Last month, a chromosomal abnormality detected in a single patient prompted the FDA to discontinue the testing of an allogeneic CAR-T product in clinical trial.¹ This news shook the biotech/pharma scene and stimulated fierce discussion surrounding the topic of genetic safety. Earlier this year, Bluebird Bio suffered a similar blow after its candidate LentiGlobin caused suspected serious adverse reactions (SUSARs) during clinical trial. Wary of safety, this observation compelled the FDA to halt the company's’ medicinal product targeting sickle cell disease (SCD).² Added to the mix,  Chinese biopharma Legend Biotech saw the same fate a few days ago, as their BCMA-directed CAR-T have now been placed on hold.³ Unarguably, these events are a hard reality check for the nascent field and reinforces the necessity for thorough genetic characterization of all advanced manufactured products.

I. In-depth genetic characterization: an uncompromising requirement by the FDA and EMA

To date, the field of Cell & Gene Therapy (CGT) is unmistakably burgeoning. In fact, its rapid growth is attested by the surge in advanced therapy medicinal products (ATMP) reported in clinical trials.⁴ With a thriving market, it is not surprising to witness increased scrutiny from regulatory authorities.⁵ Therefore, safeguarding the quality and safety of products remains one of FDA and EMA’s highest priorities.

To elaborate, whether on- or off-target insertions might have accompanied your strategy, you and your colleagues will need to robustly and comprehensively QC the outcome of your genetic engineering. Conscious of this demand, one of the groups at the Memorial Sloan Kettering Cancer Center (MSKCC) - headed by prof. dr. Michel Sadelain (Director of Center for Cell Engineering at MSKCC)⁶ - harnessed TLA-based assays to confirm correct targeting of a CAR to the TRAC locus and to quantify the specificity of their targeted integrations. Indeed, establishing the extent to which target site has been correctly edited is a must for regulatory authorities. Their results were eventually published in Nature.⁷ Besides targeted approach, other groups might prioritize tools that drive random integration instead. For such endeavors, the suitability of TLA-based assays (to detect random viral integration sites in heterogeneous samples) was first described in Nature Biotechnology, where over 1400 breakpoints (i.e. HIV integration sites) were uncovered in a patient’s blood sample.⁸

With that said, regardless of the actual strategy that you will ultimately choose to implement, scrutinizing the identity and potency of your batch will be requested by regulatory agencies. In fact, the FDA/EMA will ask you (among other things) to⁹ :

• Demonstrate intended genetic modification at the DNA level
• Precisely identify integration sites in the host genome
• Verify that no unintended sequence variants have occurred in the integrated vector sequence(s)
• Evaluate any potential insertional mutagenesis.

Such crucial genetic characteristics are sometimes referred to as Critical Quality Attributes (CQA).

II. Uniquely capturing all critical quality attributes in a single experiment

Similarly, the upstream manufacturing process has well-defined genetic QC requirements. In fact, scientists involved in cell line development constantly seek to adopt robust analytical tools that will allow them to monitor important quality parameters, while de-risking R&D decisions and minimizing time-to-clinic. The often-exploited mass spectrometric approaches such as LC-MS/MS and LC-MS - to examine the location, identity as well as to quantify sequence variants at the protein level – is not only known to be laborious but also low throughput, especially when detection of low-level sequence variants is required. Furthermore, as reflected by this comparison table, many conventional technologies also present their own set of limitations, thereby rendering them suboptimal in revealing all relevant genetic characteristics, let alone providing such information in a cost-attractive and time-effective manner. As such, NGS proves to be an attractive alternative from a cost, throughput, and sensitivity perspective. On this note, Novartis describes our proprietary method as “a powerful and versatile analytical screening platform to support the CHO cell line development process for biopharmaceutical companies."¹⁰ Although this statement specifically alludes to our Chinese hamster ovaries-work, there are undeniable similarities between the use of our patented solutions in CHO and CGT. As a case in point, FDA and NIH researchers applied our genomic screening solutions to QC their modified vector-producing cells. In this joint project, SV40 T antigen-encoding sequences were removed from HEK293T cells via CRISPR/Cas9 to mitigate the risk of activating oncogenes.¹¹ Another example includes GSK’s paper, where a rapid method to generate vector producer cell lines via stable transfection of a single DNA construct encoding all lentiviral vector components was described. Here, our genetic QC assay was leveraged to identify the exact integration sites and showcased its ability to sequence large vector constructs (i.e. bacterial artificial chromosome, BAC).¹²

