A Full GMP Process to Select and Amplify Epitope-Specific T Lymphocytes for Adoptive Immunotherapy of Metastatic Melanoma

Table 2

Validation runs for the selection and amplification of Melan-A- and MELOE-1- specific T lymphocytes from HLA-A2 melanoma patient-derived PBMC.

Run ID	≠1	≠2	≠3	≠1	≠2	≠3
Peptide	Melan-AA27L			MELOE-136-44
Number of stimulated PBMC	3×10^7	3×10^7	3×10^7	4×10^7	4×10^7	4×10^7
Number of sorted cells and fraction of specific T cells	10^7	5×10^6	5×10^6	5×10^6	5×10^6	10^7
	(9.3%)	(34%)	(38%)	(11%)	(16%)	(9%)
Theoretical number of specific T cells^1	9.3×10^5	1.7×10^6	1.9×10^6	5.5×10^5	8×10^5	9×10^5
Recovered number of sorted specific T cells	3.9×10^5	6.6×10^5	2.1×10^5	1.2×10^5	4.8×10^5	1.7×10^5
Sorting yield^2	42%	39%	11%	22%	60%	19%
Final number of amplified specific T cells and amplification yield^3	1.7×10^10	6.9×10^9	1.3×10^10	2×10^9	9.2×10^8	4.4×10^9
	(×43500)	(×10000)	(×62000)	(×16700)	(×1900)	(×25900)
Average number of cell divisions	15	13	16	14	10	15
Purity of amplified T cells^4	95%	99%	92%	90%	91%	93%
Activity of amplified T cells on the cognate peptide^5	82%	71%	72%	75%	53%	50%

1The theoretical number of specific cells is estimated from the % of tetramer-positive cells in the sorted population. 2Sorting yields are calculated by dividing the number of rosetted T cells after sorting by the theoretical number of specific T cells in the sorted populations. 3Amplification factors are estimated from the final number of amplified T cells divided by the number of rosetted T cells seeded for amplification on feeder cells. 4Purity of sorted and amplified T cells is assessed by tetramer/CD8 double staining. 5% of TNF producing T cells among tetramer positive cells : reactivity of amplified T cells is assessed by tetramer/TNF double staining, after peptide activation.