High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens

Abstract

The alpha- and beta-chains of the TCR from a highly avid anti-gp100 CTL clone were isolated and used to construct retroviral vectors that can mediate high efficiency gene transfer into primary human lymphocytes. Expression of this TCR gene was confirmed by Western blot analysis, immunocytometric analysis, and HLA Ag tetramer staining. Gene transfer efficiencies of >50% into primary lymphocytes were obtained without selection for transduced cells using a method of prebinding retroviral vectors to cell culture vessels before the addition of lymphocytes. The biological activity of transduced cells was confirmed by cytokine production following coculture with stimulator cells pulsed with gp100 peptides, but not with unrelated peptides. The ability of this anti-gp100 TCR gene to transfer high avidity Ag recognition to engineered lymphocytes was confirmed in comparison with highly avid antimelanoma lymphocytes by the high levels of cytokine production (>200,000 pg/ml IFN-gamma), by recognition of low levels of peptide (<200 pM), and by HLA class I-restricted recognition and lysis of melanoma tumor cell lines. CD4(+) T cells engineered with this anti-gp100 TCR gene were Ag reactive, suggesting CD8-independent activity of the expressed TCR. Finally, nonmelanoma-reactive tumor-infiltrating lymphocyte cultures developed antimelanoma activity following anti-gp100 TCR gene transfer. In addition, tumor-infiltrating lymphocytes with reactivity against non-gp100 melanoma Ags acquired gp100 reactivity and did not lose the recognition of autologous melanoma Ags following gp100 TCR gene transfer. These results suggest that lymphocytes genetically engineered to express anti-gp100 TCR may be of value in the adoptive immunotherapy of patients with melanoma.

FIGURE 1

TCR expressing retroviral vectors. A, Diagrams of the two retroviral vectors used to transfer and express the TCR gene from CTL clone R6C12. In vector GCsamgp100APB (APB), expression of the α-chain is mediated by the vector LTR, while the β chain expression is driven by an internal PGK gene promoter. In vector Gcsapgp100BIA (BIA), expression of both chains is mediated by the LTR, with translation coupled by the use of an IRES element. Location of retroviral vectorpackaging signal (ψ) and splice donor (sd) and splice acceptor (sa) sites is as indicated. B, FACS histogram of CD3-reactive cells from Sup T1 cells, or Sup T1 cells transduced with the APB or BIA vectors. The percentage of positive staining cells was as indicated. C, FACS profile of Sup T1 cells double stained with anti-CD3 Ab plus gp100 tetramer. Profile of Sup T1 cells, or Sup T1 cells transduced with the APB or BIA vectors is shown with the corresponding percentage of positive cells, as indicated.

FIGURE 2

TCR vector-transduced primary human PBL. FACS profiles of PBL from mock transduced (no vector, NV) or transduced with TCR vectors APB or BIA. Cells were double stained with anti-Vβ8 plus anti-CD3, percentage of positive cells, as indicated. A, Patient 1. B, Patient 2.

FIGURE 3

Sensitivity of cytokine secretion to dilutions of gp100 peptide. Shown is the resultant cytokine secretion of TCR vector-transduced PBL exposed to T2 cells pulsed with dilutions of gp100 peptide. A and B, Data from PBL from patient 1. C and D, Data for patient 2. Samples were: no vector-transduced cells (NV), control YFP vector-transduced cells (YFP), anti-gp100 TCR vector-transduced cells APB and BIA, anti-gp100 CTL (R6C12), and antimelanoma TIL (TIL).

FIGURE 4

Intracellular cytokine staining of transduced CD8 cells. Shown are the resultant FACS histograms for control PBL from patient 1, or PBL transduced with TCR vectors APB or BIA. Cells were cocultured with T2 cells pulsed with either HLA-A2-specific influenza virus peptide (Flu) or gp100 peptide 209–217 (210M). Cells were gated for CD3- plus CD8-positive cells and then analyzed for CD69 plus intracellular IFN-γ expression. The percentage of positive cells in the gated boxes was as shown.

FIGURE 5

Lysis of melanoma cell lines. PBL from patient 1 (A and B) or 2 (C and D) were tested in a ⁵¹Cr release assay. Transduced or control PBL (see legend) were cocultured with HLA-A2-positive melanoma cell line 526 (A and C) or non-HLA-A2 888 melanoma line (B and D) that had been labeled with ⁵¹Cr. Cells were incubated at the indicated E:T ratios for 4 h, after which the percentage of lysis of the target cells was calculated. The indicated CTL lines served as controls; see text for details. Samples were: no vector-transduced cells (NV), control YFP vector-transduced cells (YFP), anti-gp100 TCR vector-transduced cells APB and BIA, and anti-gp100 CTLs (R6C12, CK3H6, and D4F12).

FIGURE 6

Cytokine production by TCR-transduced CD4 cells. A, PBL from patient 1 were depleted for CD8 cells using magnetic beads and CD4⁺ cells cocultured with T2 cells pulsed with influenza peptide (Flu) or gp100 peptide 209–217 (210M). Twenty-four hours later, IFN-γ production was determined. B, FACS profile of PBL from patient 2 following exposure to T2 cells pulsed with gp100:209–217 (210M). Untransduced cells (control) or TCR vector-transduced cells (APB and BIA) were gated for CD3 plus CD4-positive cells and then analyzed for CD69 plus intracellular IFN-γ expression. The percentage of positive cells in the gated boxes was as shown.

FIGURE 7

Rescue of nonreactive TIL cultures. TIL cultures from three melanoma patients were tested and determined to be nonreactive to HLA-matched melanoma cell lines. TIL were transduced with control YFP vector or TCR vector APB and then cocultured with peptide-pulsed T2 cells. Shown are representative cytokine productions for each patient following coculture. Peptides used were HLA-A2-specific peptides for influenza virus (Flu) melanoma Ag MART-1 (MART) or gp100:209–217.