193 The Achilles VELOSTM Process 2 boosts the dose of highly functional clonal neoantigen-reactive T cells for precision personalized cell therapies

BackgroundAdoptive transfer of ex-vivo expanded tumor-infiltrating lymphocytes (TIL) has shown promise in the clinic. However, the non-specific expansion of TIL and the lack of understanding of the active component of TIL has resulted in poor correlation between clinical response and dose as well as poor understanding of response and resistance mechanisms. The VELOSTM manufacturing process generates a precision and personalized treatment modality by targeting clonal neoantigens with the incorporation of an antigen-specific expansion step to enrich the product for these specificities. Achilles has developed a second generation manufacturing process (VELOSTM Process 2) to boost the neoantigen-reactive cell dose while maintaining key qualitative features associated with function. Here we report the in-depth characterization of clonal neoantigen-reactive T cells (cNeT) products expanded using the two VELOSTM processes.MethodsMatched tumors and peripheral blood from patients undergoing routine surgery were obtained from patients with primary NSCLC or metastatic melanoma (NCT03517917). TIL were expanded from tumor fragments and peptide pools corresponding to the clonal mutations identified using the PELEUSTM bioinformatics platform were synthesized. cNeT were expanded by co-culture of TIL with peptide-pulsed autologous dendritic cells, with an optimized cytokine cocktail and co-stimulation for Process 2. Neoantigen reactivity was assessed using our proprietary potency assay with peptide pool re-challenge followed by intracellular cytokine staining. Single peptide reactivities were identified using ELISPOT and flow cytometric analysis for in-depth phenotyping of cNeT was performed.ResultsCD3+ T cells displayed higher fold expansion in Process 2 (median 77.4) compared to Process 1 (median 3.8)(n=5). Both processes showed similar CD3+ T cell content (median Process 1=91.3%, Process 2=96.9% n=5) and contained both CD4+ and CD8+ T cells showing reactivity to clonal neoantigens. Proportion of cells responding to neoantigen re-challenge was similar across both processes (median Process 1=19.9% and Process 2=18.2%) leading to higher reactive dose when coupled with higher T cell doses in Process 2. Phenotypically T cells were predominantly effector memory for both processes and Process 2 had lower frequencies of terminally differentiated T cells.ConclusionsAchilles’ proprietary potency assay enables the optimization of new processes that deliver high cNeT doses to patients by detecting the active drug component. We have generated proof of concept data that supports the transfer of the VELOSTM Process 2 to clinical manufacture for two first-in-human studies for the treatment of solid cancers.Ethics ApprovalThe samples for the study were collected under an ethically approved protocol (NCT03517917)

The Essential Need for a Validated Potency Assay for Cell-Based Therapies in Cardiac Regenerative and Reparative Medicine. A Practical Approach to Test Development

Biological treatments are one of the medical breakthroughs in the twenty-first century. The initial enthusiasm pushed the field towards indiscriminatory use of cell therapy regardless of the pathophysiological particularities of underlying conditions. In the reparative and regenerative cardiovascular field, the results of the over two decades of research in cell-based therapies, although promising still could not be translated into clinical scenario. Now, when we identified possible deficiencies and try to rebuild its foundations rigorously on scientific evidence, development of potency assays for the potential therapeutic product is one of the steps which will bring our goal of clinical translation closer. Although, highly challenging, the potency tests for cell products are considered as a priority by the regulatory agencies. In this paper we describe the main characteristics and challenges for a cell therapy potency test focusing on the cardiovascular field. Moreover, we discuss different steps and types of assays that should be taken into consideration for an eventual potency test development by tying together two fundamental concepts: target disease and expected mechanism of action. Graphical Abstract Development of potency assays for cell-based products consists in understanding the pathophysiology of the disease, identifying potential mechanisms of action (MoA) to counteract it and finding the most suitable cell-based product that exhibits these MoA. When applied, the potency assay needs to correlate bioactivity of the product, via a measurement related to the MoA, with treatment efficacy. However, in the cardiovascular field, the process faces several challenges and high requirements.

A micro-organosphere potency assay for adoptive T-cell therapy.

e15027 Background: Lung cancer is the leading cause of death worldwide. Metastatic lung cancer patients often relapse or grow refractory to chemotherapy, radiotherapy, and checkpoint inhibitors. Adoptive T cell therapy (ACT) has been garnering more attention due to longitudinal complete responses. Tumor resections from patients harbor tumor-infiltrating lymphocytes (TILs) which can be cultured ex vivo and assessed for tumor reactivity before infusion. This generally follows a patient lymphodepletion regimen which allows the transferred T cells an optimal environment to proliferate and survive in the patient. TIL ACT can produce complete responses – rarely ever observed using traditional onco-therapy – in metastatic melanoma patients. However, while TILs specific to the neo-antigens expressed by tumor cells can be expanded ex vivo, this observed specificity is low in the clinic. Researchers have attempted to solve this by priming ex vivo expanded T cells with antigen-presenting cells previously pulsed with peptides representative of the neo-antigen repertoire of the matched tumor, but have rarely observed complete responses, likely due to current biomarkers for T cell activity poorly predicting anti-tumor cytotoxicity. To date there has been no acceptable potency assay for manufactured TILs, a requirement by the FDA for approval to use them in the clinic. Thus, the need to assess potency of ex vivo engineered T-cells against matched tumor cells is evident. Methods: We have developed a novel diagnostic immune-oncology (IO) pipeline, which uses a membrane-microfluidic platform to culture patient-derived tumor micro-organospheres (MOs) in extracellular matrix droplets. MOs can be rapidly established following patient tumor sample acquisition through biopsies or resection, and preserve the stromal cell populations in the original tumor microenvironment, as characterized by both flow cytometry and single-cell RNA-seq. An automated imaging assay was further established to robustly quantify the amount of immune-induced apoptosis of tumor cells in the MOs by patient-matched TILs, which is highly specific and yields minimal background. Results: We find that this method is not only amenable to high-throughput microscopy, but the larger surface-area-to-volume ratio of micro-organospheres also allows greater TIL infiltration and interaction with tumor cells. The resulting highly-sensitive assay requires far fewer input immune and tumor cells to achieve robust, clinical grade sensitivity response, making it the first clinically feasible assay for testing personalized TIL potency. Conclusions: The MO IO technology is currently being used for assessing clinical efficacy of manufactured TIL products for an upcoming ACT trial for non-small cell lung cancer patients. This technology also provides a companion to TIL ACT, CAR T therapy, and other immunotherapies, for which the ability to predict clinical potency is generally lacking.