo proof of concept data
Poster abstracts
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is presented utilizing a humanized mouse model in immunocompromised mice and the SK-OV-3 cell line as a HER2-positive xenograft. When tumors had reached a predefined size, mice received human PBMC and weekly injections of PRS-343 for three weeks. An IgG4 isotype antibody served as the negative control, while a CD137-targeting benchmark antibody and trastuzumab with an engineered IgG4 backbone (“tras-IgG4”) served as controls for monospecific targeting of CD137 and HER2, respectively. Results: PRS-343 activity was investigated at four different weekly doses, ranging from 4 mg to 200 mg. We found that PRS-343 dose-dependently led to strong tumor growth inhibition compared to treatment with the isotype control, and that the tumor response was accompanied by a significantly higher tumor infiltration with human lymphocytes (hCD45+ ). Both these activities were absent in the anti-CD137 benchmark. The tras-IgG4 control was also devoid of lymphocyte infiltration into the tumor, but displayed a tumor growth inhibition comparable to PRS-343. The animal model also allowed investigating the potential safety of PRS-343: While the anti-CD137 benchmark accelerated the onset of graft-versus-host-disease and led to stronger expansion of CD8+ T cells in the peripheral blood compared to the isotype control group, both of these effects were absent for PRS-343, indicating tumor-localized activation of CD137 only. Conclusion: We report potent costimulatory T-cell engagement of the immunoreceptor CD137 in a HER2-dependent manner, utilizing the CD137/HER2 bispecific PRS-343. PRS-343 displays dual activity in vivo based on monospecific HER2-targeting and bispecific, tumor-localized costimulation of CD137. Compared to known CD137-targeting antibodies in clinical development, this approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity. The positive functional ex vivo and in vivo data of PRS343 as well as the excellent developability profile support investigation of its anti-cancer activity in clinical trials. Conflict of interest: Corporate-sponsored Research: Marlon J. Hinner, Rachida-Siham Bel Aiba, Corinna Schlosser, Thomas Jaquin, Andrea Allersdorfer, Sven Berger, Gabriele Matschiner, Ulrich Moebius, Christine Rothe and Shane A. Olwill are currently employed by Pieris Pharmaceuticals GmbH. Alexander Wiedenmann is a former employee of Pieris Pharmaceuticals GmbH. Julia Schuler ¨ is an employee of Oncotest GmbH which carried out the animal model experiment described in the abstract, which was sponsored by Pieris Pharmaceuticals GmbH. 302 Poster/Poster in the spotlight (Board P128) First-in-human dose-escalation study of VCN-01, a selective oncolytic adenovirus with hyaluronidase activity in patients with advanced or metastatic cancer M. Hidalgo1 , M. Gil2 , R. Garcia-Carbonero3 , R. Alvarez1 , B. Laquente2 , R. Moreno4 , A. De Martino5 , R. Alemany4 , G. Capella6 , E. Blasi7 , ´ Clara I. Viaplana7 , M. Cascallo7 , R. Salazar2 . 1 Centro Integral Oncologico Campal, Oncology, Madrid, Spain; 2 Catalan Institute of Oncology ICO, Bellvitge Biomedical Research Institute IDIBELL, Department of Medical Oncology, L’Hospitalet de Llobregat, Spain; 3 Hospital Universitario 12 de Octubre, Medical Oncology Division, Madrid, Spain; 4 Catalan Institute of Oncology ICO, Bellvitge Biomedical Research Institute IDIBELL, Translational Research Laboratory, L’Hospitalet de Llobregat, Spain; 5 Spanish National Cancer Research Center CNIO, Histopathology Unit, Madrid, Spain; 6 Catalan Institute of Oncology ICO, Bellvitge Biomedical Research Institute IDIBELL, Hereditary Cancer Program, L’Hospitalet de Llobregat, Spain; 7 VCN Biosciences, Research, Sant Cugat del Valles, Spain Background: VCN-01 is a selective oncolytic adenovirus with hyaluronidase activity. This study aimed to determine the safety, pharmacokinetics (PK) and anti-tumor activity of a single intravenous injection of VCN-01, either alone or combined with nab-paclixatel/gemcitabine (AG) and to define the recommended phase II dose (RP2D) of VCN-01 alone or in combination with AG. Material and Methods: VCN-01 was administered IV to patients with advanced solid tumors with