III. Genetic characterization from vector producer cells until medicinal product

To date, information on the precise molecular characterization of insert integrity of CAR or TCR transgenes on the nucleotide level throughout the complete production chain - as requested by authorities - remain scarce. If available, such detailed reports and investigations usually take place once side effects have been observed and evaluations seem to only be confined to the expression of the introduced transgene in the final cell product. As such, dr. Trudy Straetemans and her colleagues decided to QC the transgene integrity and vector integration profile of their TEG001 throughout manufacturing.¹³ Unlike linear-amplification-mediated PCR (LAM-PCR) techniques and tagmentation PCR (tag-PCR), assistant professor Straetemans explains that by “uniquely combining integrity assessment of the entire vector with integration site analysis, TLA helped us QC insert integrity throughout the entire production chain and these data completed our clinical study dossier for TEG001. As such, TLA played a key role in helping us establish a valuable framework for future GTMPs.”

IV. A comprehensive solution for ATMP testing

The tremendous potential of CGT, in providing significant and durable health gains to patients suffering from debilitating and devastating diseases, have impelled many to venture into this space. The field has therefore been raking in hefty investments, which in turn, have spurred a buoyant biotech landscape that is currently developing at a rapid pace. Companies betting on non-viral gene therapies instead, are also experiencing a boost in funding. There, our genetic QC solutions are certainly not unheard of either. Likewise, demonstrating that sequences have inserted correctly (i.e. without unintended disruption of other critical genome sites) will be vital, if your end-goal is to use your genetically modified (immune) cells for therapeutic purposes. Among others, Theodore Roth adopted TLA-based method in one of his projects, which culminated in a Nature publication.¹⁴ Director of the Gladstone-UCSF Institute of Genomic Immunology¹⁵ and senior author of the above-mentioned study, Alexander Marson clarifies “we perform gene editing in primary human immune cells for basic research, drug discovery, and to develop new cell therapies. Cergentis has helped us characterize the on- and off-target integrations when non-virally targeting new genetic material into human T cells with CRISPR, aiding our push towards clinical translation."

Unmatched genetic insights that meet regulatory expectations

Troubleshooting genetic QC pains is our expertise. In fact, our proprietary comprehensive genomic analysis platform has been recognized as one of the most innovative and promising technologies capable of improving genetic characterization and quality control.¹⁶ Given our ambition to improve the quality of genetic research worldwide, we have also recently enrolled in the National Institute of Standards and Technology (NIST) Genome Editing consortium.¹⁷

With CGT field still in its infancy, it will be key to think about genetic risk and safety early on to safeguard the success of your ATMPs in the long run.¹⁸ Besides, genetic QC are - unequivocally - being closely monitored by officials and therefore, carries a significant weight in FDA/EMA’s decision.⁹ For this reason, gathering sufficient genetic characterization evidence of your product(s) will be an unavoidable task as part of your filing(s).

With a decade-long experience, our proprietary genetic QC solutions are ideally positioned to help you and your team navigate and address those stringent genetic QC hurdles laid by the FDA and EMA. Therefore, if you are on a hunt for a genetic service provider that (1) offers a one-stop shop solution (2) generates high quality and resolution data and (3) provides reporting that meet regulatory authorities’ expectations, then consider adopting our robust TLA-based solutions.

Webinar

In our most recent webinar, Alexander Jansma zeroes in on the genetic characterizations requested by the FDA/EMA and demonstrates - through specific case studies - how our TLA-based solutions help our customers guarantee the (genetic) quality of their ATMP/immunotherapy products. Click below to watch the recording.

In case you would like to learn more about our services, we invite you to visit our cell & gene therapy page below.

