MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial

Emanuela Romano, Olivier Michielin, Verena Voelter, Julien Laurent, Hélène Bichat, Athina Stravodimou, Pedro Romero, Daniel E Speiser, Frédéric Triebel, Serge Leyvraz, Alexandre Harari, Emanuela Romano, Olivier Michielin, Verena Voelter, Julien Laurent, Hélène Bichat, Athina Stravodimou, Pedro Romero, Daniel E Speiser, Frédéric Triebel, Serge Leyvraz, Alexandre Harari

Abstract

Background: Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear.

Methods: We investigated in 12 patients with advanced stage IV melanoma the effect of multiple MART-1 analog peptide vaccinations with (n = 6) or without (n = 6) IMP321 (LAG-3Ig fusion protein) as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs at day (D) 0 (Trial registration No: NCT00324623). All patients were selected on the basis of ex vivo detectable MART-1-specific CD8 T-cell responses and immunized at D0, 8, 15, 22, 28, 52, and 74 post-reinfusion.

Results: After immunization, a significant expansion of MART-1-specific CD8 T cells was measured in 83% (n = 5/6) and 17% (n = 1/6) of patients from the IMP321 and control groups, respectively (P < 0.02). Compared to the control group, the mean fold increase of MART-1-specific CD8 T cells in the IMP321 group was respectively >2-, >4- and >6-fold higher at D15, D30 and D60 (P < 0.02). Long-lasting MART-1-specific CD8 T-cell responses were significantly associated with IMP321 (P < 0.02). At the peak of the response, MART-1-specific CD8 T cells contained higher proportions of effector (CCR7⁻ CD45RA⁺/⁻) cells in the IMP321 group (P < 0.02) and showed no sign of exhaustion (i.e. were mostly PD1⁻CD160⁻TIM3⁻LAG3⁻2B4⁺/⁻). Moreover, IMP321 was associated with a significantly reduced expansion of regulatory T cells (P < 0.04); consistently, we observed a negative correlation between the relative expansion of MART-1-specific CD8 T cells and of regulatory T cells. Finally, although there were no confirmed responses as per RECIST criteria, a transient, 30-day partial response was observed in a patient from the IMP321 group.

Conclusions: Vaccination with IMP321 as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs induced more robust and durable cellular antitumor immune responses, supporting further development of IMP321 as an adjuvant for future immunotherapeutic strategies.

Figures

Figure 1
Figure 1
Kinetic of MART-1-specific CD8 T-cell responses following immunization. A. Representative example of the increase in the frequency of circulating MART-1-specific CD8 T cells in Pt#1185 and Pt#1013 following infusion. MART-1-specific CD8 T cells are identified using peptide-MHC class I multimer complexes and plots are gated on viable CD3+CD8+ T cells. B. Prototypic examples of the kinetic of MART-1-specific CD8 T cells in patients from the IMP321 or no IMP321 groups. Dotted lines represent the initial frequency of MART-1-specific CD8 T cells prior to lymphodepletion. C. Proportion of patients from each group with a peak in the frequency of MART-1-specific CD8 T cells. Peaks were defined as a frequency of MART-1-specific CD8 T cells > three-fold higher than baseline, i.e. D-15 (n = 6 for each group, *P < 0.02). D. Cumulative analysis of the fold increase of MART-1-specific CD8 T cells overtime in the different groups. Mean + SEM are shown (n = 6 for each group, *P < 0.02).
Figure 2
Figure 2
Analysis of T-cell differentiation of MART-1-specific CD8 T-cell responses following immunization. A. Representative example of the expression of CD45RA and CCR7 on CD8 T cells in Pt#205 and Pt#1144. Total CD8 T cells appear in grey whereas blue dots represent MART-1-specific CD8 T cells. B. Cumulative analysis of the expression of CD45RA and CCR7 on MART-1-specific CD8 T cells in patients from the IMP321 or no IMP321 groups. All possible combinations of the distinct markers are shown on the x axis, whereas the percentages of the distinct cell subsets within MART-1-specific CD8 T cells are shown on the y axis. The pie charts summarize the data, and each slice corresponds to a certain combination of molecules. Stars (+) indicate a significant difference using a two-tailed unpaired student t test.
Figure 3
Figure 3
CD8 T cells at peak of response secrete effector cytokines against viral and tumor Ags. A. Representative example of the ability of CD8 T cells at peak of response to produce IFN-γ, IL-2 and/or TNF-α and to express perforin by ICS and polychromatic flow cytometry. Profiles are gated on viable CD3+CD8+ T cells. B. PBMCs from melanoma patients at peak of response following ACT and peptide vaccination were restimulated in vitro by T2 cells pulsed with MART-126–35 peptide. Following 1 weekly restimulation, ELISpot assays measured IFN-γ secretion after overnight exposure to the respective peptide-loaded (≡), unloaded T2 (░), or Me205 (□) targets. Limited cell numbers precluded isolation of CD3+CD8+ T cells for the ELISpot assays. Shown are the averaged data (means ± SEM) from 4 independent experiments each one performed in triplicate for the number of IFN-γ spot forming cells (SFC) per triplicate of 105 input cells at 30:1 (E:T) ratio.
Figure 4
Figure 4
Analysis of T-cell exhaustion markers of MART-1-specific CD8 T-cell responses following immunization. A. Representative example of the expression of 2B4, CD160, PD-1, and TIM-3 on CD8 T cells in Pt#205 and Pt#1022. Total CD8 T cells appear in grey whereas blue dots represent MART-1-specific CD8 T cells. B. Cumulative analysis of the expression of 2B4, CD160, LAG3, PD-1, and TIM-3 on MART-1-specific CD8 T cells in patients from the IMP321 or no IMP321 groups. All possible combinations of the distinct markers are shown on the x axis, whereas the percentages of the distinct cell subsets within MART-1-specific CD8 T cells are shown on the y axis. The pie charts summarize the data, and each slice corresponds to a certain combination of molecules. Stars (+) indicate a significant difference using a two-tailed unpaired student t test.
Figure 5
Figure 5
Analysis of the expansion of regulatory T cells following immunization. A. Representative examples (top panels) of the frequency of CD25highFOXP3+ on viable CD3+CD4+ T cells in patients from the IMP321 or no IMP321 groups. Bottom panels show the expression of CD45RO and CD127 on viable CD3+CD4+CD25highFOXP3+ regulatory T cells in a representative patient from the IMP321 group. B. Cumulative analysis of the expansion of CD25highFOXP3+ CD4+ T in patients from the IMP321 or no IMP321 groups. The star (+) indicates a significant difference using a two-tailed unpaired student t test. C. Correlation between the relative expansion of MART-1-specific CD8 T cells and of CD4+CD25highFOXP3+ regulatory T cells (R2 = 0.11; P < 0.05).

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