Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions - PubMed (original) (raw)

Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions

Marybeth S Hughes et al. Hum Gene Ther. 2005 Apr.

Abstract

The genes for the alpha and beta chains of a highly reactive anti-MART-1 T-cell receptor were isolated from T-lymphocytes that mediated in vivo regression of tumor in a patient with metastatic melanoma. These genes were cloned and inserted into MSCV-based retroviral vectors. After transduction, greater than 50% gene transfer efficiency was demonstrated in primary T-lymphocytes stimulated by an anti-CD3 antibody. The specificity and biologic activity of TCR gene-transduced T-cells was determined by cytokine production after coculture of T-cells with stimulator cells pulsed with MART-1 peptide. The production of interferon-gamma and granulocyte macrophage-colony stimulating factor (GM-CSF) was comparable to highly active MART-1 specific peripheral blood lymphocytes (PBL) in the amount of cytokine produced and transduced cells recognized peptide pulsed cells at dilutions similar to cytotoxic T lymphocyte (CTL) clones. Human leukocyte antigen (HLA) class I restricted recognition was demonstrated by mobilization of degranulation marker CD107a, by cell lysis, by cytokine production, and by proliferation in the presence of HLA-A2-positive but not HLA-A2-negative melanoma cell lines. Similar data was obtained when tumor-infiltrating lymphocytes (TIL) were transduced with the TCR genes, converting previously nonreactive cells to tumor reactive cells. TCR-transduced T-cells are thus attractive candidates for evaluation in cell transfer therapies of patients with cancer.

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Figures

FIG. 1

FIG. 1

TCR expressing retroviral vectors. A: Diagrams of four biscistonic retroviral vectors used to transfer and express the TCR gene from cytotoxic T lymphocyte (CTL) clone M1F2. The first gene in each vector was driven by the vector long-terminal repeat (LTR), while the second gene was preceded by an I internal ribosome entry site (IRES) sequence or a PGK promoter. B: FACS profile of PG-13 packaging cells intracellularly stained with anti-Vβ-12 antibody. The percent positive staining was as indicated in parenthesis using antibody isotype controls for gating.

FIG. 2

FIG. 2

Transduction of human SupT1 T cell line. A: Fluorescence-activated cell sorter (FACS) histogram of CD3+ cells after transduction of Sup T1 cells by AIB or BIA retroviral vectors using supernatant form producer cell populations. The percentage positive cells were as indicated. B: FACS profile of MART-1 tetramer staining of Sup T1 cells transduced with no vector (NV), or supernatants from PG13 producer cell clones AIB 18 and AIB 54. The corresponding percent positive cells were as shown in parenthesis. Data are representative of multiple (> 10) analyses of MART-1 TCR vector-transduced Sup T1 cell populations.

FIG. 3

FIG. 3

Transduction of primary human lymphocytes. A: Fluorescence-activated cell sorter (FACS) histogram of peripheral blood lymphocytes (PBL) transduced with either AIB 18 or AIB 54 retroviral vectors or untransduced and stained for Vβ12 48 hr posttransduction. Percent positive cells were as indicated. B: FACS histogram of the same transduced cells but stained with MART-1 tetramer. Data are representative of four independent patient transductions.

FIG. 4

FIG. 4

Recognition of melanoma cell lines. Shown are cytokine production values (interferon [IFN]-γ top graph, granulocyte macrophage-colony stimulating factor [GM-CSF] bottom graph) of transduced peripheral blood lymphocytes (PBL) after coculture with melanoma cell lines. Vectors used for transduction of PBL were MSGIN (a control green fluorescent protein [GFP] vector), anti-MART-1 TCR vectors (AIB 18, BIA 71 and BPA 34), or a vector containing an anti-gp100 TCR (gp100). JB2F4 is a cytotoxic T lymphocyte clone that is highly MART-1-reactive. MEL 526 and 624 are HLAA2-positive melanoma cell lines and 888 and 938 are HLA-A2-negative. Data are representative of four independent coculture experiments.

FIG. 5

FIG. 5

CD107a mobilization by TCR transduced peripheral blood lymphocytes (PBL). Fluoresence-activated cell sorter (FACS) analysis for degranulation marker protein CD107a was determined after coculture of AIB 18 vector-transduced PBL with no melanoma cells (No Mel), HLA-A2-negative melanoma line 888 (Mel 888), or HLA-A2-positive melanoma line 624 (Mel 624). Transduced cells were analyzed by gating for Vβ12 (left panels) followed by staining for CD3 and CD107a. The percentage of cell staining for both CD3 and CD107a were as indicated.

FIG. 6

FIG. 6

Lysis of melanoma cells by transduced peripheral blood lymphocytes (PBL). 51Cr release assay was performed on PBL either transduced with AIB18 (anti-MART-1 TCR) or a no vector (NV) control population. After 4-hr incubation with 51Cr-labeled melanoma cell lines the percent lysis was determined. MEL 526 and 624 are HLA-A2-positive and 938 is HLA-A2-negative. The effector to target ratio (E:T ratio) was as depicted on the x-axis. JB2F4 is a highly MART-1 reactive A2 restricted cytotoxic T lymphocyte clone. Data are representative of three independent experiments.

FIG. 7

FIG. 7

Lysis of melanoma cells by transduced tumor-infiltrating lymphocytes (TIL) TIL transduced with the anti-MART-1 AIB vector or mock transduced (NV) were tested for their ability to lyse 51Cr-labeled melanoma cells. After a 4-hr incubation with 51Cr-labeled melanoma cell lines the percent lysis was determined. JB2F4 is a highly MART-1-reactive cytotoxic T lymphocyte. Effector to target ratio (E:T ratio) was as depicted on the x-axis. MEL 526 and 624 are HLA-A2-positive and 938 is an HLA-A2-negative melanoma cell line. Transduced TIL were also tested for their ability to lyse a fresh (not cultured) HLAA2-positive melanoma tumor explant (fresh A2+ tumor).

FIG. 8

FIG. 8

Proliferation of TCR-transduced peripheral blood lymphocytes (PBL) in melanoma cocultures. Shown are resultant fluorescence-activated cell sorter (FACS) histograms of carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labeled PBL cocultured with melanoma lines. PBL were either not transduced (NV) or transduced with MART-1 TCR (AIB) and then cocultured with melanoma lines 526 (A2+) or MEL 888 (A2-). Histograms of CFSE-stained and -unstained populations at time zero were as shown (top). Below are three columns each containing histograms from cocultures after 4 days in varying amounts of interleukin (IL)-2. Percent of cell proliferation was as indicated. AIB-transduced cells proliferated only in the presence of an HLA-A2-positive melanoma line.

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