Analytical Method Development To Determine CAR-T Therapy Potency Without Altering Drug Formulation
Introduction
The determination of a cell and gene therapy product’s potency is a critical regulatory component required prior to patient infusion. Potency measurements ensure consistency between different lots of a manufactured drug product by establishing a set of criteria relevant to the product’s biological properties. Importantly, according to FDA guidelines, measurement of potency should reflect the cell product’s mechanism of action to correlate to clinical outcomes. Though current commercialized anti-CD19 chimeric antigen receptor (CAR)-T cell therapies mechanistically function through cytolysis of cancer cells, their potency is determined through interferon gamma ELISA assays, which have been argued as surrogate assays for killing assays. However, recent research indicates that interferon gamma production by CAR-T cells is not required for their antitumor activity in hematologic cancers, implying other methods should be explored for potency measurements. Furthermore, an ELISA assay is a multistep method which includes altering the cryopreserved cell product’s formulation through wash steps and incubation prior to the assay, thereby introducing variability. Here, we develop an analytical method that can be used as a potency assay to determine the killing activity of cryopreserved CAR-T cell products without changing the product formulation from how it is administered to patients.
Methods
To develop a killing assay for CAR-T cell products, we started with a cancer cell line positive for CD19 surface antigen which we genetically modified with firefly luciferase to allow live direct measurement of bioluminescent cell growth over time with minimal disruption to culture conditions. To develop parameters for this killing assay, we manufactured 2nd generation anti-CD19 CAR-T cells, as well as negative control CAR-T cells which recognize the CD19 antigen but are unable to signal cytolytic activity internally. All manufactured CAR-T products were cryopreserved in a 10% DMSO formulation (CS10). Given this, we sought a culture device that would allow sufficient culture volume to support an assay in which the cryopreserved T cell product is directly added without any washes or buffer exchange. The cryopreserved CAR-T products were then thawed and directly added at defined volumes to culture devices containing the CD19 positive bioluminescent target cancer cells and bioluminescent substrate. Changes in bioluminescence, which correlated to changes in target cancer cell growth, during co-culture were monitored over time.
Results
With no washing of cryopreservation media prior to the start of this assay, 2nd generation anti-CD19 CAR-T cells demonstrated significant killing activity compared to negative control CAR-T cells at an equal dose, signifying the assay is specific for cytolytic activity induced by the CAR. Negative control CAR-T Cells had no significant impact on lymphoma cell growth, suggesting the killing assay was not directly impacted by the cryopreservation medium itself. Importantly, once co-cultures of CAR-T cells and target cells were initiated, no changes to the assay conditions (media change, bioluminescent substrate addition) were required to enable week-long live assessment of killing. CAR-T killing activity was successfully assessed based on a dose titration as well as the percentage of T cells transduced with the CAR. Surprisingly, the conditions of the assay allow for a near two-week killing assessment of CAR-T cell products, revealing that 2nd generation CAR-T cells display more persistence than 1st generation CAR-T cells.
Conclusion
Here, we show successful development of an assay to directly assess killing activity of cryopreserved CAR-T cells without the requirement to change the product’s formulation from how it is infused into patients. We have developed a method that requires minimal manipulation of cryopreserved CAR-T, greatly simplifying the assessment of CAR-T cell therapy potency, a key requirement for drug products.