Cell and Gene Therapy Lesson 3: A Developing, Growing and Changing Regulatory Landscape

Heather Myler, Ph.D., director, PPD Laboratories, bioanalytical lab, discusses regulatory changes in the field.

The Food and Drug Administration (FDA), European Medicines Agency (EMA), International Council for Harmonization (ICH) and other regional regulatory agencies provide guidance covering traditional pharmacokinetic (PK) and immunogenicity (IMG) methods ^{1, 2, 3, 4, 5}. However, cell and gene therapies (CGT) have unique nuances that are not yet fully addressed. As cited in Husain 2015, due to the diverse range of investigational gene therapy products and associated biological complexity, the FDA encourages a flexible, data-driven approach based on the novel features of these products. Trial endpoints must be carefully chosen and frequently include measures of clinical and/or biological activity of the product. Sponsors are strongly encouraged to discuss these endpoints with health authorities in preparation for pivotal trials.

Guidelines on bioanalytical method validation (BMV) describe what is expected from bioanalytical assays that are submitted to support regulatory submissions^2,3. These guidelines are applicable to methods used to measure concentrations of chemical and biological drug(s) and their metabolite(s) in biological samples (e.g., blood, plasma, serum, other bodily fluids or tissues) obtained during pivotal nonclinical toxicokinetic (TK) and PK studies. The data obtained from these studies are used to make regulatory decisions in all phases of clinical trials. These guidelines apply to quantitative analysis by ligand-binding assays (LBAs) and chromatographic methods. BMV guidelines do not yet include sections applicable to flow cytometry or molecular assays, nor do they sufficiently cover the validation criteria required for cell and gene therapy methods used to assess exposure.

CGT bioanalytical methods frequently measure an analyte that occurs as a downstream product of the administered therapeutic with bioanalytical strategies that are analogous to the practices used to support biomarker methods. Consequently, both CGT and biomarker assays often lack a true reference material. The lack of true reference material necessitates the evaluation of parallelism between the surrogate reference material used for calibration and the expressed protein product as well as endogenous protein.

Conversely, oligonucleotide drug products do have reference materials available, allowing oligonucleotide methods to comply with much of the BMV and IMG guidance. Moreover, viral vectors usually have an adequate reference material for the determination of immunogenicity while the assessment of exposure is assessed through vector copy number and viral shedding.

While chromatography and LBA are long standing and mature technologies routinely used in the bioanalytical space, experts in flow cytometry and molecular genomics are required to address the needs of new CGT modalities. Most of the experience in these two areas currently exist in medical diagnostics and research laboratories. It will take time for the industry to achieve the level of applied scientific and compliance experience that currently exists in the bioanalytical community today.

“Flow cytometry methods are employed not only to measure cellular kinetics of the infused cells in adoptive cell therapy but also to evaluate immune activation and T-cell exhaustion,” says Vellalore Kakkanaiah, director biomarker labs, with more than 25 years of flow cytometry experience says. “Flow cytometry methods are more frequently being used to support all stages of drug development and updates to the current regulatory guidelines based upon technological expertise, best practices and traditional validation principles will greatly aid in the harmonization of validation expectations for this platform.”

Katie Matys, director biomarker labs, molecular genomics division, adds: “Many of the molecular methods used in nonclinical and clinical studies are not yet recognized as standardized bioanalytical methods and there are gaps in associated regulatory guidance. Molecular methods used in support of cell and gene therapy are evolving based upon program-specific needs and a core understanding of the technologies. While there is no specific guidance that describes the validation expectations, it is understood that performance should be demonstrated by thorough method optimization and validations, evaluating all critical parameters.”

As with transgene-expressed proteins measured by LBA or LCMS and cellular constructs measured using flow cytometry, RNA and DNA constructs measured using PCR and next generation sequencing methods can be complicated by a surrogate reference standard that may be a modified downstream product of the administered product. 

The bioanalytical community is actively learning and adapting and has even begun to publish concept papers and case studies ^{6, 7, 8, 9}. Ma et al. (2017) summarizes current practices and trends in the bioanalytical industry concerning the design, development, qualification/validation and implementation of bioanalytical methods used to assess exposure as well as immunogenicity for gene therapies. Gorovits and Koren (2019) address the various types of immune responses to chimeric antigen receptor CAR-T cell immunotherapies, the impact on clinical outcomes, and the methods used to assess these responses. They go on to describe the induction risk factors as being related to the presence of non-human or partially human sequences in the CAR construct, suicide domain, or other components of the CAR-T, residual viral proteins and other non-human origin proteins generated by the genetic editing. Both humoral and cellular responses are routinely monitored due to demonstrated impact on exposure. And Kavita et al. (2019) have described a method to detect antibodies to surface exposed components of a pegylated lipid nanoparticle gene delivery vehicle.

1. Gene therapy for cancer: regulatory considerations for approval. Cancer Gene Therapy. Dec; 22(12): 554–563. 2015.
2. International Council for Harmonization (ICH) harmonized guideline on Bioanalytical Method Validation M10. Draft February 2019.
3. FDA. Bioanalytical Method Validation Guidance for Industry. U.S. Department of Health and Human Services Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Center for Biologics Evaluation and Research (CBER); May 2018; Available from: https://www.fda.gov/media/70858/download.
4. Committee for Medicinal Products for Human Use C. Guideline on Immunogenicity assessment of therapeutic proteins 2017; Available from: https://www.ema.europa.eu/documents/scientific-guideline/guideline-immunogenicity-assessment-therapeutic-proteins-revision-1_en.pdf.
5. FDA. Immunogenicity Testing of Therapeutic Protein Products —Developing and Validating Assays for Anti-Drug Antibody Detection. Guidance for Industry. U.S. Department of Health and Human Services Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Center for Biologics Evaluation and Research (CBER); January 2019; Available from: https://www.fda.gov/ucm/groups/fdagov-public/@fdagov-drugs-gen/documents/document/ucm629728.pdf.
6. Ma M, Balasubramanian N, Dodge R and Zhang Y. Challenges and opportunities in bioanalytical support for gene therapy medicinal product development. Bioanalysis. 2017 Sep;9(18):1423-1430. doi: 10.4155/bio-2017-0116.
7. Steveson L, Richards S, Pillutla R, et al. 2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 – LBA/cell-based assays: immunogenicity, biomarkers and PK assays). Bioanalysis. 2018 Nov 29. doi: 10.4155/bio-2018-0287.
8. Gorovits B and Koren E. Immunogenicity of Chimeric Antigen Receptor T-Cell Therapeutics. BioDrugs. 2019 Jun;33(3):275-284. doi: 10.1007/s40259-019-00354-5.
9. Kavita U, Miller W, Ji QC, and Pillutla RC. A Fit-for-Purpose Method for the Detection of Human Antibodies to Surface-Exposed Components of BMS-986263, a Lipid Nanoparticle-Based Drug Product Containing a siRNA Drug Substance. AAPS J. 2019 Jul 22;21(5):92. doi: 10.1208/s12248-019-0360-8.