ECOG 0−1 at doses ranging from 1E11 to 1E13 viral particles per patient (vp) as a single agent and at from 3.3E12 to 1E13 vp + AG at standard doses. Blood VCN-01 DNA was measured at different time-points after treatment. Viral shedding was assessed in sputum, urine and stool. IL-6 and IL-12 levels were also measured. Viral DNA and lymphocytic populations were analyzed in tumor biopsies. Response was assessed using RECIST v1.1 criteria. Results: Twenty three patients have been treated to date including 16 patients who received VCN-01 alone (patients with gastrointestinal solid tumors, except 1 head & neck tumor) and 7 patients who received combined treatment (all having pancreatic adenocarcinoma). Neutralizing antibodies (NAb) levels against adenovirus of 1/320 were detected in all patients at screening. The most common related toxicities after injection of VCN-01 alone were: pyrexia (81%), arthralgia & myalgia (25%), AST elevation (18%), anorexia and vomiting (18%). One dose-limiting toxicity consisting
Poster abstracts
(rash, sweating), pain (headache and other pain), sore throat, rhinitis and endocrine (hot flashes). All were grade 1 or 2 in severity except for 1 patient with G3 fatigue (DL2) and 1 patient with G3 nausea, vomiting and dehydration (DL3), both of which occurred at the time of disease progression. PK were dose dependent and comparable to the predicted PK from other studies. NKG2A saturation was at 100% following two doses of monalizumab and pharmacodynamic changes were seen at all DLs. Response and pharmacodynamic data will be presented. Conclusion: Monalizumab is generally well-tolerated therapy with little toxicity. Based on PK and PD and toxicity profiles, the RP2D is 10 mg/kg given i.v. every 2 weeks. The study has been expanded to 4 cohorts including (1) platinum-resistant ovarian cancer, (2) platinum-sensitive ovarian cancer, (3) epithelial endometrial cancer, and (4) squamous cell carcinoma of the cervix. No conflict of interest. 297 Poster (Board P123) Pre-clinical efficacy in multiple syngeneic models with oral immune checkpoint antagonists targeting PD-L1 and TIM-3 P. Sasikumar1 , N. Sudarshan1 , R. Ramachandra1 , N. Gowda1 , D. Samiulla1 , P. Bilugudi1 , S. Adurthi1 , J. Mani1 , R. Nair1 , N. Gowda1 , M. Ramachandra1 . 1 Aurigene Discovery Technologies Ltd., Pre-clinical Biology, Bangalore, India Background: Antibody-mediated blockade of PD1 and PD-L1 has transformed the cancer therapy paradigm by eliciting durable antitumor responses and long-term remissions in a subset of patients with a broad spectrum of cancers. Interestingly, in an endeavor to enhance the response rate, a combination of antibodies targeting PD1 and CTLA-4 has resulted in significantly higher patient response rate. However, such combination has suffered from increased immune-related adverse events (irREs) due to the breaking of immune self-tolerance. Sustained target inhibition as a result of a long half-life (>15−20 days) and >70% target occupancy for months are likely contributing to irAEs observed. Towards addressing these shortcomings, we are developing small molecule agents targeting more than one immune checkpoint pathway to increase the response rate and dosing by oral route with relatively shorter pharmacokinetic exposure for better management of irREs. Material and Methods: Herein we report the pharmacological evaluation of the first-in-class small molecule antagonists capable of targeting both PD-L1 and TIM-3 immune checkpoint pathways. The design hypothesis for generating a dual antagonist is primarily based on the pockets of sequence similarity of PDL-1 and TIM-3 proteins. A focused library of compounds mimicking the interaction of checkpoint proteins was designed and synthesized. Screening and analysis of the resulting library led to the identification of hits capable of functional disruption of the PDL-1 and TIM-3 signaling pathways. Further optimization resulted in compounds displaying equipotent antagonism towards PD-L1 and TIM-3 with desirable physicochemical properties and exposure upon oral administration. Results: Potent functional activity comparable to that obtained with an antiPD1 or anti-TIM-3 antibody in rescuing lymphocyte proliferation and effector functions