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. 2019 Mar 13;10:319.
doi: 10.3389/fimmu.2019.00319. eCollection 2019.

Peptide Super-Agonist Enhances T-Cell Responses to Melanoma

Affiliations
Free PMC article

Peptide Super-Agonist Enhances T-Cell Responses to Melanoma

Sarah A E Galloway et al. Front Immunol. .
Free PMC article

Abstract

Recent immunotherapeutic approaches using adoptive cell therapy, or checkpoint blockade, have demonstrated the powerful anti-cancer potential of CD8 cytotoxic T-lymphocytes (CTL). While these approaches have shown great promise, they are only effective in some patients with some cancers. The potential power, and relative ease, of therapeutic vaccination against tumour associated antigens (TAA) present in different cancers has been a long sought-after approach for harnessing the discriminating sensitivity of CTL to treat cancer and has seen recent renewed interest following cancer vaccination successes using unique tumour neoantigens. Unfortunately, results with TAA-targeted "universal" cancer vaccines (UCV) have been largely disappointing. Infectious disease models have demonstrated that T-cell clonotypes that recognise the same antigen should not be viewed as being equally effective. Extrapolation of this notion to UCV would suggest that the quality of response in terms of the T-cell receptor (TCR) clonotypes induced might be more important than the quantity of the response. Unfortunately, there is little opportunity to assess the effectiveness of individual T-cell clonotypes in vivo. Here, we identified effective, persistent T-cell clonotypes in an HLA A2+ patient following successful tumour infiltrating lymphocyte (TIL) therapy. One such T-cell clone was used to generate super-agonist altered peptide ligands (APLs). Further refinement produced an APL that was capable of inducing T-cells in greater magnitude, and with improved effectiveness, from the blood of all 14 healthy donors tested. Importantly, this APL also induced T-cells from melanoma patient blood that exhibited superior recognition of the patient's own tumour compared to those induced by the natural antigen sequence. These results suggest that use of APL to skew the clonotypic quality of T-cells induced by cancer vaccination could provide a promising avenue in the hunt for the UCV "magic bullet."

Keywords: T-cell receptor; altered peptide ligand; cancer immunotherapy; cancer vaccine; melanoma; tumour infiltrating lymphocyte.

