462. Automated Manufacturing Process for the Production of Genetically Modified Lymphocytes

Abstract

The MM-TK is a medicinal product constituted of T lymphocytes ex vivo genetically modified with the γ retroviral vector SFCMM-3 Mut2 #48 to express a mutated form of the Herpes Simplex Virus Thymidine kinase (HSV-TK Mut2) and a truncated form of the human Low affinity Nerve Growth Factor Receptor (ΔLNGFR) genes. It has development as adjunctive therapy in leukaemia patients or undergone bone marrow transplantation. The current manufacturing process is performed following semi-automated procedure using instrument and tubing set not marketing compliant. In order to optimize this and to standardize cellular therapeutic production for clinical trial and for the market, MolMed have strengthened an alliance with Miltenyi Biotec for tailoring of CliniMACS Prodigy platform for MM-TK DS manufacturing.A closed and automated cell process was developed using the new device with a specific software designed in order to manufacture a robust, safe and consistent product.The manufacturing process essentially consists in the stimulation of a large number of PBMCs (about 3×109 cells) isolated from an haploidentical donor, with anti-CD3 monoclonal antibody and IL-2, transduction of the stimulated cells with the SFCMM-3 Mut2 #48 retroviral vector in RetroNectin® coated bags and immunoselection of positively transduced cells using the CD271-LS Microbeads reagent (Miltenyi Biotec). Selected TK cells are then further expanded for 7 days before final formulation in freezing medium. The whole manufacturing process lasts 13 days.The following steps have been automated: cell concentration and cell washing at different manipulation steps and immunomagnetic selection of the transduced cells.Four development full-scale runs demonstrate that automated cell processing for the genetic manipulation of T lymphocytes can be performed in the closed- and automated-system designed by MolMed, reaching good results for all in-process parameters evaluated: cell recovery of 80-90%; cell viability more than 80% at each step; total process yield of 72%. The DS/DP product has been evaluated for the quality attributes as product and process related impurities (CD14% + CD16% + CD19% and residual retronectin, residual anti-CD3, residual CD271 microbeads) and potency of the product (ΔLNGFR expression; sensitivity to Ganciclovir; Vector Copy Number; immunophenotype as CD3%, CD4%, CD8%, CD45%,CD25, CD69, HLA-DR, CD95; CD45RA and CD62L). The results will support the discussion with Regulatory Autorities and permit the introduction of the new optimized closed manufacturing process in the late phase of the clinical trial and for the market.