192. Deep Phenotyping of Manufactured Enhanced-Affinity NY-ESO-1-Specific T Cells Reveals a Pattern of Effector and Memory Programming That Correlates with Clinical Outcome in Observed Cancer Indications
Authors: Luca Melchiori, Daniel E. Lowther, Gwendolyn K. Binder-Scholl, Daniel D. Williams, Yoav Peretz, Marylene Fortin, Willian D. Tap, Aaron P. Rapoport, Crystal L. Mackall, Bent K. Jakobsen
Journal: Molecular Therapy (2016)

It is well established that human tumors express unique antigens; however, immunosuppressive mechanisms prevent natural immune responses. We hypothesized that infusion of T cells that have been genetically modified to express affinity-optimized tumor antigen-specific TCRs may overcome these barriers. We initiated several ongoing clinical studies to evaluate T cells engineered with an affinity-enhanced TCR specific for the NY-ESO-1 and LAGE-1 cancer testis antigens (NY-ESOc259-T), in synovial sarcoma (SS) and multiple myeloma (MM) patients with antigen-positive tumors. Here we report updated phenotyping results from two MM and SS cohorts of patients. Manufactured product (MP) and baseline samples were analyzed by flow cytometry evaluating memory and activation markers (e.g. CD45RA, CCR7, ICOS, OX40, etc) and polyfunctionality markers (e.g. IFN-γ and IL-2). NY-ESOc259-T was detected by pentamer staining and its phenotype was correlated with clinical response. Upon culture NY-ESOc259-T generated different memory phenotypes. The phenotype at baseline (day -50) did not predict the phenotype acquired at the end of culture. Interestingly, in MM a positive trend with clinical response was observed in MPs bearing a higher percentage of Central Memory (CM) cells secreting IL-2 and IFN-γ, while a negative trend was found in Effector Memory (EM) and Effectors (EMRA)-dominated MPs secreting high levels of TNF-α. This trend was confirmed by analysing the absolute number of CM cells infused in a small number of patients, hinting at the existence of an "effective CM cells dose" of around 3×108 CM cells necessary to achieve clinical response. Of note, this trend was observed also in the SS cohort. Upon stimulation T cells proliferate and differentiate in vitro, but at the same time cells need time in culture to reach an effective dose. To understand these dynamics we retrospectively analysed the evolution of CM cells in relation to the length of culture. We observed a negative correlation between percentage of CM cells and culture time, while EM and EMRA cells increased with the duration of culture. This suggests the importance of reconciling time of culture with phenotype and number of cells in order to achieve that "sweet spot" critical for clinical response. Activation/costimulation markers in the SS cohort were also analysed in relation to culture conditions. MPs consistently displayed up-regulation of ICOS, CD40L and OX40, suggesting that an activated phenotype is achieved even after several days of culture. Of note, markers of immunological memory like CD27 and CD28 were also maintained up to the end of culture highlighting the fitness of the product despite the duration of the culture. These data suggest that NY-ESOc259-T may acquire a CM phenotype that positively correlates with clinical response in the cancer indications observed. Additionally, we show how the manufacturing process successfully produces a population of activated cells that express important activation markers without losing markers of fitness and memory - properties that are key to driving the anti-tumour response observed in cancer patients.