were observed with a lead compound, AUPM-327. AUPM-327 showed selectivity against other immune checkpoint proteins including CTLA-4, VISTA, LAG-3 and BTLA as well as in a broad panel of receptors and enzymes. In syngeneic preclinical models of melanoma, breast and colon cancers, AUPM-327 showed significant efficacy in inhibition of both primary tumor growth and metastasis upon once a day oral dosing with excellent tolerability. Anti-tumoral activity correlated well with drug exposure and activation of CD4+ and CD8+ T cells. Conclusions: The findings demonstrating the dual inhibition of PD-L1 and TIM-3 pathways resulting in activation of T cells and anti-tumor activities support further development of these orally bioavailable agents. Conflict of interest: Ownership: All authors are employees of Aurigene Discovery Technologies Limited. 298 Poster (Board P124) T cell activation and anti-tumor efficacy of anti-Lag3 mAbs are independent of Lag-3–MHC Class II blocking capacity R. De Waal Malefyt1 , S. Zhao2 , B. Haines3 , M. Chenard2 , J. Laskey3 , L. Cui3 , W. Blumenschein4 , T. Churakova5 , R. Shukla5 , E. Hsieh5 , M. Beaumont5 , L. Fayadat-Dilman5 , S. Cemerski2 . 1 MRL Palo Alto, Immunology & IMR, Palo Alto, USA; 2 MRL-Boston, Immunology & IMR, Boston, USA; 3 MRL-Boston, In Vivo Pharmacology, Boston, USA; 4 MRL Palo Alto, Gene expression and profiling, Palo Alto, USA; 5 MRL Palo Alto, Protein Sciences, Palo Alto, USA LAG-3 has been shown to act as an inhibitory molecule in the regulation of T cell activation, proliferation and homeostasis. Exhausted T cell
Poster Session – Immunotherapy, Wednesday 29 November 2016 populations that evolve in the tumor microenvironment or during chronic viral infections show coordinated expression of LAG3 and PD-1. LAG-3 has been identified as a protein structurally related to CD4 and which also binds to MHC-II. Anti-LAG-3 antibodies have shown preclinical efficacy in several disease models in particular when combined with anti-PD-1 antibodies. It has been demonstrated that LAG-3 blockade is efficacious in both CD4+ and CD8+ T cells despite the lack of significant MHCII levels on CD8+ T cell. We describe studies that evaluated if anti-LAG-3 efficacy is dependent on the ability of the antibody to inhibit the binding of LAG-3 to MHCII. We have compared, the anti-mLAG-3 antibody (C9B7W) which does not block LAG-3–MHCII interaction and an in-house generated anti-LAG-3 antibody (28G10) that strongly inhibits the interaction between LAG-3 and its ligand (MHCII) in a series of in vitro assays. Biophysical characterization confirmed the differences of these antibodies to disrupt LAG-3–MHCII binding and epitope mapping indicated that this may be associated with the different regions of LAG-3 that these antibodies bind to. Furthermore, in in vitro functional assays performed using TCR transgenic CD4+ T cells, no differences were seen between the two anti-LAG-3 antibodies to enhance antigen-specific T cell responses. In addition, their ability to synergize with an anti-PD-1 antibody was comparable. To understand if the overall enhancement in CD4+ T cell activation obtained with C9B7W and 28G10 was achieved through different mechanisms of action, we evaluated gene induction following anti-LAG-3 antibody treatment and did not identify any significant differences between the gene signatures induced by these antibodies. Consistent with this observation, we did not see an additive effect of C9B7W and 28G10 when used together in vitro. Neither antibody demonstrated a significant effect on the activity of TCR transgenic CD8+ T cells in in vitro functional assays. Studies in mouse syngeneic tumor models also showed comparable antitumor efficacy of the LAG-3–MHCII blocking and non-blocking when dosed in combination with anti-PD-1. In conclusion, we demonstrate that efficacy of LAG-3-targeting antibodies is not associated with their ability to disrupt LAG-3–MHCII interactions. Conflict of interest: Corporate-sponsored Research: All authors are employees of Merck, Sharpe and Dome. 299 Poster (Board P125) APG101 protects immune cells from activation-induced cell death by blocking pro-apoptotic CD95/CD95L signaling C. Merz1 , J. Sykora1 , D.M. Richards2 , H. Fricke3 , C. Gieffers4 . 