Table 3.

Proposed QC tests and cutoffs for end-of-expansion, clinical canine CAR T cellular products

Assessment	Method	Cutoff
Identity
•CAR expression	Flow cytometrya	Positive
Purity
•Transduction efficiency	Flow cytometryb	> 3%
•Cell viability (%)	Trypan Blue, flow cytometryb	> 90% viable
•Non-T cells	Flow cytometryb	None
•Residual dynabeads	Microscopy	None
Safety
•Sterility	Gram stainingc	Negative
•Mycoplasma test	MycoAlert (biochemical) testd	Negative
•Replication-Competent Retrovirus	RD114 qPCRe	Decreased to undetectable
Potency
•Release of IFNγ	ELISAf	Positive

^aCAR expression could be also confirmed by PCR.

^bThe same flow cytometry panel used for day-4 and end-of-expansion assessment may be sufficient to determine the proportion of CAR⁺ cells, T cells and viable cells.

^cGram staining can be performed in-house using commercially available kits, e.g., Remel™ Gram Staining Kit, Thermo Scientific™, cat# R40080, or at commercial microbiology laboratories, i.e., the microbiology laboratory at the University of Pennsylvania’s School of Veterinary Medicine.

^dMycoplasma contamination can be quickly detected using a biochemical, luciferase-based assay that selectively detects the activity of mycoplasmal enzymes, e.g., MycoAlert Mycoplasma Detection Kit (Lonza).

^eStrategies to monitor for the emergence of Replication-Competent Retrovirus (RCR), including RD114 pseudotyped retrovirus in canines, have been previously described (Narushima et al., 2011). These may entail qPCR assays and should be performed on CAR T cell samples during manufacturing and after infusion on patient samples obtained at defined time points.

^fTo assess IFNy release, we use the Canine IFN-gamma DuoSet ELISA kit, RnDSystems Cat# DY781B, according to the manufacture’s recommendations Canine-IFN-gamma-Duoset-ELISA. For an example of assay setup, please refer to (Panjwani et al., 2020).