Isolation of T cell receptors specifically reactive with mutated tumor associated antigens
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POSTER PRESENTATION
Open Access
Isolation of T cell receptors specifically reactive with mutated tumor associated antigens Maria Parkhurst*, Paul Robbins, Steven Rosenberg From Society for Immunotherapy of Cancer 29th Annual Meeting National Harbor, MD, USA. 6-9 November 2014 Background and hypotheses The adoptive transfer of tumor infiltrating lymphocytes (TIL) can mediate the regression of metastatic melanoma [1]. In addition, the adoptive transfer of lymphocytes expressing T cell receptors (TCRs) specifically reactive with antigens expressed on melanoma cells can mediate tumor
regression [2]. Many T cells from TIL recognize mutated antigens expressed only on the autologous patient’s tumor cells [3]. Therefore, we have attempted to isolate TCRs reactive with unique mutated antigens so that we may eventually treat patients with autologous T cells that have been genetically modified to express those TCRs.
Table 1 IFNg secretion (pg/ml) by TCR transduced PBL TCR sourcea,b
TCR a/bc
media
T2+ HBVd peptide
peptide stimulated PBLa peptide stimulated PBL peptide stimulated PBL
TRAV12-2*01/ TRBV2*01 TRAV19*01/ TRBV12-4*01 TRAV3*01/ TRBV2*01
118
CD137 FACS sorted TILb
TRAV29/DV5*01/ TRBV5-6*01
T2+ wild type AHNAK peptide 698
T2+ mutated SRPXf peptide 220
T2+ wild type SRPX peptide 244
Allogeneic melanoma (A2 +)
Autologous melanoma (A2 +)
167
T2+ mutated AHNAKe peptide >10000
85
9784
90
100
150
154
>10000
246
60
9715
84
84
121
105
>10000
217
34
>10000
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 IFNg 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, a-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 a and b 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 IFNg secretion after overnight coculture. d HBV: hepatitis B core virus peptide used as a negative control with
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