1 Apogenix AG, Cellular Analytics, Heidelberg, Germany; 2 Apogenix AG, Immunology, Heidelberg, Germany; 3 Apogenix AG, CMO, Heidelberg, Germany; 4 Apogenix AG, Protein Chemistry/Analytics, Heidelberg, Germany Background: Inhibitory receptors on immune cells like PD-1 or CTLA-4 − now termed immune checkpoints − are used by cancers expressing cognate checkpoint ligands in order to escape immune surveillance. The classical death receptor CD95 is a regulator of immune homeostasis and is consequently upregulated following activation of immune cells. Initiation of apoptosis in activated immune cells upon contact with CD95 ligand (CD95L) makes CD95 another putative immune checkpoint. Like other checkpoint ligands, the CD95 ligand is frequently overexpressed in cancers and tumor-associated blood vessels, which possibly facilitates killing or inactivation of immune cells (i.e., tumor counter-attack). Here we examined the mode-of-action of APG101, a drug designed to neutralize CD95L, in protection of immune cells from activation induced cell death and subsequent effects on tumor cell killing. Methods: Monocytes isolated from healthy-donor blood samples were differentiated in vitro into either M1- or M2-type macrophages, which was confirmed by multicolor-flow cytometry for subtype markers. Subsequently, we analyzed the respective M1 and M2-type macrophages regarding apoptosis induction upon CD95L exposure. Analogous experiments were performed using purified T-cells. Analytical FACS was used to monitor apoptosis by appearance of cleaved PARP. Real-time cell analysis was performed on direct co-cultures of activated T-Lymphocytes and monocyte-depleted PBMCs with tumor cell lines to monitor tumor cell killing by immune cells. Results: Differentiated M1- and M2-type macrophages express specific marker antigens including CD95. Exposure of macrophages to soluble recombinant CD95L induces apoptosis in both populations. Higher sensitivity of M1- compared to M2-macrophages is observed, coinciding with higher expression of CD95 on M1-macrophages. Addition of APG101 dose-dependently protects macrophages from CD95L-induced cell death. Similar results are obtained after treatment of T-cells with CD95L and APG101 in apoptosis-assays. In direct co-culture, purified T-Lymphocytes and monocyte-depleted PBMCs are able to induce apoptosis in several tumor cell lines. Exogenously added APG101 does not interfere with chemical-induced activation or stimulation of immune cells by anti-CD3 and -CD28 antibodies and, importantly, tumor cell killing is not impaired. Conclusion: APG101 is a potent inhibitor of pro-apoptotic CD95/CD95L signaling and protects activated immune cells from AICD by undergoing
Poster Session – Immunotherapy, Wednesday 29 November 2016 apoptosis through exposure to CD95 ligand. Importantly, APG101 does not interfere with macrophage differentiation or activation of the cytolytic functions of T-Lymphocytes and PBMCs in vitro. Conflict of interest: Other Substantive Relationships: All authors are employees and/or stock holders of Apogenix AG. 300 Poster (Board P126) Immune cell activation by novel hexavalent CD40 agonist APG1233 compared to trimeric formats or agonistic anti-CD40 antibodies D.M. Richards1 , C. Merz2 , J. Sykora2 , M. Thiemann3 , T. Beyer2 , S. Kuehn3 , H. Fricke4 , C. Gieffers3 , O. Hill5 . 1 Apogenix AG, Immunology, Heidelberg, Germany; 2 Apogenix AG, Cellular Analytics, Heidelberg, Germany; 3 Apogenix AG, Protein Chemistry/Analytics, Heidelberg, Germany; 4 Apogenix AG, CMO, Heidelberg, Germany; 5 Apogenix AG, Molecular Biology, Heidelberg, Germany Introduction: The co-stimulatory receptor CD40 is strongly expressed on B cells, monocytes and antigen-presenting cells (APC). By promoting their maturation, activation and survival, CD40 signaling greatly contributes to anti-tumor responses of the immune system. The HERA-Technology developed by Apogenix is a powerful engineering platform for the production of modular fusion proteins targeting the TNF-receptor superfamily. Here we compared the efficacy of different CD40 agonist formats, including the novel hexavalent scCD40L-RBD-Fc (APG1233), and the functional consequences of differential receptor clustering. Materials and Methods: