Vaccination to improve the persistence of CD19CAR gene-modified T cells in relapsed pediatric acute lymphoblastic leukemia.

Trials with second generation CD19 chimeric antigen receptors (CAR) T-cells report unprecedented responses but are associated with risk of cytokine release syndrome (CRS). Instead, we studied the use of donor Epstein-Barr virus-specific T-cells (EBV CTL) transduced with a first generation CD19CAR, relying on the endogenous T-cell receptor for proliferation. We conducted a multi-center phase I/II study of donor CD19CAR transduced EBV CTL in pediatric acute lymphoblastic leukaemia (ALL). Patients were eligible pre-emptively if they developed molecular relapse (>5 × 10-4) post first stem cell transplant (SCT), or prophylactically post second SCT. An initial cohort showed poor expansion/persistence. We therefore investigated EBV-directed vaccination to enhance expansion/persistence. Eleven patients were treated. No CRS, neurotoxicity or graft versus host disease (GVHD) was observed. At 1 month, 5 patients were in CR (4 continuing, 1 de novo), 1 PR, 3 had stable disease and 3 no response. At a median follow-up of 12 months, 10 of 11 have relapsed, 2 are alive with disease and 1 alive in CR 3 years. Although CD19CAR CTL expansion was poor, persistence was enhanced by vaccination. Median persistence was 0 (range: 0-28) days without vaccination compared to 56 (range: 0-221) days with vaccination (P=0.06). This study demonstrates the feasibility of multi-center studies of CAR T cell therapy and the potential for enhancing persistence with vaccination.

A full GMP process to select and amplify epitope-specific T lymphocytes for adoptive immunotherapy of metastatic melanoma.

A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved. We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting. In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1. All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification. The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 108 pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients. This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.

Comparison of three culture media for the establishment of melanoma cell lines.

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols.

Induction of apoptosis in mouse thymocytes by cyclosporin A: an in vitro study.

The effect of cyclosporin A (CsA) on mouse thymocytes was investigated in vitro. Ultrastructural examination and DNA electrophoresis of 24hr-CsA-treated thymocytes showed chromatin condensation, cellular shrinkage and nuclear fragmentation in oligonucleosomal fragments. DNA labeling of CsA-treated-thymocytes with propidium iodide (PI) showed an increase in the number of apoptotic nuclei compared to untreated thymocytes. Furthermore, flow cytometric analysis using monoclonal antibodies (mAbs) specific to particular thymocyte subsets showed that CsA induces a decrease in the relative number of immature double positive (DP) CD4+CD8+ thymocytes in direct proportion to the concentration of CsA. No significant changes were observed in mature single positive (SP) CD4+CD8- and CD8+CD4- cells. Moreover, the cell viability of CsA-treated thymocytes was decreased. These results suggest that CsA induces the programmed cell death of thymocytes in vitro. Taken together with our previous study on the in vivo effect of CsA on mouse thymus and thymocytes, the present study confirms that, in addition to its effect on the thymic epithelium, CsA acts directly on thymocytes by inducing their programmed cell death. We postulate that immature DP thymocytes are the most likely targets of CsA.

Effects of PGE2 upon differentiation and programmed cell death of suspension cultured CD4-CD8- thymocytes.

Recently, several works have focused on the modulation of the immune response by arachidonic acid metabolites. Some of these metabolites, such as prostaglandins, have been shown to influence thymocyte "education" in vitro. However, the effect of one of them, prostaglandin E2 (PGE2), in the education of CD4-CD8- double negative immature thymocytes (DN cells) remained unclear. Using a flow cytometry analysis of DN cells cultured for 24 h in the presence of PGE2, we observed, compared with DN thymocytes cultured without PGE2, an increase in the CD4+CD8-CD3- immature thymocytes and in the CD4+CD8- and CD8+CD4- mature single positive thymocytes and a decrease in the DN and CD4highCD8high double positive thymocytes. Other differentiation thymocyte surface markers, such as CD3, CD5, TCR alpha beta, TCR delta gamma and HSAg, revealed an increasing number of thymocytes bearing these first four markers and a lower expression of the HSAg. Furthermore, we observed an accumulation of CD4lowCD8low thymocytes and an increasing proportion of hypodiploid nuclei. These two findings have been shown to be markers of the programmed cell death process. These findings suggest that PGE2 probably acts on thymocyte differentiation through at least two distinct pathways. On the one hand, PGE2 seems to promote differentiation of DN cells into CD4+CD8-CD3- immature cells and drive CD4+CD8+CD3+ thymocyte to a CD4+CD8- and CD8+CD4- mature phenotype. On the other hand, PGE2 is probably implicated directly or indirectly in the increase or the acceleration of the programmed cell death process of immature CD4+CD8+CD3+ thymocytes, which is linked to the positive and/or negative selection.

Induction of apoptosis in mouse thymocytes by cyclosporin A: in vivo study.

We investigated the in vivo effect of cyclosporin A (CsA) on mouse thymus and thymocytes. Administration of CsA (10 mg/kg of body weight) was found to induce a marked reduction in the size, weight and consistency of the thymus. These modifications were associated with thymic reticulo-epithelial cells (TREC) and thymocyte damage. Some of the damaged thymocytes displayed characteristic of cells undergoing apoptosis. Ultrastructural study of thymocytes and thymic tissue, as well as DNA electrophoresis of thymocytes, showed chromatin condensation, cellular shrinkage, and nuclear fragmentation in oligonucleosomal fragments. DNA labeling with propidium iodide (PI) of thymocytes from CsA treated mice cultured for 24 hrs showed an increased number of apoptotic nuclei. Furthermore, flow cytometric analysis using monoclonal antibodies (mAbs) specific for thymocyte subsets confirmed that CsA induces a large decrease in the relative number of mature single positive (SP) CD4+CD8- and CD8+CD4- thymocytes expressing high densities of CD3 and T cell receptor ab (TCR alpha beta) surface molecules, but also a decrease in the absolute number of the other thymocyte subsets. These results suggest that CsA causes macroscopic and ultrastructural modifications of the thymus, associated with an active process of cell death in mouse thymocytes in vivo. In line with these results we formulate a hypothesis concerning the stage of T-cell development at which CsA induces apoptosis.