These findings suggest that exogenous IL-15 may have a potential role in adoptive immunotherapy by both enhancing proliferation and modulating functionality during ex vivo T-cell expansion.

Immunotherapy with ex vivo cultured T cells depends on a large supply of biologically active cells. Understanding the effects of culture parameters is essential for improving the proliferation and efficacy of the expanded cells. Low oxygen tension (5% pO(2)) was previously reported to improve T-cell expansion and alter cellular phenotypic characteristics compared to T cells cultured at 20% pO(2). Here we report the use of DNA-array based transcriptional analysis coupled with protein-level analysis to provide molecular insights into pO(2) and patient-variability effects on expanded primary human T cells. Analysis of seven blood samples showed that reduced pO(2) results in higher expression of genes important in lymphocyte biology, immune function, and cell-cycle progression. 20% pO(2) resulted in higher expression of genes involved in stress response, cell death, and cellular repair. Expression of granzyme A (gzmA) was found to be significantly regulated by oxygen tension with cells at 5% pO(2) having greater gzmA expression than at 20% pO(2). Protein-level analysis of gzmA was consistent with transcriptional analysis. Granzyme K (gzmK) was coexpressed with gzmA, whereas Granzyme B (gzmB) expression was found to precede the expression of both gzmA and gzmK in 15-day cultures. Temporal gene expression patterns for seven blood samples demonstrate that most genes are expressed by all patient samples in similar temporal patterns. However, several patient-specific gene clusters were identified, and one cluster was found to correlate well with cell proliferation and may potentially be used to predict patient-specific T-cell expansion.

Background Lisocabtagene maraleucel (liso-cel) is an investigational, CD19-directed, genetically modified, autologous cellular immunotherapy administered as a defined composition of CD8+ and CD4+ components to deliver target doses of viable chimeric antigen receptor (CAR) T cells from both components. The CAR comprises a CD19-specific scFv and 4-1BB-CD3ζ endodomain. Liso-cel is being developed for the treatment of multiple B cell malignancies, including relapsed/refractory large B cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). The liso-cel manufacturing process design includes controls that enable robustness across heterogeneous patient populations and disease indications, minimizing between-lot variability. This is highlighted by consistency in process duration, reduction of terminally differentiated T cells present in the T cell starting material, and consistency in T cell purity across B cell NHL and CLL/SLL indications. Methods The liso-cel manufacturing process involves selection of CD8+ and CD4+ T cells from leukapheresis, followed by independent CD8+ and CD4+ activation, transduction, expansion, formulation, and cryopreservation. Liso-cel was manufactured in support of the TRANSCEND NHL 001 (NCT02631044) and TRANSCEND CLL 004 (NCT03331198) clinical trials. Phenotypic analysis of T cell and B cell composition from leukapheresis, T cell starting material, and CAR T cell product was performed by flow cytometry. Molecular characterization of T cell receptor (TCR) clonality was estimated from the T cell starting material and CAR T cell product through transcriptional profiling. Results Liso-cel manufacturing process optimizations have been implemented in advance of commercialization. These optimizations have significantly improved process duration consistency (Figure 1; F test P=4.1×10−36). Both phenotypic and molecular TCR clonality analyses demonstrated a significant reduction in terminally differentiated CD8+ T cells across the manufacturing process. Frequencies of CD45RA+ CCR7− populations were measured by flow cytometry in CD8+ T cell starting material (median=35.1%) and CAR T cell product (median=11.7%; Wilcoxon rank sum P=3.1×10−25). Characterization of TCR clonality showed a significant decrease in clonality in the CAR T cell product compared with T cell starting material (Wilcoxon rank sum P=5.6×10−6), suggesting selective expansion of clonally diverse, less differentiated T cell populations. These findings are supported by the predominant memory T cell composition observed in liso-cel. Manufacturing process robustness enabled by in-process T cell selection is further demonstrated by the capability to produce highly pure T cell products across heterogeneous patient populations and different disease indications. T cell and B cell composition were characterized in the leukapheresis, selected T cell material, and CAR T cell product, demonstrating consistent clearance of non-T cells, including CD19+ B cells in both B- cell NHL and CLL/SLL patient cohorts. Although the CD19+ B cell composition is significantly higher in leukapheresis from patients with CLL/SLL (median=10.0% of leukocytes) compared with B cell NHL patients (median=0.0% of leukocytes, Wilcoxon rank sum P=1.6×10−9), CAR T cell products manufactured from both CLL/SLL and B cell NHL patient populations consistently demonstrated clearance of non-T cells, including CD19+ cells, to below levels of quantitation. Conclusion Despite variation between B cell NHL and CLL/SLL patient leukapheresis, T cell enrichment before activation and transduction enables consistent downstream process performance and T cell purity, and a substantially reduced risk of transducing residual tumor cells. In addition, the reduction of terminally differentiated effector T cells and capacity to retain T cell diversity further improved consistency in product quality. Taken together, process modifications have enabled consistent manufacturing duration and quality of liso-cel product, which support operational efficiency and scalability for commercial production. Disclosures Teoh: Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Johnstone:Juno Therapeutics, a Celgene Company: Employment, Patents & Royalties: Author on a number of patent applications and invention disclosures relating to cell therapy and immunosequencing. Christin:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Yost:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Haig:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Mallaney:Juno Therapeutics, a Celgene Company: Employment. Radhakrishnan:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gillenwater:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Albertson:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Guptill:Juno Therapeutics, a Celgene Company: Employment. Brown:Juno Therapeutics, a Celgene Company: Employment. Ramsborg:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership, Patents & Royalties: Numerous patents. Hause:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Larson:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership.

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