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Tetramer guided, cell sorter assisted production of clinical grade autologous NY-ESO-1 specific CD8(+) T cells.

Pollack SM, Jones RL, Farrar EA, Lai IP, Lee SM, Cao J, Pillarisetty VG, Hoch BL, Gullett A, Bleakley M, Conrad EU, Eary JF, Shibuya KC, Warren EH, Carstens JN, Heimfeld S, Riddell SR, Yee C - J Immunother Cancer (2014)

Bottom Line: NY-ESO-1 specific T cells were generated from all 6 patients.The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8(+), tetramer(+) T cells with a memory phenotype that recognized endogenous NY-ESO-1.This study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy.

View Article: PubMed Central - PubMed

Affiliation: Clinical Research Division, D3-100 Fred Hutchinson Cancer Research Center, 1100 Fairview Ave, Seattle, WA 98109 USA ; Department of Medicine, University of Washington, Seattle, WA USA.

ABSTRACT

Background: Adoptive T cell therapy represents an attractive modality for the treatment of patients with cancer. Peripheral blood mononuclear cells have been used as a source of antigen specific T cells but the very low frequency of T cells recognizing commonly expressed antigens such as NY-ESO-1 limit the applicability of this approach to other solid tumors. To overcome this, we tested a strategy combining IL-21 modulation during in vitro stimulation with first-in-class use of tetramer-guided cell sorting to generate NY-ESO-1 specific cytotoxic T lymphocytes (CTL).

Methods: CTL generation was evaluated in 6 patients with NY-ESO-1 positive sarcomas, under clinical manufacturing conditions and characterized for phenotypic and functional properties.

Results: Following in vitro stimulation, T cells stained with NY-ESO-1 tetramer were enriched from frequencies as low as 0.4% to >90% after single pass through a clinical grade sorter. NY-ESO-1 specific T cells were generated from all 6 patients. The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8(+), tetramer(+) T cells with a memory phenotype that recognized endogenous NY-ESO-1.

Conclusion: This study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy. This approach, when used to target more broadly expressed tumor antigens such as WT-1 and additional Cancer-Testis antigens will enhance the scope and feasibility of adoptive T cell therapy.

No MeSH data available.


Related in: MedlinePlus

Functional avidity of NY-ESO-1 specific T cells. A. NY-ESO-1 specific T cell products from patients #1-#6. The left panel for each patient shows the lysis of T2 cells pulsed with various concentrations of NY-ESO-1 peptide by NY-ESO-1-specific T cells at an effector to target (E:T) ratio of 20:1. The right panel shows lysis of the NY-ESO-1 expressing tumor cell line MelA375 at various E:T ratios. The tumor cell line Mel526 (NY-ESO-1 negative, gp100 positive) is a negative control. B. Lysis of NY-ESO-1 peptide pulsed T2 cells and tumor cells by a high affinity NY-ESO-1 T cell clone isolated by our lab, T cells transfected with the αLY TCR and sorted to >80% purity with NY-ESO-1 tetramer, and a gp100154-162 specific T cell clone.
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Fig2: Functional avidity of NY-ESO-1 specific T cells. A. NY-ESO-1 specific T cell products from patients #1-#6. The left panel for each patient shows the lysis of T2 cells pulsed with various concentrations of NY-ESO-1 peptide by NY-ESO-1-specific T cells at an effector to target (E:T) ratio of 20:1. The right panel shows lysis of the NY-ESO-1 expressing tumor cell line MelA375 at various E:T ratios. The tumor cell line Mel526 (NY-ESO-1 negative, gp100 positive) is a negative control. B. Lysis of NY-ESO-1 peptide pulsed T2 cells and tumor cells by a high affinity NY-ESO-1 T cell clone isolated by our lab, T cells transfected with the αLY TCR and sorted to >80% purity with NY-ESO-1 tetramer, and a gp100154-162 specific T cell clone.

Mentions: We evaluated the function of the NY-ESO-1-specific T cell products by assaying specific lysis of T2 (HLA-A2+) targets pulsed with titrated concentrations of NY-ESO-1 peptide as well as the NY-ESO-1+ tumor cell line Mel A375. All cell products exhibited specific lysis of T2 cells pulsed with <0.01 μg/ml of NY-ESO-1 peptide and of the Mel A375 tumor cells that endogenously expressed NY-ESO-1 (Figure 2A). The lytic ability of NY-ESO-1 specific CTL generated from the sarcoma patients in this study was comparable to a high affinity NY-ESO-1-specific T cell clones that we previously isolated [23], and to T cells transduced with the high affinity mutant αLY NY-ESO-1 specific TCR and sorted to >80% purity (Figure 2A and B). In response to T2 cells pulsed with NY-ESO-1 peptide, the T cell products from all patients secreted IFN-γ (mean 305 pg/mL, range 143 to 425 pg/mL) and TNF alpha (mean 674.9 pg/mL, range 313.4 to 1113.9 pg/mL) (Additional file 1: Figure S4). In each case, the NY-ESO-1 specific CTL lines were also confirmed to lyse a SS tumor line (SYO-1) and a MRCL tumor line (402) which had been transfected with the gene for A*0201 (Additional file 1: Figure S5).Figure 2


Tetramer guided, cell sorter assisted production of clinical grade autologous NY-ESO-1 specific CD8(+) T cells.

