Table 1.

Pre-clinical testing options for therapeutic TCR candidates.

Parameter to consider when choosing a candidate TCR for therapy	Testing option/s
Could the candidate TCR cross-react with a peptide presented by an autologous classical and non-classical MHC molecule?	Scan the candidate TCR across different primary cell subset targets (monocytes, DCs, B-cells, T-cells) sorted from prospective patients.
	Scan the candidate TCR across PBMC and cell lines (monocytes, DCs, B-cells, T-cells, fibroblast, epithelial) from a library of HLA allele matched healthy donors.
	Scan the candidate TCR across peptide length-matched CPL to establish a metric of cross-reactivity potential.
Could the candidate TCR alloreact with a peptide presented by a mismatched MHC molecule?	Scan the candidate TCR across an extensive, fully HLA haplotyped cell line library. The cell line library should contain HLA alleles found at high frequency in the target population.
Are the germline sequences for the candidate TCR donor/patient matched?	Compare the TRAV, TRAJ, TRBV, and TRBJ sequences of the candidate TCR with patient TR loci. Polymorphisms in these genes may alter the effectiveness of the therapeutic TCR in vivo. Additionally, if the donor/patient TR alleles do not match, or if the patient has a key TR allele deleted, there is a possibility that a patient-derived immune response could be mobilized against the “foreign” TCR.
Could the candidate TCR steer functional phenotype of recipient T-cells when used in gene therapy?	Transduce the candidate TCR in naive T-cells in vitro or into mice with human immune system components. Prime the cultures with differing concentrations of cognate Ag and monitor cell fate decisions. Note temporal and final ratios in effector, memory and Tfh differentiation.
	Transduce the candidate TCR in memory T-cells in vitro. Prime the cultures with differing concentrations of cognate Ag and monitor if cell fate is altered when compared to phenotype pre-transduction.