Immune cells were isolated from healthydonor blood samples and profiled by multicolor flow cytometry (MC-FC). Subsequently, immune cells were cultured in growth media containing various forms of CD40 agonists. Upregulation of activation markers on B cells and monocytes (e.g. CD69, CD86, HLA-DR) and T cellinduced killing of tumor cells in direct co-culture was assessed by MC-FC and employing a real-time cell analysis system (xCELLigence), respectively. Secretion of cytokines in response to CD40 ligation and the pharmacokinetic properties of the fully human APG1233 and the chimeric murine/human APG1274 were determined by ELISA. Results: In vivo stability of APG1233 was demonstrated in a single dose mouse PK study revealing a terminal half-life of 84 hours. The chimeric surrogate molecule APG1274 which binds murine CD40 is eliminated much quicker (t1/2 of 4 hours) demonstrating the specificity of both compounds. In vitro only the hexavalent APG1233 efficiently stimulated B cells, monocytes and PBMCs. In contrast, neither trimeric CD40L nor an agonistic antibody against CD40 were able to upregulate expression of activation markers. Similarly, the secretion of proinflammatory cytokines such as IL-12, CD95L and IFNg by PBMCs was only stimulated after exposure to APG1233 and not in the presence of other CD40 agonists. In functional co-culture assays, after exposure to APG1233, in vitro generated M2-macrophages underwent conversion and acquired M1-type surface markers which strongly enhanced proliferation of na¨ıve CD4+ T cells. Consistent with these data, only APG1233 efficiently increased direct cytotoxic activity of immune cells against tumor cells measured by a realtime cell analysis assay. Conclusion: The CD40 agonist APG1233 is a member of a novel class of hexavalent TNFRSF agonists which binds its target with high specificity, exhibits excellent in vivo stability and superior biological activity over other agonistic formats (e.g. agonistic antibodies to CD40). Conflict of interest: Other Substantive Relationships: All authors are employees and/or stock holders of Apogenix AG. 301 Poster (Board P127) Costimulatory T-cell engagement by PRS-343, a CD137 (4-1BB)/HER2 bispecific, leads to tumor growth inhibition and TIL expansion in a humanized mouse model M.J. Hinner1 , R.S. Bel Aiba1 , C. Schlosser1 , T. Jaquin1 , A. Allersdorfer1 , ¨ 2, S. Berger1 , A. Wiedenmann1 , G. Matschiner1 , J. Schuler U. Moebius1 , C. Rothe1 , S.A. Olwill1 . 1 Pieris Pharmaceuticals GmbH, Immuno-Oncology, Freising, Germany; 2 Oncotest GmbH, In vivo operations, Freiburg, Germany Background: CD137 (4-1BB) is a key costimulatory immunoreceptor and a highly promising therapeutic target in cancer. To overcome limitations of current mAb-based approaches which monospecifically target CD137, we generated PRS-343, a CD137/HER2 bispecific designed to promote CD137 clustering by bridging CD137-positive T cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T cells. Methods: We have previously described the generation of PRS-343 as a fusion of a CD137-specific Anticalin® protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone. PRS-343 was found to efficiently activate T cells ex vivo in the presence of HER2-positive cells. Here, in vivo proof of concept data
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is presented utilizing a humanized mouse model in immunocompromised mice and the SK-OV-3 cell line as a HER2-positive xenograft. When tumors had reached a predefined size, mice received human PBMC and weekly injections of PRS-343 for three weeks. An IgG4 isotype antibody served as the negative control, while a CD137-targeting benchmark antibody and trastuzumab with an engineered IgG4 backbone (“tras-IgG4”) served as controls for monospecific targeting of CD137 and HER2, respectively. Results: PRS-343 activity was investigated at four different weekly doses, ranging from 4 mg to 200 mg. We found that PRS-343 dose-dependently led to strong tumor growth inhibition compared to treatment with the isotype control, and that the tumor response was accompanied by a significantly higher tumor infiltration with human lymphocytes (hCD45+ ). Both these activities were absent in the anti-CD137 benchmark. The tras-IgG4 control