Despite advancements in outcomes with the introduction of anti-CD20 monoclonal antibody therapy, a substantial proportion of B-cell Non-Hodgkin's Lymphoma (B-NHL) patients do not sustain a durable response to standard of care treatment. T-cell bispecific antibodies (TCBs) represent a new class of disease-targeting agents shown to activate T-cells to kill cancer cells, offering this exciting mechanism of action with 'off the shelf' availability. CD20-TCB (RG6026) is a novel T-cell-engaging bispecific antibody whose "2:1" format possesses two CD20 binders in addition to a CD3 binder, enabling increased tumor antigen avidity, rapid T-cell activation, and enhanced tumor cell killing. NP30179 is a multicenter phase I dose escalation trial investigating the safety, tolerability, pharmacokinetics (PK), biomarker responses, and antitumor activity (assessed by overall response rate [ORR] and complete response rate [CR] per Lugano 2014 criteria) of single agent CD20-TCB. Patients receive escalating doses of CD20-TCB as intravenous infusions with dose escalation guided by a model implementing the Bayesian continuous reassessment method with overdose control. In order to reduce the potential risk of cytokine release syndrome (CRS) induced by CD20-TCB mediated systemic T-cell activation, a single dose of 1000 mg obinutuzumab (G) pretreatment (Gpt) is administered seven days prior to start of CD20-TCB and has preclinically been shown to debulk peripheral B cells and reduce systemic cytokine release (Bacac et al. Clin Canc Res 2018). As of July 29th 2018 a total of 64 patients (pts) with aggressive r/r B-NHL (DLBCL/PMBCL/trFL/Richter´s transformation (n=47)) and indolent r/r B-NHL (FL(n=17)) received CD20-TCB doses ranging from 5 µg to 1800 µg in a Q2W schedule. Median age was 64 years (range 28-82), 61% were male, median prior lines of therapy was 3 (range 1-10). A single DLT (myocardial infarction) was observed at 220 µg and until the maximum tested dose CD20-TCB was well tolerated. The most reported AEs were pyrexia (n=14, all Gr 1 and 2), neutropenia (all grades n=17, Gr 3 and 4 n=14) and cytokine release syndrome (CRS, n=14, all Grade 1-2 according to the criteria by Lee et al. Blood 2014). Two patients received tocilizumab for CRS management all CRS events were manageable and resolved, without leading to dose reduction or study withdrawal. No CNS toxicity was reported so far in this study. Fifty-five pts had at least one post-baseline response assessment and were eligible for efficacy analysis. Responses were observed from 15 µg onwards, and complete responses (CR) occurred from 300 µg onwards following two cycles of treatment. At doses of 300 µg or above (n=29) the ORR and CR rate by investigator assessment was 38% and 24%, respectively (FL: 3/5 pts and 2/5 pts respectively, aggressive B-NHL: 8/24 pts and 5/24 pts respectively). All CRs are sustained thus far with a median follow up 96 days (range 26-152). Responses were seen across various NHL subtypes and across prognostic factors such as prior lines of therapy, refractoriness to the most recent line of therapy, tumor burden, and IPI or FLIPI. CD20-TCB exposure and receptor occupancy increased dose-dependently across the investigated dose range; dose-escalation is continuing to optimize these factors. No anti-drug-antibodies have occurred. CD20-TCB is a novel 2:1 format T-cell-engaging bispecific antibody which already at suboptimal doses displays promising clinical activity in heavily-pretreated B-NHL. In addition, Gpt has shown clinical proof of principle as an approach to efficiently mitigate CRS. An update on safety and efficacy as well as biomarker data will be presented. Disclosures Hutchings: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Iacoboni:Celgene: Other: Travel funding; Roche: Honoraria. Morschhauser:Janssen: Other: Scientific Lectures; BMS: Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sureda:Takeda: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Sanofi: Honoraria. Salles:Janssen: Honoraria; Merck: Honoraria; Gilead Sciences: Honoraria; Epizyme: Honoraria; Amgen: Honoraria; Acerta: Honoraria; Abbvie: Honoraria; Celgene: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Morphosys: Honoraria; Pfizer: Honoraria; Servier: Honoraria; Takeda: Honoraria. Carlo-Stella:Rhizen Pharmaceuticals: Research Funding; Boehringer Ingelheim: Consultancy; Sanofi: Consultancy; MSD: Other: Advisory Board; Genenta Science: Other: Advisory Board; BMS: Other: Advisory Board; Amgen: Other: Advisory Board; Janssen: Other: Advisory Board; AstraZeneca: Other: Advisory Board; ADC Therapeutics: Other: Advisory Board. Martinez Lopez:Celgene: Research Funding, Speakers Bureau; BMS: Research Funding, Speakers Bureau; Jansen: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Thomas:Roche: Employment, Equity Ownership. Morcos:Roche: Employment, Equity Ownership. Quackenbush:Roche: Employment. Ferlini:Roche: Employment. Bacac:F. Hoffmann-La Roche Ltd.: Employment, Other: stock, Patents & Royalties. Broeske:Roche: Employment, Equity Ownership. Dimier:Roche: Employment, Equity Ownership. Moore:Roche: Employment. Weisser:Roche: Employment, Equity Ownership, Patents & Royalties. Dickinson:GSK: Consultancy.

