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Characterizing insert integrity throughout GTMP manufacturing as requested by the FDA and EMA

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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, αβT cells were genetically modified 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

 

Table 1

 

Next, our genome-wide coverage plot revealed 9 integration sites (intact vector copies) in the MCB (Figure 1B).6

 

Fig 1B

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

 

Fig 4

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

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References

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[18] 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

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