Figures

Figure 1
Figure 1
CD8 T-cell clone ST8.24 derived from patient TIL persists in patient blood after complete remission. (A) HLA A2-EAAGIGILTV Melan-A restricted CD8 T-cell clone ST8.24 was isolated from the TIL of a stage IV metastatic melanoma patient MM909.24 who successfully underwent tumour infiltrating lymphocyte (TIL) therapy. The TCR β chain of ST8.24 was present in the TIL infusion product, and in the blood 6 months post complete remission. (B) in vitro culture of TIL MM909.24 with autologous melanoma leads to expansion of Melan-A tetramer+ cells. TILs were stained prior to culture and at day 10, with irrelevant (preproinsulin, ALWGPDPAAA) and Melan-A (EAAGIGILTV) PE conjugated tetramers, using an optimised protocol (protein kinase treatment + anti-PE 1° antibody + PE conjugated 2° antibody). Percentage of cells residing in each gated population is shown. ST8.24 was amongst the expanded EAAGIGILTV tetramer+ T-cells. (C) Recognition by MM909.24 TIL of EAAGIGILTV peptide or super-agonist FATGIGIITV after 5 h using T2 cells as antigen presenting cells. The percentage of cells producing IFNγ (intracellular staining) is plotted (minus background IFNγ production by TILs alone) vs. peptide concentration. (D) MIP-1β ELISA of EAAGIGILTV reactive clones ST8.24 and MEL5 vs. EAAGIGILTV and FATGIGIITV peptides at the concentration range shown.
Figure 2
Figure 2
Combinatorial peptide library (CPL) screening of T-cell clone ST8.24 allowed candidate super-agonist peptides to be designed. (A) Decamer CPL screen with melanoma and EAAGIGILTV peptide reactive CD8 clone ST8.24, showing the preferred amino acid residue landscape (EAAGIGILTV residues shown in green). (B) Data from the CPL was used to generate a list of potential super-agonists. Candidate super-agonist peptides are ranked from 1 to 10 with number 1 predicted to be the most likely to be recognised by ST8.24. Amino acids residues shared with the Melan-A peptide are shown in green. (C) Peptide titrations of 10 candidate super-agonist and EAAGIGILTV peptides using clone ST8.24. Results expressed as MIP-1β in ng/mL, minus the MIP-1β value of T-cells incubated alone. Non-linear curves of best-fit are shown.
Figure 3
Figure 3
Melan-A tetramer staining of CD8 T-cells primed from healthy HLA A2+ donors with candidate super-agonist peptides. (A) CD8 T-cells isolated from five healthy donors were primed with wild-type (WT) EAAGIGILTV Melan-A peptide, or one of 10 candidate super-agonists (sequences shown in C), and cultured for 2 weeks. Primed T-cells were stained with EAAGIGILTV PE-conjugated tetramers, using an optimised protocol (protein kinase inhibitor treatment + tetramer + anti-PE 1° antibody). Percentage of EAAGIGILTV-tetramer positive cells is shown in bar charts. The dashed line aligns with the percentage of tetramer+ cells for the EAAGIGILTV peptide primed cells. Line graphs show reactivity to WT peptide EAAGIGILTV (at the range of concentrations indicated) of CD8 T-cells primed with EAAGIGILTV and MTSAIGILPV from donors 1 and 2 in an IFNγ ELISpot. Results are shown as number of spot forming units (SFU) per 50,000 T-cells. (B) Percentage of EAAGIGILTV-tetramer positive cells primed in three further donors (as in A). (C) Heat bars were generated using MATLAB R2017b and based on the percentage of EAAGIGILTV-tetramer positive cells (A&B), with a heat bar for all donors combined shown on the right. The colour indicates the relative magnitude of priming, according to the key. Priming with peptides MTSAIGILPV generated significantly more EAAGIGILTV tetramer positive cells when compared to priming with EAAGIGILTV (**p < 0.01). Associated flow cytometry data are shown in Supplementary Figure 1.
Figure 4
Figure 4
Priming CD8 T-cells from healthy donors with super-agonist peptide MTSAIGILPV elicits significantly more EAAGIGILTV-tetramer positive cells. (A) CD8 T-cells from seven additional healthy donors (6-12) were primed with wild type EAAGIGILTV Melan-A peptide or super-agonist MTSAIGILPV. The magnitude of reactive cells was tested at day 14 and 28 by EAAGIGILTV-tetramer staining, with percentage tetramer+ cells expressed graphically (associated flow cytometry plots shown in Supplementary Figures 2, 3). The difference in magnitude of EAAGIGILTV-tetramer positive cells elicited by the super-agonist peptide was significantly greater than EAAGIGILTV primed cells at both time points (paired one-tailed t-test). (NS ≥0.05 *p ≤ 0.05 **p ≤ 0.01). (B) Flow cytometry plots for one donor from (A) with percentage EAAGIGILTV-tetramer+ cells shown. (C) CFSE proliferation of T-cells primed with wild-type EAAGIGILTV, heteroclitic ELAGIGILTV, or super-agonist peptide MTSAIGILPV. T-cells were sampled at multiple time points and background proliferation (CFSElow) in the no peptide (DMSO) condition subtracted from the CFSElow cells in the peptide conditions to give percentage proliferation.
