Adoptive cell therapy (ACT) has the potential to reinstate T cell-mediated immunity, to combat infectious diseases as well as cancer.1,2 Although lenti- or retroviral systems are commonly exploited for TCR editing3-8, these conventional strategies lack control over the quantity and the site of transgenic TCR integration events, thereby putting safety and functionality in jeopardy. To increase the safety profile of the engineered T cell products, both FDA and EMA recommend low vector copy number (VCN).9,10 Nevertheless, a low VCN could also compromise functionality by limiting TCR transcription levels and protein surface expression.7,11,12
In this study, researchers at the Technical University of Munich (Technische Universität München) leveraged orthotopic TCR replacement (OTR) method to eliminate endogenous TCR chains and place an antigen-specific receptor under endogenous TCR transcriptional control. With this, they sought to investigate the differential consequences on the magnitude, variability, and interrelatedness of transgenic TCR surface expression and functionality. More precisely, the scientists in question made a systematic comparison between OTR and conventional editing outcomes, by scrutinizing: TCR transgene integration site, transcription, surface expression and T cell product functionality.13
Editing method influences T cell product function and safety
As part of this investigation, 51 different antigen-specific TCRs were analyzed.13
To determine the exact genomic position of TCR transgene insertion sites, the scientists had recourse to TLA-based assays. Our data revealed 2 integration sites in OTR samples (Figure 1, left). More specifically, the entire TCR transgene was found at the intended site (i.e. first exon of TRAC) and a homology-independent partial transgene integration was also spotted at the intended double-strand break in the TRBC1/2 locus (Figure 1, left). On the other hand, the integration profile following conventional editing appeared to be highly variable (Figure 1, right).13
Figure 1. TLA genome-wide coverage plot across the human genome for integration of TCR 1-4. Chromosomes are listed on the y-axis, whereas the chromosomal position is indicated on the x-axis. Circles indicate the positions of integration sites whereas arrows depict the integrations that are less abundant. TRAC locus is located in chromosome 14 and TRBC locus is in chromosome 7.13
Taken together, results from this study suggest that conventional editing - via viral transduction – give rise to variable copy numbers and untargeted transgene integration. In turn, these occurrences are responsible for the variability in TCR expression and functionality. In sharp contrast, targeted editing seems to incite more homogeneous TCR expression, akin to physiological T cells.13
Increased T cell product homogeneity correlates with predictable T cell responses in vivo
The enhanced predictability of T cell product function as well as the increased safety profile following targeted TCR editing, truly positions OTR engineering as an attractive alternative strategy for future immunotherapies. As such, targeted TCR editing may offer unique therapeutic opportunities and could potentially revolutionize immunotherapy as a whole in the future.
Improved analytic QC solutions that meet regulatory expectations
To briefly touch upon our genetic screening capabilities - with regards to the identification of integration events - our unique TLA-based solutions will be able to support your and/or your colleagues’ work regardless if an on- or off-target strategy has been employed. Furthermore, our TLA-based assays will be able to identify any potential sequence variants (i.e. single nucleotide or structural variations) in integrated vectors sequences and insertion sites, unlike PCR-based approaches which are mostly hypothesis-driven.14
Given the meteoric rise of cell and gene therapy products over the last few years, we have had the pleasure to witness a growing adoption of our TLA-based solutions by leading pharma and biotech companies across the globe as well as world-renowned academic research institutions. In fact, the unique genetic insights of our proprietary technology have been described by the likes of Memorial Sloan Kettering Cancer Center, Cellectis and FDA/NIH through peer-reviewed publications.
For more information regarding our TLA-based services within Cell & Gene Therapy application, we invite you to visit the following webpage.
 Billingham, R.E., Brent, L., and Medawar, P.B. (1954). Quantitative studies on tissue transplantation immunity. II. The origin, strength and duration of actively and adoptively acquired immunity. Proc. R. Soc. London. Ser. B, Biol. Sci 143, 58–80.
 June, C.H., Riddell, S.R., and Schumacher, T.N. (2015). Adoptive cellular therapy: a race to the finish line. Sci. Transl. Med. 7, 280ps7.
 Morgan, R.A., Dudley, M.E., Wunderlich, J.R., Hughes, M.S., Yang, J.C., Sherry, R.M., Royal, R.E., Topalian, S.L., Kammula, U.S., Restifo, N.P., et al. (2006). Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126–129.
 Robbins, P.F., Morgan, R.A., Feldman, S.A., Yang, J.C., Sherry, R.M., Dudley, M.E., Wunderlich, J.R., Nahvi, A.V., Helman, L.J., Mackall, C.L., et al. (2011). Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J. Clin. Oncol. 29, 917–924.
 Chodon, T., Comin-Anduix, B., Chmielowski, B., Koya, R.C., Wu, Z., Auerbach, M., Ng, C., Avramis, E., Seja, E., Villanueva, A., et al. (2014). Adoptive transfer of MART-1 T-cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma. Clin. Cancer Res. 20, 2457–2465.
 van Loenen, M.M., de Boer, R., Hagedoorn, R.S., van Egmond, E.H.M., Falkenburg, J.H.F., and Heemskerk, M.H.M. (2011). Optimization of the HA-1-specific T-cell receptor for gene therapy of hematologic malignancies. Haematologica 96, 477–481.
 Okamoto, S., Mineno, J., Ikeda, H., Fujiwara, H., Yasukawa, M., Shiku, H., and Kato, I. (2009). Improved expression and reactivity of transduced tumor-specific TCRs in human lymphocytes by specific silencing of endogenous TCR. Cancer Res. 69, 9003–9011.
 Ahmadi, M., King, J.W., Xue, S.-A., Voisine, C., Holler, A., Wright, G.P., Waxman, J., Morris, E., and Stauss, H.J. (2011). CD3 limits the efficacy of TCR gene therapy in vivo. Blood 118, 3528–3537.
 Aiuti, A., Cossu, G., de Felipe, P., Galli, M.C., Narayanan, G., Renner, M., Stahlbom, A., Schneider, C.K., and Voltz-Girolt, C. (2013). The committee for advanced therapies’ of the European Medicines Agency reflection paper on management of clinical risks deriving from insertional mutagenesis. Hum. Gene Ther. Clin. Dev. 24, 47–54.
 Sadelain, M. (2004). Insertional oncogenesis in gene therapy: how much of a risk? Gene Ther. 11, 569–573.
 Kustikova, O.S., Wahlers, A., Kuhlcke, K., Stahle, B., Zander, A.R., Baum, C., and Fehse, B. (2003). Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. Blood 102, 3934–3937.
 Viola, A., and Lanzavecchia, A. (1996). T cell activation determined by T cell receptor number and tunable thresholds. Science 273, 104–106.
 Müller TR, Jarosch S, Hammel M, Leube J, Grassmann S, Bernard B, Effenberger M, Andrä I, Chaudhry MZ, Käuferle T, Malo A, Cicin-Sain L, Steinberger P, Feuchtinger T, Protzer U, Schumann K, Neuenhahn M, Schober K, Busch DH. Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy. Cell Rep Med. 2021 Aug 17;2(8):100374. doi: 10.1016/j.xcrm.2021.100374. PMID: 34467251; PMCID: PMC8385324.
 de Vree, P., de Wit, E., Yilmaz, M. et al. Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol 32, 1019–1025 (2014). https://doi.org/10.1038/nbt.2959