with R/R DLBCL, 2/11 (18%) had responses at doses between 5-12 mg, 6/11 (55%) had responses at doses between 18-40 mg, and 2/2 (100%) had responses at the 80 mg dose, with both of these latter being CRs. Elevated levels of serum cytokines were observed w/ dosing; however, no correlation was observed w/ clinical efficacy.Immunohistological analysis of malignant lymph node tissue demonstrated that pts w/ high and low CD20 achieved clinical response.Relapse among responders was seen w/ either maintenance or loss of CD20 expression, suggesting antigen-dependent and independent disease escape mechanisms. Conclusions: R1979 was well tolerated in pts w/ R/R B-NHL. No DLTs and no significant neurological toxicity were observed. Tx w/ R1979 showed impressive efficacy w/ 100% ORR in R/R FL starting at doses ≥5 mg. More resistant tumors such as R/R DLBCL are showing benefit w/ increasing doses. Based on these efficacy findings, a Phase 2 study in R/R FL Gr 1-3a, R/R DLBCL, and other R/R B-NHL subtypes is planned.Introduction: CD20-TCB (RG6026) is a T-cell-engaging bispecific antibody with a novel "2:1" molecular format which preclinically showed greater avidity for CD20 antigen, combinability with other anti-CD20 antibodies, and greater efficacy as compared to other CD20-CD3 bispecific formats. NP30179 is an ongoing multicenter phase I dose escalation trial investigating the safety, tolerability, pharmacokinetics (PK), biomarkers and antitumor activity of CD20-TCB. Methods: Patients receive escalating doses of CD20-TCB as intravenous infusions guided by a model implementing a Bayesian CRM method with overdose control. To reduce the risk of cytokine release syndrome (CRS), a single dose of obinutuzumab as pretreatment (Gpt) is administered seven days prior to CD20-TCB to debulk B cells in peripheral blood and normal tissues (Bacac et al Clin Canc Res 2018).Updated data at clinically-relevant doses of 600 μg and above are reported.Results: As of February 13 th 2019 a total of 87 pts with r/r aggressive (a)NHL (DLBCL/PMBCL/trFL/Richter´s transformation; n=79) and r/r FL (n=8) received CD20-TCB doses ranging from 600 μg to 25mg in a Q2W or Q3W schedule. Median age was 61 years (range 22-84), 92 ABSTRACT median prior lines of therapy was 3 (range 1-13), and 86% pts were refractory to prior therapy. CRS (according to the criteria by Lee et al.Blood 2014) occurred in 45 patients (23% G1, 24% G2, 3% G3, 1% G4). At the highest dose cohort (25 mg) >G3 CRS occurred in 3/8 pts at cycle 1, thereby preventing further dose escalation. One patient in the 25 mg cohort died of upper GI bleeding after an episode of severe CRS. All other CRS events were manageable and resolved, and patients were retreated without delay at the same dose at cycle 2, where only one additional CRS event was seen (G1).In the efficacy evaluable pts (n=84) the ORR and CR rate by investigator assessment (Lugano 2014 criteria) in aNHL (n=76) was 46% and 29%, and in FL (n=8) 63% and 50%, respectively. In the highest dose cohorts (10-25 mg) the ORR and CR rate in aNHL (n...