was also devoid of lymphocyte infiltration into the tumor, but displayed a tumor growth inhibition comparable to PRS-343. The animal model also allowed investigating the potential safety of PRS-343: While the anti-CD137 benchmark accelerated the onset of graft-versus-host-disease and led to stronger expansion of CD8+ T cells in the peripheral blood compared to the isotype control group, both of these effects were absent for PRS-343, indicating tumor-localized activation of CD137 only. Conclusion: We report potent costimulatory T-cell engagement of the immunoreceptor CD137 in a HER2-dependent manner, utilizing the CD137/HER2 bispecific PRS-343. PRS-343 displays dual activity in vivo based on monospecific HER2-targeting and bispecific, tumor-localized costimulation of CD137. Compared to known CD137-targeting antibodies in clinical development, this approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity. The positive functional ex vivo and in vivo data of PRS343 as well as the excellent developability profile support investigation of its anti-cancer activity in clinical trials. Conflict of interest: Corporate-sponsored Research: Marlon J. Hinner, Rachida-Siham Bel Aiba, Corinna Schlosser, Thomas Jaquin, Andrea Allersdorfer, Sven Berger, Gabriele Matschiner, Ulrich Moebius, Christine Rothe and Shane A. Olwill are currently employed by Pieris Pharmaceuticals GmbH. Alexander Wiedenmann is a former employee of Pieris Pharmaceuticals GmbH. Julia Schuler ¨ is an employee of Oncotest GmbH which carried out the animal model experiment described in the abstract, which was sponsored by Pieris Pharmaceuticals GmbH. 302 Poster/Poster in the spotlight (Board P128) First-in-human dose-escalation study of VCN-01, a selective oncolytic adenovirus with hyaluronidase activity in patients with advanced or metastatic cancer M. Hidalgo1 , M. Gil2 , R. Garcia-Carbonero3 , R. Alvarez1 , B. Laquente2 , R. Moreno4 , A. De Martino5 , R. Alemany4 , G. Capella6 , E. Blasi7 , ´ Clara I. Viaplana7 , M. Cascallo7 , R. Salazar2 . 1 Centro Integral Oncologico Campal, Oncology, Madrid, Spain; 2 Catalan Institute of Oncology ICO, Bellvitge Biomedical Research Institute IDIBELL, Department of Medical Oncology, L’Hospitalet de Llobregat, Spain; 3 Hospital Universitario 12 de Octubre, Medical Oncology Division, Madrid, Spain; 4 Catalan Institute of Oncology ICO, Bellvitge Biomedical Research Institute IDIBELL, Translational Research Laboratory, L’Hospitalet de Llobregat, Spain; 5 Spanish National Cancer Research Center CNIO, Histopathology Unit, Madrid, Spain; 6 Catalan Institute of Oncology ICO, Bellvitge Biomedical Research Institute IDIBELL, Hereditary Cancer Program, L’Hospitalet de Llobregat, Spain; 7 VCN Biosciences, Research, Sant Cugat del Valles, Spain Background: VCN-01 is a selective oncolytic adenovirus with hyaluronidase activity. This study aimed to determine the safety, pharmacokinetics (PK) and anti-tumor activity of a single intravenous injection of VCN-01, either alone or combined with nab-paclixatel/gemcitabine (AG) and to define the recommended phase II dose (RP2D) of VCN-01 alone or in combination with AG. Material and Methods: VCN-01 was administered IV to patients with advanced solid tumors with ECOG 0−1 at doses ranging from 1E11 to 1E13 viral particles per patient (vp) as a single agent and at from 3.3E12 to 1E13 vp + AG at standard doses. Blood VCN-01 DNA was measured at different time-points after treatment. Viral shedding was assessed in sputum, urine and stool. IL-6 and IL-12 levels were also measured. Viral DNA and lymphocytic populations were analyzed in tumor biopsies. Response was assessed using RECIST v1.1 criteria. Results: Twenty three patients have been treated to date including 16 patients who received VCN-01 alone (patients with gastrointestinal solid tumors, except 1 head & neck tumor) and 7 patients who received combined treatment (all having pancreatic adenocarcinoma). Neutralizing antibodies (NAb) levels against adenovirus of 1/320 were detected in all patients at screening. The most common related toxicities after injection of VCN-01 alone were: pyrexia (81%), arthralgia & myalgia (25%), AST elevation (18%), anorexia and vomiting (18%). One dose-limiting toxicity consisting