[1] WCG FDAnews. (2021, October 12). FDA Halts Allogene’s CAR-T Therapy Studies. WCG FDAnews. https://www.fdanews.com/articles/204795-fda-halts-allogenes-car-t-therapy-studies

[2] WCG FDAnews. (2021, February 26). FDA Stops Bluebird Bio’s Sickle Cell Study After Serious Side Effects. WCG FDAnews. https://www.fdanews.com/articles/201550-fda-stops-bluebird-bios-sickle-cell-study-after-serious-side-effects

[3] Adams, B. (2021, November 02). Legend's status put on hold as FDA delays Janssen-partnered CAR-T decision. FIERCE Biotech. https://www.fiercebiotech.com/biotech/legend-s-status-put-hold-as-fda-delays-janssen-partnered-car-t-decision 

[4] Pagliarulo, N. (2021, May 19). FDA seeking more consistency from cell, gene therapy developers, top official says. Biopharma Dive. https://www.biopharmadive.com/news/fda-marks-gene-therapy-consistency/600445/

[5] Pacific Biolabs. Gene therapy and cell therapy CMC requirements for IND applications. Pacific Biolabs. https://pacificbiolabs.com/gene-therapy-cmc-requirements

[6] ASGCT Staff. (2021, April 22). Congratulations to Our 2021 Annual Meeting Award Winners. American Society of Gene + cell Therapy. https://asgct.org/research/news/april-2021/2021-annual-meeting-award-winners

[7] 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

[8] 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

[9] FDA. (2013, November). Preclinical assessment of investigational cellular and gene therapy products. U.S Food & Drug Administration. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/preclinical-assessment-investigational-cellular-and-gene-therapy-products

[10] Aeschlimann, S.H., Graf, C., Mayilo, D., Lindecker, H., Urda, L., Kappes, N., Burr, A.L., Simonis, M., Splinter, E., van Min, M. and Laux, H. (2019), Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next-Generation Sequencing Technologies for Cell Line Development. Biotechnol. J., 14: 1800371. https://doi.org/10.1002/biot.201800371

[11] Bae DH, Marino M, Iaffaldano B, Fenstermaker S, Afione S, Argaw T, McCright J, Kwilas A, Chiorini JA, Timmons AE, Reiser J. Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region. Mol Ther Methods Clin Dev. 2020 Jul 9;18:631-638. doi: 10.1016/j.omtm.2020.07.006. PMID: 32775497; PMCID: PMC7397404.

[12] Chen YH, Pallant C, Sampson CJ, Boiti A, Johnson S, Brazauskas P, Hardwicke P, Marongiu M, Marinova VM, Carmo M, Sweeney NP, Richard A, Shillings A, Archibald P, Puschmann E, Mouzon B, Grose D, Mendez-Tavio M, Chen MX, Warr SRC, Senussi T, Carter PS, Baker S, Jung C, Brugman MH, Howe SJ, Vink CA. Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct. Mol Ther Methods Clin Dev. 2020 Aug 14;19:47-57. doi: 10.1016/j.omtm.2020.08.011. PMID: 32995359; PMCID: PMC7501408.

[13] 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.

[14] Roth, T.L., Puig-Saus, C., Yu, R. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405–409 (2018). https://doi.org/10.1038/s41586-018-0326-5

[15] Langelier, J. (2020, may 07). Two new research institutes in the Bay Area. Gladstone Institutes. https://gladstone.org/news/two-new-research-institutes-bay-area?utm_campaign=CGT&utm_content=169152599&utm_medium=social&utm_source=linkedin&hss_channel=lcp-3942757

[16] Raper, V. (2021, April 02). Gene Therapy Adopts New Tools to Guarantee Quality. Genetic Engineering & Biotechnology News. https://www.genengnews.com/insights/gene-therapy-adopts-new-tools-to-guarantee-quality/

[17] Cergentis. (2021, June 01). Cergentis joins the NIST Genome Editing Consortium to further improve the quality of genetic research. Cergentis. https://news.cergentis.com/news/cergentis-joins-the-nist-genome-editing-consortium-to-further-improve-the-quality-of-genetic-research

[18] Albert, H. (2021, October 13). Thinking About Risk Early on Is Key for Cell Therapy Success. Labiotech. https://www.labiotech.eu/interview/solentim-cell-therapy/?itm_source=Bibblio-module-desktop&itm_medium=recommended-content&itm_campaign=content-recirculation