Development of a large-scale H3.3K27M-specific TCR-transduced T cell manufacturing method for adoptive cell therapy in HLA-A2+ patients with diffuse midline glioma

Abstract

Background & Aim Diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG), constitutes up to 20% of pediatric brain cancer and has a median survival of less than one year. Genetic studies have revealed that greater than 70% of DMG cases harbor an amino acid substitution from lysine (K) to methionine (M) at position 27 of histone 3 variant 3 (H3.3). We recently reported that a peptide encompassing the H3.3K27M mutation (amino acid position 26-35) is presented by HLA-A*0201+ human glioma cells and induces a specific cytotoxic responses in CD8+ T lymphocytes. Furthermore, we cloned a high-affinity T cell receptor (TCR) that specifically recognizes the H3.3K27M epitope. Alanine scanning demonstrated that the key amino-acid sequence motif in the epitope for the TCR reactivity is not shared by any known human protein. Based on these data, we plan to develop a phase I study of adoptive transfer therapy using autologous T cells transduced with H3.3K27M-specific TCR. To this end, we have initiated developing processes for manufacturing the TCR-transduced T cells. Methods, Results & Conclusion We optimized the codon usage of full-length cDNA for α- and β-chains of the TCR, and cloned them into a TCR retroviral vector system, which incorporates small interfering (si)RNA targeting constant regions of the endogenous TCR α- and β- chains to avoid mispairing between endogenous and transgene TCR chains (Takara Bio, Inc.). PG13 packaging cells were transfected with siTCR retroviral vector and established as the producer cell line. Using Miltenyi CliniMACS Prodigy system, which allows closed and automated manufacturing of T cell products, we activated human T cells (starting number 1 × 10e8 T cells) with MACS GMP T Cell TransActTM(Miltenyi), and subsequently transduced with the siTCR retroviral vector and expanded the cells in the presence of IL-7 and IL-15. We achieved over 20-fold expansion of the T cells with at least 20% transduction efficiency. Furthermore, the vast majority of TCR-transduced T cells showed central memory phenotype represented by co-expression of CCR7 and CD62L. To further optimize manufacturing conditions, we are currently evaluating other parameters, such as cytokine doses and combinations, as well as starting T cell populations. These data suggest that the Prodigy system may represents a feasible system for manufacturing TCR-transduced T cells for adoptive transfer therapy.