Background aims: The generation of gene-modified T cells for clinical adoptive T-cell therapy is challenged by the potential instability and concomitant high financial costs of critical T-cell activation and transduction components. As part of a clinical trial to treat patients with metastatic renal cell cancer with autologous T cells engineered with a chimeric antigen receptor (CAR) recognizing carboxy-anhydrase-IX (CAIX), we evaluated functional stability of the retroviral vector, T-cell activation agent Orthoclone OKT3 (Janssen-Cilag, Beerse, Belgium) monoclonal antibody (mAb) and the transduction promoting agent RetroNectin (Takara, Otsu, Japan). Methods: Carboxy-anhydrase-IX chimeric antigen receptor retrovirus-containing culture supernatants (RTVsups) were generated from two packaging cell lines, Phoenix-Ampho (BioReliance, Sterling, UK) and PG13, and stored at -80°C over 10 years and 14 years. For Orthoclone OKT3 and RetroNectin, aliquots for single use were prepared and stored at -80°C. Transduction efficiencies of both batches of RTVsups were analyzed using the same lots of cryopreserved donor peripheral blood mononuclear cells, Orthoclone OKT3 and RetroNectin over time. Results: We revisit here an earlier report on the long-term functional stability of the RTVsup, observed to be 9 years, and demonstrate that this stability is at least 14 years. Also, we now demonstrate that Orthoclone OKT3 and RetroNectin are functionally stable for periods of at least 6 years and 10 years. Conclusions: High-cost critical components for adoptive T-cell therapy can be preserved for ≥10 years when prepared in aliquots for single use and stored at -80°C. These findings may significantly facilitate, and decrease the financial risks of, clinical application of gene-modified T cells in multicenter studies.

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Keywords RetroNectin, anti-CD3 mAb, chimeric antigen receptor, clinical trial, human T lymphocytes, retroviral vectors, stability
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Journal Cytotherapy
Lamers, C.H.J, van Elzakker, P.M.M.L, van Steenbergen, S.C.L, Luider, B.A, Groot, C, van Krimpen, B.A, … Gratama, J.W. (2013). Long-term stability of T-cell activation and transduction components critical to the processing of clinical batches of gene-engineered T cells. Cytotherapy, 15(5), 620–626. doi:10.1016/j.jcyt.2012.12.006