IT-35cGMP MANUFACTURING OF EX VIVO EXPANDED TUMOR-SPECIFIC T CELLS AND AMPLIFIED TUMOR RNA-PULSED DC VACCINES TARGETING GLIOBLASTOMA AND MEDULLOBLASTOMA

摘要

BACKGROUND: Adoptive cellular therapy using transfer of tumor-specific lymphocytes has emerged as a potent strategy for treatment of advanced and refractory malignancies. We have employed the use of total tumor RNA (TTRNA)-pulsed DCs in the ex vivo expansion of tumor-specific lymphocytes for use in adoptive cellular therapy targeting pediatric and adult malignant brain tumors. METHODS: Total tumor RNA was extracted and amplified from resected brain tumor specimens using previously published methods. Efforts to improve recovery, fidelity of amplification, and integrity of RNA included comparative analysis of free extraction methods, SMART ^TM-based cDNA synthesis, and PCR primer modifications. DC generation and T cell expansion under varying culture conditions and cytokine milieus were evaluated to improve functional yields of autologous cellular products. Validation runs of multi-site collection of tumor cells and leukapheresis products for DC and T cell generation under GLP and GMP conditions were performed using standardized operating procedures. RESULTS AND CONCLUSIONS: We have optimized high quality (RNA integrity, RIN ≥ 8) TTRNA amplification from resected brain tumor specimens using as little as 1 ng of input TTRNA through incorporation of solid-phase paramagnetic bead technology for cDNA synthesis and optimized PCR conditions. We founded that TTRNA transfection up-regulates the expression of CD80, CD83, CD86, HLA-DR in mature DCs. The feasibility of external site collection and delivery of tumor cells and leukapheresis products for tumor RNA amplification, DC generation, and T cell expansion has been validated by our laboratory. We have recently demonstrated the safety and feasibility of this adoptive cellular therapy platform in a phase I trial of pediatric patients with relapsed medulloblastoma and PNETs in a single institutional setting. A multi-institutional phase 2 clinical trial is underway (Re-MATCH trial: FDA IND BB-14058, UF IRB 128-2013).