High-affinity TCRs generated by phage display provide CD4+ T cells with the ability to recognize and kill tumor cell lines

Abstract

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 microM to 26 pM). CD8(+) T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (K(D) value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, K(D) 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range-with K(D) values of 450 nM and 4 microM-demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8(+) T cells, introduction of these TCR dramatically increased the reactivity of CD4(+) T cells to tumor cell lines.

Figures

FIGURE 1

High-affinity TCRs and tetramer staining. A, Summary of high-affinity TCRs used in this study. Amino acid sequences are indicated in single-letter code with changes in sequence relative to the wt 1G4 TCR shown in bold. KD were obtained using Biacore SPR with soluble version of the selected TCRs (nd, not determined). B, Specific HLA-tetramer binding of high-affinity TCR-transfected CD8+ T cells. Flow cytometry analysis of HLA-tetramer and anti-Vβ13.1 Ab staining for high-affinity NY-ESO-1/HLA-A2-restricted TCR genes electroporated as IVT RNA into CD8+ T cells. Controls were NY-ESO-1 TCR ET-8F and TIL clones L2D8 and JKF6 that are specific for gp100 and MART-1, respectively. The numbers in each quadrant indicates the percentage (%) of stained cells, living cells gated for by propidium iodide (PI) exclusion and CD8 gated, data representative of five experiments.

FIGURE 2

Nonspecific lysis of high-affinity TCR-transfected CD8+ T cells. Purified CD8+ T cells were electroporated with IVT TCR RNA for wt 1G4, wt ET-8F, and four mutated TCRs; c58/c61 (26 pM), c5/c100 (5 nM), c10/c1 (84 nM), or wt α/c59 (4 μM) TCR. T cells were cocultured with 51Cr-labeled peptide-pulsed T2 cells; 1 μM NY-ESO-1 or 1 μM Flu-MP and the amount of lysis was determined after 4 h. GFP RNA-transfected cells served as control. E:T was as shown; data are representative of two experiments.

FIGURE 3

Specificity of high-affinity TCR-transfected CD4+ T cells. A, CD8+ or CD4+ purified T cells were electroporated with IVT RNA from the TCRs as indicated and cocultured with T2 pulsed with serially diluted NY-ESO-1 peptide, or 100 nM MART-1 or gp100 peptide. IFN-γ production for both CD8+ and CD4+ cells, and IL-2 production by CD4+ cells is shown (nd, not determined; data are representative of three experiments). B, TCR-transfected CD4+ T cells were cocultured with 51Cr-labeled peptide-pulsed T2 cells; 1 μM NY-ESO-1 or 1 μM gp100 and the amount of lysis was determined after 4 h. E:T was as shown. GFP IVT RNA-transfected cells served as control; data are representative of two experiments.

FIGURE 4

Effect of CD8 coreceptor on T cell activation of high-affinity TCR-transfected T cells. A, IFN-γ production by TCR, or GFP control, transfected CD8+ T cells were incubated with Abs as indicated and then cocultured with 100 nM NY-ESO-1 or gp100 peptide-pulsed T2 cells. Cytokine levels determined following overnight coculture; data are representative of two experiments. B, IFN-γ production by CD4+ T cells retrovirally transduced with TCR genes then electroporated with IVT RNA for CD8α, CD8α/CD8β or GFP. Engineered CD4+ T cells were cocultured overnight with peptide-pulsed T2 cells (100 nM NY-ESO-1 or gp100) and cytokine levels were determined. GFP-expressing retroviral vector MSGIN served as control.

FIGURE 5

Increased tumor cell line recognition specificity by high-affinity TCR-transfected CD4+ T cells. Purified CD8+ (A) and CD4+ (B) T cells were electroporated with IVT TCR RNA for 1G4 wt, three mutant high-affinity TCRs; c58/c61 (26 pM), c5/c100 (5 nM), and c10/c1 (84 nM), or the medium-affinity c12/c2 (450 nM) TCR. T cells were cocultured with tumor cell lines overnight and production of IFN-γ determined. HLA-A2 and NY-ESO-1 expression by the melanoma cell lines were as indicated. Graphs truncated as shown with absolute values of cytokine production for TCR c58/c61 shown above each bar; data are representative of four experiments.

FIGURE 6

Density of MHC on the surface of APC affects the specificity of recognition by high-affinity TCR-expressing T cells. A, Flow cytometry analysis of HLA-A2 expression on C1R cells transfected with increasing amount of HLA-A2 IVT RNA. B, IFN-γ production by high-affinity TCR c10/c1 (84 nM) IVT RNA-transfected CD8+ T cells incubated with NY-ESO-1 peptide-pulsed or nonpulsed C1R cells expressing HLA-A2 at different levels. Data normalized to 100% for 1.3 μg of HLA-A2 RNA (values; 1234 pg/ml no peptide, 2553 pg/ml NY-ESO-1 peptide, representative of two experiments). C, IFN-γ production by purified CD8+ or CD4+ T cells cocultured with NY-ESO-1 or MART-1 SCT IVT RNA-transfected melanoma cell line 888mel with HLA-A2 IVT RNA or no RNA controls. D, HLA-A2 expression of SCT or HLA-A2 RNA-transfected 888mel was shown (percentage of stained cells are shown with mean fluorescent intensity in parentheses). Data representative of three experiments.