Figure 5
Figure 5
CD8 T-cells isolated from healthy HLA A2+ donors primed with super-agonist MTSAIGILPV exhibit superior melanoma killing. CD8 T-cells from both donors 11 and 14 were primed with MTSAIGILPV and EAAGIGILTV peptides and the magnitude of EAAGIGILTV-tetramer positive cells determined on day 14 and 28 for donor 11 (Figure 4, and associated flow cytometry plots in Supplementary Figures 2, 3) and on day 28 only for donor 14 (this figure). (A) Chromium release cytotoxicity assay performed for the T-cell lines generated from donor 11, with melanoma from patient MM909.24. (B) Peptide priming data (left panel) and flow cytometry based killing of melanoma tumours FM79 (+HLA A2 transgene) and MM909.24 with the CD8 T-cell lines from donor 14. A ratio of 1 T-cell to 1 tumour cell was used for both assays.
Figure 6
Figure 6
HLA A2-MTSAIGILPV is a structural mimic of HLA A2-EAAGIGILTV that exhibits improved binding to HLA. (A) T2 cells (TAP deficient HLA A2+) were incubated overnight in serum-free media with EAAGIGILTV and MTSAIGILPV at 1, 10, and 100 μM, alongside a panel of control peptides including HLA A2 binding peptides ELAGIGILTV (heteroclitic L at positon 2), GILGFVFTL (from M1 of influenza), HLA B*2705 binding peptide DRASFIKNL (from collagen) and HLA B*3501 HPVGEADYFEY (from EBV). Experiment was carried out in duplicate and results are expressed as the mean fluorescence intensity (MFI) of HLA A2 expression. The dashed line shows the MFI staining in the absence of exogenously supplied peptide. Associated flow cytometry plots are displayed in Supplementary Figure 5. (B) Structural analysis of the HLA A2-MTSAIGILPV complex compared to HLA A2-EAAGIGILTV. The HLA α1 helix is shown as grey cartoon with the MTSAIGILPV (orange sticks) and EAAGIGILTV (green sticks) superimposed. Black arrows demonstrate the upward facing solvent exposed and downward facing buried residues in each peptide. Boxes to the left show the interaction between the HLA A2 B-pocket (grey surface) and EAAGIGILTV (green cartoon and sticks) and MTSAIGILPV (orange cartoon and sticks).
Figure 7
Figure 7
Super-agonist tetramer binds EAAGIGILTV specific T-cells with greater fluorescent intensity. (A) pMHC-tetramer staining of five EAAGIGILTV clones derived from healthy donors (MEL5, CACTUS, EDDY and MANUELA) or a metastatic melanoma patient (ST8.24). The mean fluorescence intensity of staining (MFI) is displayed for irrelevant (ALWGPDPAAA from preproinsulin), EAAGIGILTV and MTSAIGILPV tetramers. Tetramer staining was carried out using a standard protocol (MEL5, CACTUS, and EDDY), with PKI (ST8.24) or with PKI + anti-PE 1° antibody + PE conjugated 2° antibody (MANUELA) in order to give clear EAAGIGILTV tetramer staining (22). Line graphs indicate sensitivity to EAAGIGILTV peptide or MTSAIGILPV super-agonist at the concentrations indicated in an overnight activation assay followed by MIP-1β ELISA. (B) pMHC tetramer staining of EAAGIGILTV or MTSAIGILPV peptide primed polyclonal CD8 T-cell populations isolated from healthy donors 13 and 14. T-cells were stained using an optimised protocol: PKI + PE tetramer + 1° + 2° antibodies. The percentage of gated cells and their MFI is displayed. (C) Accumulated mean florescent intensity (MFI) of all EAAGIGILTV-CD8 T-cell clones and polyclonal CD8 T-cell populations used. The difference in MFI between EAAGIGILTV-tetramer and MTSAIGILPV-tetramer staining was statistically significant (P = 0.003, paired one-tailed T-test).
Figure 8
Figure 8
CD8 T-cells from melanoma patients primed with super-agonist MTSAIGILPV resulted in superior autologous melanoma killing. (A) 4 × 105 CD8 T-cells from metastatic melanoma patients MM909.37 and MM1413.12 were primed with WT EAAGIGILTV or super agonist MTSAIGILPV peptides. MM909.37 is now deceased having not responded to therapy. EAAGIGILTV and irrelevant (ALWGPDPAAA from preproinsulin) tetramer staining was performed at day 28, with percentage of tetramer+ cells displayed. (B) Chromium release cytotoxicity assay performed for the T-cell lines from MM909.37 using autologous melanoma. The T-cell line to melanoma cell ratio displayed is based on total T-cell number. Insufficient cells were available from patient MM1413.12 to perform the killing assay. (C) Cytotoxicity assay as in (B), but with cell numbers adjusted according to EAAGIGILTV tetramer positivity shown in (A), to give two EAAGIGILTV tetramer+ cell per three autologous melanoma cells, for both the EAAGIGILTV and MTSAIGILPV primed T-cell lines (thus a different number of total cells was used from each primed line). P-values are displayed for an unpaired one-tailed t-test.

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