Isolation of T cell receptors specifically reactive with mutated tumor associated antigens

Parkhurst, Maria; Robbins, Paul; Rosenberg, Steven

doi:10.1186/2051-1426-2-S3-P33

Table 1 IFNγ secretion (pg/ml) by TCR transduced PBL

From: Isolation of T cell receptors specifically reactive with mutated tumor associated antigens

TCR source^a,b	TCR α/β^c	media	T2+ HBV^d peptide	T2+ mutated AHNAK^e peptide	T2+ wild type AHNAK peptide	T2+ mutated SRPX^f peptide	T2+ wild type SRPX peptide	Allogeneic melanoma (A2+)	Autologous melanoma (A2+)
peptide stimulated PBL^a	TRAV12-201/ TRBV201	118	167	>10000	698	220	244	85	9784
peptide stimulated PBL	TRAV1901/ TRBV12-401	90	100	150	154	>10000	246	60	9715
peptide stimulated PBL	TRAV301/ TRBV201	84	84	121	105	>10000	217	34	>10000
CD137 FACS sorted TIL^b	TRAV29/DV501/ TRBV5-601	126	134	139	189	>10000	183	55	>10000

^a CD8+ PBL from patient 3713 were stimulated in vitro with mature autologous DCs pulsed with mutated peptides predicted to bind with high affinities to HLA-A*0201 and were restimulated twice with autologous peptide-pulsed PBMCs. Recognition of relevant target cells was evaluated on the basis of IFNγ secretion after overnight coculture. 2 peptides, one derived from a mutation in the AHNAK protein and one derived from a mutation in the SRPX protein, stimulated T cells that specifically recognized peptide, COS7 cells expressing HLA-A*0201 that had been transfected with the relevant minigene, and the autologous tumor cell line.
^b TIL from patient 3713 were cocultured overnight with autologous DCs electroporated with in vitro transcribed (IVT) RNA encoding a fragment of the mutated SRPX protein. CD3+ CD8+ 41BB+ cells were sorted by FACS and expanded in the presence of allogeneic feeder cells, α-CD3, and IL2. The resulting T cell population specifically recognized peptide, COS7 cells expressing HLA-A*0201 that had been transfected with the relevant minigene, and the autologous tumor cell line.
^c TCR α and β chains in T cell populations were identified by 5' RACE using degenerate constant region primers and were cloned into retroviral vectors. These were then used to transduce PBL from patient 3713, and the function of the resulting genetically modified T cells was evaluated on the basis of IFNγ secretion after overnight coculture.
^d HBV: hepatitis B core virus peptide used as a negative control with high binding affinity to HLA-A*0201.
^e AHNAK: neuroblast differentiation-associated protein, also known as desmoyokin.
^f SRPX: sushi repeat-containing protein.

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