Pollack SM, Jones RL, Farrar EA, Lai IP, Lee SM, Cao J, Pillarisetty VG, Hoch BL, Gullett A, Bleakley M, Conrad EU, Eary JF, Shibuya KC, Warren EH, Carstens JN, Heimfeld S, Riddell SR, Yee C - J Immunother Cancer (2014)

Functional avidity of NY-ESO-1 specific T cells. A. NY-ESO-1 specific T cell products from patients #1-#6. The left panel for each patient shows the lysis of T2 cells pulsed with various concentrations of NY-ESO-1 peptide by NY-ESO-1-specific T cells at an effector to target (E:T) ratio of 20:1. The right panel shows lysis of the NY-ESO-1 expressing tumor cell line MelA375 at various E:T ratios. The tumor cell line Mel526 (NY-ESO-1 negative, gp100 positive) is a negative control. B. Lysis of NY-ESO-1 peptide pulsed T2 cells and tumor cells by a high affinity NY-ESO-1 T cell clone isolated by our lab, T cells transfected with the αLY TCR and sorted to >80% purity with NY-ESO-1 tetramer, and a gp100154-162 specific T cell clone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4196009&req=5

Fig2: Functional avidity of NY-ESO-1 specific T cells. A. NY-ESO-1 specific T cell products from patients #1-#6. The left panel for each patient shows the lysis of T2 cells pulsed with various concentrations of NY-ESO-1 peptide by NY-ESO-1-specific T cells at an effector to target (E:T) ratio of 20:1. The right panel shows lysis of the NY-ESO-1 expressing tumor cell line MelA375 at various E:T ratios. The tumor cell line Mel526 (NY-ESO-1 negative, gp100 positive) is a negative control. B. Lysis of NY-ESO-1 peptide pulsed T2 cells and tumor cells by a high affinity NY-ESO-1 T cell clone isolated by our lab, T cells transfected with the αLY TCR and sorted to >80% purity with NY-ESO-1 tetramer, and a gp100154-162 specific T cell clone.
Mentions: We evaluated the function of the NY-ESO-1-specific T cell products by assaying specific lysis of T2 (HLA-A2+) targets pulsed with titrated concentrations of NY-ESO-1 peptide as well as the NY-ESO-1+ tumor cell line Mel A375. All cell products exhibited specific lysis of T2 cells pulsed with <0.01 μg/ml of NY-ESO-1 peptide and of the Mel A375 tumor cells that endogenously expressed NY-ESO-1 (Figure 2A). The lytic ability of NY-ESO-1 specific CTL generated from the sarcoma patients in this study was comparable to a high affinity NY-ESO-1-specific T cell clones that we previously isolated [23], and to T cells transduced with the high affinity mutant αLY NY-ESO-1 specific TCR and sorted to >80% purity (Figure 2A and B). In response to T2 cells pulsed with NY-ESO-1 peptide, the T cell products from all patients secreted IFN-γ (mean 305 pg/mL, range 143 to 425 pg/mL) and TNF alpha (mean 674.9 pg/mL, range 313.4 to 1113.9 pg/mL) (Additional file 1: Figure S4). In each case, the NY-ESO-1 specific CTL lines were also confirmed to lyse a SS tumor line (SYO-1) and a MRCL tumor line (402) which had been transfected with the gene for A*0201 (Additional file 1: Figure S5).Figure 2

Bottom Line: NY-ESO-1 specific T cells were generated from all 6 patients.The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8(+), tetramer(+) T cells with a memory phenotype that recognized endogenous NY-ESO-1.This study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy.

View Article: PubMed Central - PubMed

Affiliation: Clinical Research Division, D3-100 Fred Hutchinson Cancer Research Center, 1100 Fairview Ave, Seattle, WA 98109 USA ; Department of Medicine, University of Washington, Seattle, WA USA.

ABSTRACT

Background: Adoptive T cell therapy represents an attractive modality for the treatment of patients with cancer. Peripheral blood mononuclear cells have been used as a source of antigen specific T cells but the very low frequency of T cells recognizing commonly expressed antigens such as NY-ESO-1 limit the applicability of this approach to other solid tumors. To overcome this, we tested a strategy combining IL-21 modulation during in vitro stimulation with first-in-class use of tetramer-guided cell sorting to generate NY-ESO-1 specific cytotoxic T lymphocytes (CTL).

Methods: CTL generation was evaluated in 6 patients with NY-ESO-1 positive sarcomas, under clinical manufacturing conditions and characterized for phenotypic and functional properties.

Results: Following in vitro stimulation, T cells stained with NY-ESO-1 tetramer were enriched from frequencies as low as 0.4% to >90% after single pass through a clinical grade sorter. NY-ESO-1 specific T cells were generated from all 6 patients. The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8(+), tetramer(+) T cells with a memory phenotype that recognized endogenous NY-ESO-1.

Conclusion: This study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy. This approach, when used to target more broadly expressed tumor antigens such as WT-1 and additional Cancer-Testis antigens will enhance the scope and feasibility of adoptive T cell therapy.

No MeSH data available.


Related in: MedlinePlus