How STR Analysis Supports CAR-T Cell Manufacturing

Engineered T cell therapies involve the genetic modification of a patient’s own immune cells with chimeric antigen receptors (CARs). When re-introduced into the patient, these engineered CAR-T cells instruct the patient’s immune system to recognize and kill tumor cells. The first CAR-T therapy was approved by the FDA in 2017 to treat patients with B-cell precursor acute lymphoblastic leukemia that had proven resistant to other therapies or had relapsed (1). Additional CAR-T cell therapies have since been approved to treat other refractory or relapsed cancers, including some lymphomas and multiple myeloma.

CAR-T cells are generated over five basic steps. First, T cells are isolated from the patient’s blood plasma. Then, the cells are activated to accept a viral vector containing the genetic construct for the CAR. After the CAR construct is inserted into the genome of the T cells, the cells are expanded in culture, and then finally dosed to the patient intravenously. Though current approved therapies make use of patient samples, there is interest in being able to use allogeneic T cells provided from donors (2).

Because patients provide the T cells for their CAR-T cell therapy, each manufactured dose of CAR-T cells is unique, presenting quality control challenges. For example, patient samples may require modified purification methods to address the presence of various impurities, and manufacturers must ensure that the CAR construct is present in the final product’s genome and has not negatively impacted cell function. Controls must also be in place to prevent mix-ups or contamination of patients’ T cell and CAR-T cell cultures during collection, shipment and production of the cells. Manufacturers use a variety of analytical techniques to ensure the satisfactory identity, safety, purity, quality, stability and potency of the final CAR-T cell dose (3).

One important tool that can establish fidelity of cell identity throughout the manufacturing process is short tandem repeat (STR) analysis, a genetic identification technique widely employed in the forensics community to establish paternity and identity. STR analysis is also the recommended method for authenticating research cell lines (4).

The human genome contains regions of repetitive DNA—hypervariable regions consisting of a short DNA sequence repeated in tandem. These STR regions are polymorphic in that the sequence varies in the number of copies of the repeated unit. The lengths of any given STR allele will vary from individual to individual and measuring the length of multiple STR alleles can provide robust genetic fingerprints. After isolating genetic material from an individual or a sample, amplifying a standard set of STR loci with specially designed primers and separating the amplicons by mass with capillary electrophoresis, the resulting profile provides a genomic profile for the sampled individual.

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In CAR-T cell manufacturing, the STR profile of a cell-based CAR-T product can be collected at any point in the manufacturing process to confirm the cells are derived from the original patient or donor. A reference STR profile from a subset of the cells isolated from the patients is first collected. The cells are then analyzed at various stages during the manufacturing process to confirm that the resulting profile matches the original. If sample mix-ups occur or if samples become contaminated with cells from other individuals in the manufacturing facility, STR analysis can help identify and correct the error.

STR analysis can also support research into using allogeneic T cells for CAR-T cell therapy. Cells collected from healthy donors are attractive alternatives to current approaches using autologous cells for various reasons, including uniformity of samples and the reduced levels of disease-related biological dysfunction (2). As with autologous CAR-T cells, the same STR identification steps during manufacturing would ensure cell identity and purity. In a research context, STR analysis can also be used to easily determine the percentage of allogenic CAR-T cells in a mixture of CAR-T cells and native cells. In experimental therapies, this mixed sample analysis can point to the relative levels of patient and donor cells present in a sample.

References

1. June, C. H. et al. (2018) CAR T cell immunotherapy for human cancer. Science 359, 1361–1365.
2. Depil, S. et al. (2020) ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat. Rev. Drug Discov. 19, 185–199.
3. U.S. Department of Health and Human Services, Food and Drug Administration (2020) Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs): Guidance for Industry.
4.  ATCC SDO (2021) Human Cell Line Authentication: Standardization of Short Tandem Repeat (STR) Profiling. ATCC Standards Development Organization, Manassas, VA.