IM-13 * MOLECULAR ENGINEERING OF AMPLIFIED TUMOR RNA-PULSED DENDRITIC CELLS IN ADOPTIVE CELLULAR THERAPY TARGETING MEDULLOBLASTOMA AND GLIOBLASTOMA

Abstract

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 phenol-free extraction methods, SMART™-based cDNA synthesis, RT and PCR primer modifications. To characterize whole pool of cDNA libraries and their IVT RNA products obtained from tumor brain tissues we used next generation sequencing approach. DC generation and T cell expansion under varying culture conditions and cytokine milieus were evaluated to improve functional yields of autologous cellular products. 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. Massively parallel RNA sequencing analysis of TTRNA amplified from brain tumor specimens using in-house development of a complete system for cDNA amplification show similar or even better results compared to commercial available Clontech's SMARTer kit. RNA-Seq analysis detected the higher number of transcripts in brain tumor tissues compared to normal brain. We found that TTRNA transfection up-regulates the expression of CD80, CD83, CD86, HLA-DR in mature DCs. 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 multiinstitutional phase 2 clinical trial is underway.