BackgroundFibroblast activation protein alpha (FAP) is frequently over-expressed in the tumor microenvironment (TME) while exhibiting limited expression in normal tissues. FAP expression was reported to be immunosuppressive in tumor mouse models and generally associated with worse prognosis in clinical studies. Therefore, it is important to understand the context in which FAP both exhibits immunosuppressive characteristics and be a useful target for immunotherapy.MethodsComprehensive immunohistochemistry (IHC) analyses on formalin-fixed paraffin-embedded tissue specimens with emphasis on lymph nodes and primary and metastatic tumor lesions spanning a wide range of indications were undertaken in this study. FAP staining of tumor tissues was performed with an optimized IHC robust-prototype-assay (RPA) and manually scored. The area (normal stroma & neoplastic) staining positively relative to the total tumor area at each intensity level was recorded and an H-score calculated (FAP-intensity score).These were supplemented by gene expression analysis using public as well as Roche phase 1, 2 and 3 cancer immunotherapy (CIT) clinical trial data sets.ResultsAnalysing FAP expression on normal tissue confirmed the general absence of FAP apart from a subset of pancreatic islet cells. Unlike the more homogenous expression of typical protein targets on tumor cells, FAP expression in the TME is heterogeneous in both pattern and intensity, requiring the analysis of a large sample set. Therefore, we evaluated 1216 samples from 23 tumor indications and 70 sub-indications. FAP expression exhibited a significant spread ranging from indications with highly abundant expression to those with low coverage.Using data from matching IHC and gene expression samples we confirmed FAP mRNA expression to significantly correlate with RPA H-scores (Spearman correlation: 0.62) (N=289, P=1.2E-31). Gene expression data from 12 atezolizumab clinical studies, including standard of care (SOC) randomized studies, with more than 6000 samples from 4 major indications were interrogated for the association between FAP expression and clinical response as evaluated by overall and progression free survival. This analysis suggests that FAP expression is generally associated with higher hazard ratios across all atezolizumab-treated samples (OS: 95% CI 1.04–1.09; PFS: 1.04–1.08), with the highest effect observed in Renal Cell Carcinoma (OS: 95% CI 1.08–1.31; PFS: 1.05–1.21), indicating a potential role of FAP in limiting CIT.ConclusionsData from these analyses can tailor indication and patient enrichment strategies for achieving optimal FAP-targeting. We propose to select indications with FAP-levels that are high enough to enable drug accumulation, yet low enough to reduce immunosuppressive effects that can hamper successful immunotherapy.

Introduction RG7386 is a novel bispecific antibody, binding with high affinity to fibroblast activation protein (FAP) and with low affinity to death receptor 5 (DR5). Avidity-driven binding of the bispecific antibody mediates hyper-clustering of DR5 thus triggering tumor cell death. Induction of FAP dependent apoptosis translated into strong efficacy in vivo using patient derived xenografts thereby proposing RG7386 as an attractive therapeutic approach for the treatment of stroma rich FAP-positive solid tumors as well as FAP positive sarcomas. However, thus far DR5 targeting strategies failed to show clinical efficacy likely due to lack of hyperclustering and intrinsic resistance mechanisms. Preclinical translational studies were conducted to demonstrate the on-target mode of action, to ensure maximal activity and to guide pharmacodynamic (PD) analysis to unravel potential resistance mechanisms. Material and Methods Based on strong anti-tumor efficacy of RG7386 alone and in combination with irinotecan in a colorectal cancer (CRC) cell line based xenograft model (DLD-1) co-injected with fibroblasts a kinetic study was designed. Tumors were explanted 6, 16, 72 and 168 hours after RG7386 single agent treatment and harvested for immunohistochemical (IHC) and ELISA based protein analysis of apoptosis markers, such as cleaved caspase 3 (cc3), cleaved PARP and activated caspase 8 and 9. Additionally, PD effects of RG7386 treatment as single agent and in combination with doxorubicin were investigated in a FAP positive desmoplastic melanoma cell line derived model (LOX-IMVI). Results We observed significant time-dependent induction of apoptosis upon treatment with RG7386 by IHC and ELISA in xenograft tumors expressing FAP in stroma or on tumor cells directly. High, transient levels of apoptosis markers such as cc3 were observed by IHC early after treatment compared to vehicle control. Analysis of equivalent tissue lysates by ELISA revealed also rapid induction of cc3, cleaved PARP and activated caspase 8 and 9 in monotherapy in the DLD-1 CRC xenograft model which was superior when given together with doxorubicin in the LOX-IMVI desmoplastic melanoma model. Conclusion We identified that RG7386 strongly induces tumor cell apoptosis in vivo shortly after injection independently if FAP was expressed on tumor stroma or at tumor cells and discovered optimal pharmacodynamic markers and time points for sampling and analysis. As a result, early clinical trials of RG7386 will be designed to increase the therapeutic potential by choosing the right combination partner and to ensure demonstration of the postulated mode of action by pharmacodynamic data. Citation Format: Thomas Friess, Ann-Marie Broeske, Stefanie Lechner, Esther Abraham, Gabriele Hoelzlwimmer, Hadassa Sade, Peter Bruenker, Oliver Krieter. Preclinical pharmacodynamic biomarker and combination strategy of RG7386, a novel FAP-DR5 bispecific antibody for targeting solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C125.

