To date, the number of gene therapy medicinal products (GTMP) tested in clinical trials is thriving. However, there is meagre information available regarding the exact molecular characterization - of various GTMPs under investigation - throughout the entire production chain.1-3
To mend this paucity, researchers at the University Medical Center Utrecht (UMCU) applied TLA-based solutions to QC their vector-producing clone and their downstream TEG001 products. In particular, they characterized αβT cells engineered to express a defined γδT cell engineered to express a defined γδT receptor (TEG).4,5,6 As such, TLA-based method was leveraged to QC (TEG001) insert integrity throughout manufacturing.
Analyzing TEG001 insert integrity from vector producer cells until drug product
First, the integrity of TEG001 insert in their master cell bank (MCB)/viral-producing clone 73 was assessed. No structural rearrangement was observed, and no sequence variants were found in the protein encoding TCRγ5-T2A-5 DNA sequence. However, 4 point mutations and 1 insertion were picked up in the non-coding region of the TEG001 insert. Since the frequency of the mutation ranged from 8 to 14%, this suggested that the identified SNVs - in this clonal cell population - were only present in a fraction of the integrated transgene copies (Table 1).6
Next, our genome-wide coverage plot revealed 9 integration sites (intact vector copies) in the MCB (Figure 1B).6
In contrast, the vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. Furthermore, TLA data also revealed that the previously detected mutational variants - in the MCB - were also transferred to the TEG001 drug product and that the mutant allele frequencies there were also within the same ratio (8%-19%). No additional nucleotide variants were spotted here (Table 1). Additional analysis of the 5 small sequence variants showed that their presence does not lead to increased risk for malignant transformation in vivo.6
Finally, TLA-based genome-wide coverage plot on TEG001 medicinal product displayed a heterogeneous integration pattern at the genomic level (Figure 4), and heterogeneity was seen to decrease following a significant in vitro culture period.6
A valuable framework for future GTMPs
In this study, UMCU researchers reported on the development of a GMP-compliant TEG001 product.7 By leveraging TLA-based assays, they were able to reliably detect sequence variants in non-coding regions of the TEG001 insert in the MCB and TEG001 drug product. Nevertheless, these occurrences did not appear to lead to a growth advantage in long-term cultured TEG001cells in vitro.
The number of integrated TEG001 inserts in the MCB was estimated at 9 copies per cell, and although the transgene copies per cell in TEG001 cells was calculated as being a factor 0.72 below, it still exceeded the 5 copy numbers per cell in TEG001. Of note, 5 (transgene) copies per cell have been considered - for many years - as the acceptance threshold vis-à-vis GTMP copy number.8 As such, CAR T products do not exceed this limit.1,9-12 Nevertheless, high copy numbers have now become acceptable if defined reasoning is provided.
All in all, the authors indicate that this extensive reporting on the molecular characterization of TEG001 transgene integrity “resulted in the approval of a phase I clinical study that did not only allow to investigate the safety and tolerability of TEG001 in patients with relapsed/refractory AML [acute myeloid leukemia], high-risk myelodysplastic syndrome, and relapsed/refractory MM [multiple myeloma], but also will provide a valuable framework for future GTMPs.”6
Improved analytical QC tool to guarantee quality and satisfy regulatory requirements
Unlike LAM-PCR13,14, tag-PCR15 and INSPIIRED16,17, TLA-based method enables the complete targeted sequencingof any transgene toward precisely mapping (transgene) insertion sites as well as assessing the integrity of the integrated vector sequences (e.g. CAR or TCR transgene) on the nucleotide level.6,18-21 Therefore, our TLA-based approach truly represents an improved analytical tool to thoroughly QC (cellular) engineered products as well as genetically modified immune cells, throughout the complete production chain (i.e. from vector producer cells until final medicinal product).6
If you are interested in learning more about the relevance of our TLA-based solutions in cell & gene therapy manufacturing, click here to review our latest webinar:
 Kalos, M., Levine, B.L., Porter, D.L., Katz, S., Grupp, S.A., Bagg, A., and June, C.H. (2011). T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci. Transl. Med. 3, 95ra73.
 Chapuis, A.G., Egan, D.N., Bar, M., Schmitt, T.M., McAfee, M.S., Paulson, K.G., Voillet, V., Gottardo, R., Ragnarsson, G.B., Bleakley, M., et al. (2019). T cell receptor gene therapy targeting WT1 prevents acute myeloid leukemia relapse post-transplant. Nat. Med. 25, 1064–1072.
 van Loenen, M.M., de Boer, R., van Liempt, E., Meij, P., Jedema, I., Falkenburg, J.H., and Heemskerk, M.H. (2014). A Good Manufacturing Practice procedure to engineer donor virus-specific T cells into potent anti-leukemic effector cells. Haematologica 99, 759–768.
 Marcu-Malina, V., Heijhuurs, S., van Buuren, M., Hartkamp, L., Strand, S., Sebestyen, Z., Scholten, K., Martens, A., and Kuball, J. (2011). Redirecting ab T cells against cancer cells by transfer of a broadly tumor-reactive gdT-cell receptor. Blood 118, 50–59.
