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. The work of this core is supported by close collaborative relationships with the Cell Processing Service of DTM, for support of microarray analysis and development of clinical products; the ETIB Flow Cytometry Facility, for support of sorting of clinical products for research endpoints; and the laboratory of Ronald Gress, for technical support in RNA and DNA isolation and quantitative assays. Several 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 (PI: Daniel Fowler), PDCMF staff working within CPS/DTM, supported clinical production and molecular characterization of an activated donor CD4 T cell product (T.Rapa.12) administered prophylactically 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), but potential for longer persistence after infusion, was assessed for anti-tumor efficacy in lymphoma and in renal cell carcinoma patients (Fowler et al, Clin Cancer Res, 2015). (2) In ETIB protocol 09-C-0224, (P.I.: Nancy Hardy) we used cellular and molecular assays to monitor changes to test whether localized tumor death following targeted irradiation could stimulate a next-day donor lymphocyte infusion (DLI) to initiate a systemic immune response and trigger immune-mediated tumor regression at non-irradiated sites. We determined that circulating T cells in radiation-plus-DLI treated patients were activated, as compared with DLI alone. Furthermore, using a custom panel of multiplex RNA probes (Nanostring), we identified upregulation of inflammasome and TLR genes responding to cell damage, as well as genes induced by IFNa, at both irradiated and non-irradiated (abscopal) sites, consistent with systemic immune response. (3) 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. This protocol expanded autologous T cells in the presence of IFNa and rapamycin, to generate a cell product with Th1/Tc1 activity). We supported clinical translation of the T1.Rapa cells, from production protocol through IND documentation, and characterized gene expression and T cell lineage phenotype in the T1.Rapa infusion product. In the clinical trial, we assessed serial changes in effector and regulatory T cell populations in blood and in bone marrow (the main tumor site), identified the clonal expansion of specific T cells by T cell receptor sequencing and demonstrated changes in gene expression and T cell phenotype consistent with increased Th1/Tc1 activity. (4) In a collaborative study with Dr. Fowler's laboratory we used flow cytometry to characterize the effects of infusions of mesenchymal stem cells, derived from human marrow, on human T helper cells in a murine xenogeneic GVHD model (Amarnath et al, Stem Cells, 2015). (5) In collaboration with Dr. Fowler, we have assessed the effects of use of the pentostatin/cytoxan regimen he developed for lymphodepletion in support of a mesothelioma protocol using a partially humanized anti-mesothelin antibody-immunotoxin developed by Dr. Pastan's laboratory. This year we collaborated with Dr. Pastan's laboratory to investigate regulation of immunogenicity of immunotoxins by HLA-DR/DP/DQ expression (Mazor et al, AAPSJ, 2016 in press) (6) 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 have validated the process of Tscm isolation and expansion and have developed standard operating protocols and documentation for an IND submission. (7) 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). When the protocol was expanded to treat follicular lymphoma and acute lymphoblastic leukemia, we have similarly tracked anti-CD19 CAR+ T cells in peripheral blood (Brudno et al, J Clin Oncol, 2016). Furthermore, PDCMF staff in CPS developed 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 and put into clinical trials this year (16-C-0054). (8) In a clinical trial initiated last year (14-C-0168: PI. James Kochenderfer)) Dr. Kochenderfer used a new CAR construct directed against the BCMA receptor expressed on myeloma cells (Carpenter et al, Clin Cancer Res, 2013). PDCMF staff in DTM validated the procedures for transfection of the final GMP grade construct and prepared the SOP for generation of the expanded CAR product. In the PDCMF laboratory, we tracked anti-BCMA CAR+ T cells by flow cytometry demonstrating a massive expansion in blood and marrow concurrent with depletion of myeloma (Abbas et al, Blood, 2016). PDCMF has also supported biospecimen processing for Dr. Kochenderfer's involvement in a multicenter CRADA trial of another anti-BCMA CAR product (16-C-0025). Finally PDCMF staff in DTM have validated clinical-grade generation of a new anti CD30 CAR developed by Dr. Kochenderfer for implementation later this year.
