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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

Representative production of clinical grade NY-ESO-1 specific T cell products from patient 1. A. No detectable cells are observed with CD8 and Tetramer staining of untreated PBMC from patient 1. B. Small CD8+ and Tetramer+ were observed in 3 wells of three 48 well plates after 2 stimulations using peptide pulsed dendritic cells. C. The 3 positive wells were sorted using the clinical grade cell sorted and underwent 2 expansions. CD8 and tetramer staining of the final product is shown. D. The final product was able to lyse peptide pulsed targets as well as an endogenously NY-ESO-1 expressing tumor line (NY-ESO-1). Mel 526 is an HLA-A2+, NY-ESO-1− tumor line used as a control.
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Fig1: Representative production of clinical grade NY-ESO-1 specific T cell products from patient 1. A. No detectable cells are observed with CD8 and Tetramer staining of untreated PBMC from patient 1. B. Small CD8+ and Tetramer+ were observed in 3 wells of three 48 well plates after 2 stimulations using peptide pulsed dendritic cells. C. The 3 positive wells were sorted using the clinical grade cell sorted and underwent 2 expansions. CD8 and tetramer staining of the final product is shown. D. The final product was able to lyse peptide pulsed targets as well as an endogenously NY-ESO-1 expressing tumor line (NY-ESO-1). Mel 526 is an HLA-A2+, NY-ESO-1− tumor line used as a control.

Mentions: The strategy to isolate NY-ESO-1 specific T cells from PBMC is illustrated in Figure 1. For patient #1, three 48 well plates were plated with T cells and peptide pulsed DC’s. After two stimulations, each individual well was stained with the NY-ESO-1 tetramer and analyzed by flow cytometry. NY-ESO-1 tetramer positive CD8+ T cells were not observed in the starting leukapheresis product (detection threshhold <0.01%, Figure 1A), however 3 of the 144 stimulated wells contained NY-ESO-1 tetramer positive T cells at a frequency of >0.5% (Figure 1B). These three wells were pooled and the tetramer binding T cells were sorted, expanded initially in a T25 flask and subsequently in Lifecell Bags in the CPF. The final product was >94% CD8+ and NY-ESO-1 tetramer positive and specifically lysed T2 cells pulsed with NY-ESO-1157-165 peptide and the NY-ESO-1 expressing melanoma cell line, MelA375 (see Figure 1C and D).Figure 1


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)

Representative production of clinical grade NY-ESO-1 specific T cell products from patient 1. A. No detectable cells are observed with CD8 and Tetramer staining of untreated PBMC from patient 1. B. Small CD8+ and Tetramer+ were observed in 3 wells of three 48 well plates after 2 stimulations using peptide pulsed dendritic cells. C. The 3 positive wells were sorted using the clinical grade cell sorted and underwent 2 expansions. CD8 and tetramer staining of the final product is shown. D. The final product was able to lyse peptide pulsed targets as well as an endogenously NY-ESO-1 expressing tumor line (NY-ESO-1). Mel 526 is an HLA-A2+, NY-ESO-1− tumor line used as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Representative production of clinical grade NY-ESO-1 specific T cell products from patient 1. A. No detectable cells are observed with CD8 and Tetramer staining of untreated PBMC from patient 1. B. Small CD8+ and Tetramer+ were observed in 3 wells of three 48 well plates after 2 stimulations using peptide pulsed dendritic cells. C. The 3 positive wells were sorted using the clinical grade cell sorted and underwent 2 expansions. CD8 and tetramer staining of the final product is shown. D. The final product was able to lyse peptide pulsed targets as well as an endogenously NY-ESO-1 expressing tumor line (NY-ESO-1). Mel 526 is an HLA-A2+, NY-ESO-1− tumor line used as a control.
Mentions: The strategy to isolate NY-ESO-1 specific T cells from PBMC is illustrated in Figure 1. For patient #1, three 48 well plates were plated with T cells and peptide pulsed DC’s. After two stimulations, each individual well was stained with the NY-ESO-1 tetramer and analyzed by flow cytometry. NY-ESO-1 tetramer positive CD8+ T cells were not observed in the starting leukapheresis product (detection threshhold <0.01%, Figure 1A), however 3 of the 144 stimulated wells contained NY-ESO-1 tetramer positive T cells at a frequency of >0.5% (Figure 1B). These three wells were pooled and the tetramer binding T cells were sorted, expanded initially in a T25 flask and subsequently in Lifecell Bags in the CPF. The final product was >94% CD8+ and NY-ESO-1 tetramer positive and specifically lysed T2 cells pulsed with NY-ESO-1157-165 peptide and the NY-ESO-1 expressing melanoma cell line, MelA375 (see Figure 1C and D).Figure 1

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