The Preclinical Development and Clinical Monitoring Facility (PDCMF) of the Experimental Transplantation and Immunology Branch supports the development and implementation of new protocols involving adoptive immune cell therapies through preclinical development, translational implementation of clinical products and preservation and analysis of patient blood and tissues during clinical trials. Five novel protocols involving adoptive transfer of T cells have been implemented in recent years as a result of this process. (1) In ETIB protocol 04-C-0055, Daniel Fowler (ETIB) utilized donor-derived CD4 helper cells expanded with anti-CD3/anti-CD28 beads in the presence of IL-4 and rapamycin (T.Rapa.12) to enhance donor engraftment and reduce AGVHD (Fowler et al, Blood, 2013). Subsequently, a second generation product, cultured for 6 days and having a less differentiated phenotype (Castiello et al, Cytotherapy 2013) was assessed for anti-tumor efficacy in lymphoma and in renal cell carcinoma patients (Fowler et al, Clin Cancer Res, 2015). PDCMF staff, working within CPS, supported development and molecular characterization of these novel products and implementation of the clinical trials. (2) A second novel product developed in the Fowler laboratory involved CD3/CD28 expansion of autologous T cells (both CD4 and CD8) in the presence of IFNalpha and rapamycin (T1.Rapa), to generate a cell product with Th1/Tc1 activity. In ETIB protocol 11-C-0016 (P.I. Claude Sportes/Daniel Fowler), patients with multiple myeloma received an autologous hematopoietic stem cell transplant followed by infusion of T1.Rapa cells in a phase one trial of escalating T1.Rapa cell doses; through expanding the scale of product manufacturing, serial infusions of T1.Rapa cells were then implemented. Clinical production of these cells, from IND support through clinical implementation, was supported through our staff in CPS. Following initiation of this trial, we have monitored serial changes in lymphocyte populations in blood and in bone marrow (the main tumor site) and have provided plasma assays of inflammatory cytokines to monitor patient responses at each dose escalation. We are currently evaluating altered gene expression in CD4 and CD8 T cells in the T1.Rapa product. Furthermore, using flow cytometry, we are characterizing the T1.Rapa product in terms of T-helper lineage, cytokine production and rapamycin-induced metabolic changes; patient peripheral blood and marrow are being similarly evaluated at serial timepoints after T1.Rapa infusion. (3) We have supported the implementation of the first trial of the use of donor-derived anti-CD19 Chimeric Antigen Receptor (CAR) T cells in patients with relapsed or persistent lymphoma following allogeneic transplant (Protocol 10-C-0054: P.I. James Kochenderfer (ETIB)). Using multi-parameter flow cytometry, we tracked the presence of CAR+ T cells following adoptive transfer and have demonstrated the expansion of anti-CD19 CAR+ T cells in the blood concurrent with the onset of anti-tumor activity, approximately one week after adoptive transfer (Kochenderfer et al, Blood 2013). In on-going studies, as the protocol as been expanded to treat follicular lymphoma and acute lymphoblastic leukemia, we have similarly tracked anti-CD19 CAR+ T cells in peripheral blood. Currently PDCMF staff in CPS are developing the operational protocols and documentation for IND submission and implementation of a new lentiviral human-immunoglobulin-based anti-CD19 CAR vector that Dr. Kochenderfer has designed. (4) Dr. Kochenderfer has also developed a new CAR construct directed against the BCMA receptor expressed on myeloma cells (Carpenter et al, Clin Cancer Res, 2013), PDCMF staff in DTM have also worked validated the procedures for transfection of the final GMP grade construct and prepared the SOP for generation of the expanded CAR product in a clinical trial initiated this year in multiple myeloma (14C-0168: PI. James Kochenderfer). (5) Dr. Luca Gattinoni (ETIB) has demonstrated that persistent CD8+ stem central memory T cells (Tscm) are the most effective T cells for adoptive immune therapy against tumors. To support utilization of Tscm as the base cell for CAR transfection and adoptive immune therapy, PDCMF staff in CPS are validating the process of Tscm transduction and expansion and have developed standard operating protocols and documentation for an IND submission. (6) We have also used cellular and molecular assays to monitor implementation of a novel therapy utilizing irradiation of selected tumor sites followed by donor lymphocyte infusion. (09-C-0224, P.I.: Nancy Hardy (ETIB)) This protocol was developed to test whether localized tumor death following irradiation could stimulate a systemic immune response and trigger immune-mediated tumor regression at non-irradiated sites. Flow cytometry of peripheral blood T cells demonstrated systemic activation of T cells (increased proliferation) in radiation-plus-DLI treated patients. We have developed a multiplex RNA quantitation assay (Nanostring) using a custom panel of probes we designed to assess upregulation of inflammasome and TLR genes responding to cell damage as well as genes induced by interferon (IFN). Although developed to monitor immune activation in graft versus host disease (GVHD) (described in Project ZIC BC 010934), we have determined that these genes are upregulated following targeted tumor irradiation and infusions of expanded donor T cells. (7) Finally we have also participated in a collaborative study with Dr. Fowler's laboratory on the effect of infusions of marrow-derived mesenchymal stem cells (MSC) in reducing acute graft versus host disease (GVHD). In this study we used flow cytometry to characterize the effects of infusions of MSC on human Type I T helper cells in a murine xenogeneic GVHD model (Amarnath et al, Stem Cells, 2015).