 Gründer, C., van Dorp, S., Hol, S., Drent, E., Straetemans, T., Heijhuurs, S., Scholten, K., Scheper, W., Sebestyen, Z., Martens, A., et al. (2012). g9 and d2CDR3 domains regulate functional avidity of T cells harboring g9d2TCRs. Blood 120, 5153–5162.
 Straetemans T, Janssen A, Jansen K, Doorn R, Aarts T, van Muyden ADD, Simonis M, Bergboer J, de Witte M, Sebestyen Z, Kuball J. TEG001 Insert Integrity from Vector Producer Cells until Medicinal Product. Mol Ther. 2020 Feb 5;28(2):561-571. doi: 10.1016/j.ymthe.2019.11.030. Epub 2019 Dec 14. PMID: 31882320; PMCID: PMC7001055.
 Straetemans, T., Kierkels, G.J.J., Doorn, R., Jansen, K., Heijhuurs, S., Dos Santos, J.M., van Muyden, A.D.D., Vie, H., Clemenceau, B., Raymakers, R., et al. (2018). GMPGrade Manufacturing of T Cells Engineered to Express a Defined gdTCR. Front. Immunol. 9, 1062.
 Dropulic, B., Schonely, K., Slepushkin, V., Lu, X., Andre, K., Boehmer, J., Bengston, K., Doub, M., Cohen, R., Berlinger, D., et al. (2003). QC Release Testing of an HIV-1 based Lentiviral Vector Lot and Transduced Cellular Product. Bioprocess. J. 2, 39–47.
 Lock, D., Mockel-Tenbrinck, N., Drechsel, K., Barth, C., Mauer, D., Schaser, T., Kolbe, C., Al Rawashdeh, W., Brauner, J., Hardt, O., et al. (2017). Automated Manufacturing of Potent CD20-Directed Chimeric Antigen Receptor T Cells for Clinical Use. Hum. Gene Ther. 28, 914–925.
 Blaeschke, F., Stenger, D., Kaeuferle, T., Willier, S., Lotfi, R., Kaiser, A.D., Assenmacher, M., Döring, M., Feucht, J., and Feuchtinger, T. (2018). Induction of a central memory and stem cell memory phenotype in functionally active CD4+ and CD8+ CAR T cells produced in an automated good manufacturing practice system for the treatment of CD19+ acute lymphoblastic leukemia. Cancer Immunol. Immunother. 67, 1053–1066.
 Wang, X., Naranjo, A., Brown, C.E., Bautista, C., Wong, C.W., Chang, W.C., Aguilar, B., Ostberg, J.R., Riddell, S.R., Forman, S.J., and Jensen, M.C. (2012). Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale. J. Immunother. 35, 689–701.
 Brentjens, R.J., Davila, M.L., Riviere, I., Park, J., Wang, X., Cowell, L.G., Bartido, S., Stefanski, J., Taylor, C., Olszewska, M., et al. (2013). CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med. 5, 177ra38.
 Paruzynski, A., Arens, A., Gabriel, R., Bartholomae, C.C., Scholz, S., Wang, W., Wolf, S., Glimm, H., Schmidt, M., and von Kalle, C. (2010). Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing. Nat. Protoc. 5, 1379–1395.
 Schmidt, M., Schwarzwaelder, K., Bartholomae, C., Zaoui, K., Ball, C., Pilz, I., Braun, S., Glimm, H., and von Kalle, C. (2007). High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nat. Methods 4, 1051–1057.
 Hamada, M., Nishio, N., Okuno, Y., Suzuki, S., Kawashima, N., Muramatsu, H., Tsubota, S., Wilson, M.H., Morita, D., Kataoka, S., et al. (2018). Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR. EBioMedicine 34, 18–26.
 Sherman, E., Nobles, C., Berry, C.C., Six, E., Wu, Y., Dryga, A., Malani, N., Male, F., Reddy, S., Bailey, A., et al. (2016). INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes. Mol. Ther. Methods Clin. Dev. 4, 39–49.
 Berry, C.C., Nobles, C., Six, E., Wu, Y., Malani, N., Sherman, E., Dryga, A., Everett, J.K., Male, F., Bailey, A., et al. (2016). INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions. Mol. Ther. Methods Clin. Dev. 4, 17–26.
 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
 Müller TR, Jarosch S, Hammel M, Leube J, et. al. 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.
 Sachdeva M, Busser BW, Temburni S, Jahangiri B, Gautron AS, Maréchal A, Juillerat A, Williams A, Depil S, Duchateau P, Poirot L, Valton J. Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality. Nat Commun. 2019 Nov 13;10(1):5100. doi: 10.1038/s41467-019-13088-3. Erratum in: Nat Commun. 2020 May 7;11(1):2357. PMID: 31723132; PMCID: PMC6853973.
 Eyquem, J., Mansilla-Soto, J., Giavridis, T. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017). https://doi.org/10.1038/nature21405