Abstract. Immunogenicity (the development of an adaptive immune response reactive with a therapeutic) is a well-described but unwanted facet of biotherapeutic development. There are commonly applied procedures for immunogenicity risk assessment, testing strategies, and bioanalysis. With some modifications, these can be applied to new biotherapeutic modalities. For novel therapies such as antibody-drug conjugates (ADCs), the unique structural components may contribute additional complexities to both immunologic responses and bioanalytical methods. US product inserts (USPIs) for two commercially available ADCs detail the incidence of immunogenicity; however, the body of literature on immunogenicity of ADCs is limited. We recently participated in a conference session on this topic (Annual meeting of the American Association of Pharmaceutical Scientists, held November 2013 in San Antonio, TX, USA. The meeting featured the Symposium: Immunogenicity Assessment for Novel Antibody Drug Conjugates, Nonclinical to Clinical) which prompted an effort to share our perspectives on how immunogenicity risk assessment, testing strategies, and bioanalytical methods can be adapted to reflect the complexity of ADC therapeutics.

A single-center trial of CD19 directed, lentiviral transduced chimeric antigen receptor (CAR) T cells (CTL019) for relapsed and refractory (r/r) B-ALL pediatric patients showed rates of CR >90% with prolonged CAR T cell persistence/CR without further therapy in the majority of patients infused (Maude NEJM 2014). We report here the feasibility, safety and efficacy of the first multicenter global pivotal registration CAR T cell trial. Features of this trial include: i) the first trial in which industry-manufactured cells were provided to all patients; ii) enrollment across 25 centers in the US, EU, Canada, Australia, and Japan; iii) successful transfer and manufacturing of cells in a global supply chain; and iv) successful implementation of cytokine release syndrome (CRS) management across a global trial. All patients had CD19 positive B-ALL with morphologic marrow tumor involvement at registration (>5% blasts), and were either primary refractory; chemo-refractory after first relapse, relapsed after second line therapy; or ineligible for allogeneic SCT. CTL019 was manufactured from patient PBMC under GMP conditions in the US, at a centralized "sponsor-owned" manufacturing facility, and supplied to all sites. The primary endpoint of overall remission rate (CR+CRi) within 3 months and secondary endpoints (EFS, DOR, OS and safety) were assessed by an independent review committee. Based on preliminary data as of March 2016, 57 patients were enrolled. There were 3 manufacturing failures (5%), 5 patients were not infused due to death or adverse events (9%), and 15 patients were pending infusion at the data cut off. Following fludarabine/cyclophosphamide lymphodepleting chemotherapy in the majority of the patients, 34 patients (median age 11 [3-23], 50% with prior HSCT) were infused with a single dose of CTL019 at a median dose of 2.9 x106 transduced CTL019 cells/kg (0.2 to 4). Among 29 patients reaching D28 prior to the data cutoff, 83% (24/29) achieved CR or CRi by local investigator assessment, all of which were MRD-negative. Two early deaths occurred prior to initial disease assessment, one due to disease progression and one due to intracranial hemorrhage. Two patients did not respond. One patient was in CR by BM at D28, but CSF was not assessed, therefore this patient was classified as "incomplete" assessment. Safety was managed by a protocol-specified CRS algorithm with no cases of refractory CRS. Using the Penn CRS grading scale, 82% of patients experienced CRS, with 7 grade 3 (21%) and 8 grade 4 (24%) events. 44% patients with CRS required anti-cytokine therapy; all received tocilizumab with or without other anti-cytokine therapy, with complete resolution of CRS. Besides CRS, the most common grade 3 and 4 non-hematologic AEs were febrile neutropenia (29%), increased bilirubin (21%), increased AST (21%), and hypotension (21%). 21% of patients experienced grade 3 or 4 neuropsychiatric events including confusion, delirium, encephalopathy, agitation and seizure; no cerebral edema was reported. CTL019 in vivo cellular kinetics by qPCR demonstrated transgene persistence in blood in responding patients at and beyond 6 months. Overall exposure (AUC 0-28d) and maximal expansion (Cmax) of CTL019 DNA measured by qPCR was higher in responding compared with non-responding patients. In summary, this pivotal global study in pediatric and young adult patients with r/r B-ALL receiving CTL019, confirms a high level of efficacy and a similar safety profile to that shown in the prior single center experience. Safety was effectively and reproducibly managed by appropriately trained investigators. The study has completed accrual. At the meeting, updated data from a planned formal interim analysis including safety, efficacy (primary and selected secondary endpoints), cellular kinetics, and impact of anti-cytokine therapy will be presented for more than 50 patients infused at 25 global sites. Disclosures Grupp: Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy. Laetsch:Novartis: Consultancy; Loxo Oncology: Consultancy. Bittencourt:Seattle Genetics: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Educational Grant. Maude:Novartis: Consultancy. Myers:Novartis Pharmaceuticals: Consultancy. Rives:Novartis: Consultancy; Jazz Pharma: Consultancy. Nemecek:Medac, GmbH: Research Funding; Novartis: Consultancy; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Schlis:Novartis: Honoraria. Martin:Jazz Pharmaceuticals: Other: One time discussion panel; Novartis: Other: Support of clinical trials. Bader:Medac: Consultancy, Research Funding; Riemser: Research Funding; Neovii Biotech: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Peters:Novartis: Consultancy; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy; Medac: Consultancy. Biondi:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees. Baruchel:Servier: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Jazz: Consultancy; Baxalta: Research Funding. June:University of Pennsylvania: Patents & Royalties; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder . Sen:Novartis: Employment. Zhang:Novartis: Employment. Thudium:Novartis: Employment. Wood:Novartis Pharmaceuticals: Employment, Other: Stock. Taran:Novartis: Employment. Pulsipher:Chimerix: Consultancy; Jazz Pharmaceutical: Consultancy; Novartis: Consultancy, Other: Study Steering Committee; Medac: Other: Housing support for conference.

Bcl-2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3-mimetic, has been designed to specifically target and counteract anti-apoptotic Bcl-2 proteins. We evaluated the biological effects of obatoclax on the anti-tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy-sensitive (RSCL), -resistant cell lines (RRCL) and primary tumour-cells derived from patients with B-cell lymphomas (N=39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase-independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient-derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin-1 knockdown. In summary, obatoclax is an active Bcl-2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response.

The American Association of Pharmaceutical Scientists (AAPS) National Biotechnology Conference Short Course “Translational Challenges in Developing Antibody-Drug Conjugates (ADCs),” held May 24, 2012 in San Diego, CA, was organized by members of the Pharmacokinetics, Pharmacodynamics and Drug Metabolism section of AAPS. Representatives from the pharmaceutical industry, regulatory authorities, and academia in the US and Europe attended this short course to discuss the translational challenges in ADC development and the importance of characterizing these molecules early in development to achieve therapeutic utility in patients. Other areas of discussion included selection of target antigens; characterization of absorption, distribution, metabolism, and excretion; assay development and hot topics like regulatory perspectives and the role of pharmacometrics in ADC development. MUC16-targeted ADCs were discussed to illustrate challenges in preclinical development; experiences with trastuzumab emtansine (T-DM1; Genentech) and the recently approved brentuximab vedotin (Adcetris®; Seattle Genetics) were presented in depth to demonstrate considerations in clinical development. The views expressed in this report are those of the participants and do not necessarily represent those of their affiliations.