Background: Activation of the extrinsic apoptotic pathway by TRAIL is dependent on clustering of death receptors (DR) on the surface of cells. However, current TRAIL-based strategies have proven ineffective in clustering death receptors and failed to demonstrate robust therapeutic activity in clinical trials. More potent DR agonist therapies could help to overcome insufficient pathway activation and resistance to TRAIL activation. RG7386 is a novel bispecific FAP-DR5 antibody, binding with high affinity to fibroblast activation protein (FAP) and with low affinity to DR5. FAP is expressed at high prevalence on cancer associated fibroblasts (CAFs) in various tumor types as well as on tumors of mesenchymal origin, such as sarcomas. Avidity-driven binding of the bispecific antibody induces hyperclustering of DR5, which leads to potent induction of extrinsic apoptosis pathway signaling and tumor cell death. Biomarkers will be crucial in predicting sensitivity to DR5 activation and apoptosis induction and for selection of patients most likely to benefit from treatment with RG7386. Aim: The aim of the study was to explore the efficacy of RG7386 in vitro and in vivo. CRC and PDAC xenograft models expressing FAP on tumor stroma as well as sarcoma models were used to explore in vivo efficacy. Molecular profiling of sensitive and resistant tumors was also performed to identify response prediction markers. Results: RG7386 demonstrated additive efficacy in vitro with clinically relevant combinations (e.g. irinotecan, paclitaxel) in a variety of CRC and PDAC cell lines. In a xenograft model, where CRC cells (DLD-1) were co-injected with fibroblasts, RG7386 showed strong anti-tumor efficacy in combination with irinotecan. Remarkably, in a patient-derived CRC xenograft model (Co5896), the efficacy of RG7386 in combination with irinotecan induced complete tumor remission in all mice (n = 10/10). Furthermore, the combination of RG7386 with doxorubicin generated complete remissions in FAP+ sarcoma patient and desmoplastic melanoma cell line derived xenograft models such as Sarc4605 and LOX-IMVI. Finally, extensive molecular profiling of sensitive and resistance models in vitro revealed a distinct response prediction signature of DR5 sensitivity. Conclusion: RG7386 is a novel bispecific antibody inducing avidity-driven DR5 crosslinking by binding to FAP. This induces potent apoptosis of tumor cells, making it an attractive therapeutic approach for treatment of FAP+ solid tumors. Encouraging data indicate the high potential of RG7386 to treat FAP positive sarcomas. A comprehensive biomarker program will be employed in the early clinical development of RG7386 to enable selection of patients likely to benefit and to corroborate the mode of action, anti-tumor activity and potential response prediction markers. Citation Format: Thomas Friess, Stefanie Lechner, Esther Abraham, Ann-Marie Broeske, Sabine Bader, Andreas Roller, Meher Majety, Katharina Wartha, Suzana Vega-Harring, Hadassah Sade, Oliver Krieter, Peter Bruenker. Induction of avidity-driven hyperclustering of DR5 by a new FAP-DR5 bispecific antibody (RG7386) leads to strong anti-tumor efficacy. [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 952. doi:10.1158/1538-7445.AM2015-952