Abstract 3201: Generation of tumor-antigen specific T cells under highly controlled environmental conditions by utilization of a novel cGMP compliant cell culture reactor
Authors: Aigner Michael , Standar Stefanie , Moi Stefanie , Gary Regina , Gerbitz Armin , Mackensen Andreas 
Published: August 2012

Abstract
Large scale in vitro generation of tumor-antigen specific T cells for adoptive transfer under good manufacturing procedures (GMP) conditions is one of the major challenges for future clinical applications. The novel Z(R)RP cell culture system consists of high precision control units for gas and fluidics in combination with a class A micro laminar flow unit and a GMP conform culturing unit which allows for maximal control of cell culture conditions. This system is - to our knowledge - the only commercially available product that fully complies with all regulatory specifications required for advanced therapy medical products (ATMP) production in the European Union. In addition, the Z(R)RP bioreactor system also implements the guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q10 and the requirements from 21 Code of federal regulations (CFR) parts 210, 211, 600 and 1271, making this system a potential candidate for the cGMP compliant production of human cellular products for individualized medicine approaches. We studied the potency of the Z(R)RP system for the generation of MART-1 antigen-specific T cells. Mononuclear cells (MNC) from healthy donors were cultured in GMP grade media supplemented with 1% of autologous serum. MNC were repeatedly pulsed with MART-1 peptide and culture outcome was analysed with respect to cellular composition, relative expansion and expression of cytokines of MART-1 specific T cells by flow cytometry. In short term cultures of 5x10E8 cells for up to 2 weeks medium pH as well as CO2 and O2 saturation was kept constant throughout culture duration. Glucose and lactate levels were monitored daily and continuous influx of fresh medium was adapted accordingly to match nutrient depletion and metabolite production. Media glucose concentration was kept at &gt;500mg/ml and lactate concentrations at &lt;1500mg/ml. Antigen specific T cells as identified by tetramer staining were expanded up to 350 fold. In comparison, standard control closed system cultures in bags resulted in 10 to 40% lower yield of specific T cells. In addition, overall cell yield was 25 - 90% higher from bioreactor cultures than from closed bag cultures. Analysis of MART-1 specific CD8+ T cells revealed a more naive phenotype as shown by CD45RA staining (65% in reactor vs. 20% in bag cultures). Production of cytokines as demonstrated by intracellular FACS staining of interferon-gamma and interleukin-4 was not significantly different in T cell populations from bioreactor or bag cultures. Our results show the superior potential of this bioreactor principle for expansion of high numbers of tumor-antigenspecific T cells under fully cGMP compliant conditions, which is pre-requisite for future individualized anti-tumoral cellular therapies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3201. doi:1538-7445.AM2012-3201