Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8⁺ and CD4⁺ T-cell responses with multiple specificities including a novel DR7-restricted epitope

Figures & data

Table 1. Patients' characteristics.

						Tumor outcome
Study vaccination groups	Patient ID	Sex (F/M)	Age at study entry	AJCC tumor staging at study entry	Vaccines received	Study entry	Study end	Best response	Disease-free survival (months)	Overal survival (months)	Discontinuation reason
Group A (no IL-2)
	LAU 986	M	35	IIIA	12	NED	NED	NED	63.8	63.8
	LAU 205	M	36	IV	2	ED	PD	PD	73.6	8.5	PD
	LAU 331	M	45	IV	18	NED	NED	NED	80.5	80.5
	LAU 518	M	65	IV	9	ED	PD	PD	5.8	33.1	PD
	LAU 1280	M	59	IIIB	8	NED	PD	NED	9.4	11.4	PD
	LAU 1330	M	61	IIIC	8	NED	PD	PD	3.5	11.3	PD
	LAU 1293	F	65	IV	12	NED	NED	NED	49.9	49.9
	LAU 1286	F	31	IIIC	10	NED	NED	NED	71.6	71.6	Patient's choice
	LAU 1352	M	65	IIIC	12	NED	PD	NED	14.0	24.5
	LAU 1350	M	61	IV	6	ED	PD	PD	3.9	34.6	PD
	all patients	2/8	60 (31–65)		10 (2–18)				32.0	33.8
Group B (IL-2)
	LAU 1357	M	58	IIIB	12 + IL-2	NED	NED	NED	6.7	68.4
	LAU 1397	F	36	IIIC	8 + IL-2	NED	PD	NED	8.7	61.2	PD
	LAU 1408	M	64	IIIB	1	NED	PD	PD	1.1	2.2	Degraded health status
	LAU 1415	M	46	IV	5 + IL-2	NED	PD	NED	5.1	12.4	PD
	LAU 1417	M	67	IIIA	8 + IL-2	NED	PD	NED	8.0	55.9	PD
	LAU 466	F	65	IV	2 + IL-2	NED	NED	NED	41.3	41.3	Patient's choice
	LAU 1394	M	64	IIIC	4 + IL-2	NED	PD	PD	8.8	56.8	PD
	LAU 1402	F	59	IIIA	4 + IL-2	NED	NED	NED	49.1	49.1	Patient's choice
	LAU 1504	M	37	IV	12	NED	NED	NED	20.2	20.2
	all patients	3/6	59 (36–67)		6 (1–12)				8.7	49.1
All groups
	all patients	5/146	59 (31–67)	3-IIIA	8 (2–18)	16- ED	8 - NED	13-NED	9.4	41.3
				3-IIIB		3-ED
				5-IIIC		0-PD	11-D	6-PD
				8-IV

Abbreviations: AJCC, American Joint Committee on Cancer; NED, no evidence of disease; ED, evidence of disease; PD, progressive disease.

* The patient was rendered tumor-free by resection of a left axillary metastasis, after V6.

Figure 1. Study design. Vaccinations (V) consisted of three cycles (C1–C3) of four monthly subcutaneous (s.c.) injections of 0.5 mg of NY-ESO-1_79–108 long peptide. HLA-A2+ patients were also vaccinated with 0.1 mg of Melan-A_26–35 native peptide and 20 μg of Mage-A10_254–262 peptide in the first cycle, followed by 0.1 mg Melan-A_{26–35(A27L)} analog peptide and 0.1 mg of Mage-A10_254–262 peptide in the following cycles. In addition, Group B patients were treated with low dose rh-IL-2. Peptides for HLA-A2⁺ patients were emulsified in 1 mL Montanide® ISA-51 and 2 mg CpG-7909/PF-3512676, peptides for HLA-A2⁻ patients were emulsified in 0.5 mL Montanide® ISA-51 and 1 mg CpG-7909/PF-3512676. The three vaccines of the cycle 3 were formulated without Montanide. Blood samples were withdrawn and PBMC were prepared at baseline (100 mL), after two vaccinations (two samples at 7 d interval: 30 and 100 mL) and after four vaccinations (two samples at 7 d interval: 30 and 100 mL) for the assessment of immune responses.

Figure 2. Specific CD8⁺ T-cell responses before and after vaccination with NY-ESO-1 LSP. (A) Representative example of a NY-ESO-1-specific CD8⁺ T-cell response 14 d after IVS. Cytokine secreting cells are enumerated after 6-hchallenging of the expanded cells with the NY-ESO-1 pool of overlapping peptides, or without any peptide as control. (B) Details of longitudinal NY-ESO-1-specific CD8⁺ T-cell responses (IFNγ, TNFα, and IL-2) measured individually in each patient before and during vaccination. (C) Polyfunctionality of NY-ESO-1-specific CD8⁺ T-cell responses assessed as IFNγ⁺TNFα⁺ or IFNγ⁺TNFα⁺IL-2⁺ cells, measured individually in each patient before and during vaccination. (D) Quantification of the contribution of each individual cytokine (IFNγ, TNFα, and IL-2) to the NY-ESO-1-specific CD8⁺ T-cell response, before and during vaccination. The mean of the response for each cytokine is shown for all patients grouped as % of the total response (that is defined as 100%). The magnitude (mean for all patients grouped) of the total response at each time point is indicated on the bottom of each pie.

Figure 3. Specific CD4⁺ T-cell responses before and after vaccination with NY-ESO-1 LSP. (A) Representative example of a NY-ESO-1-specific CD4⁺ T-cell response 14 d after IVS. Cytokine secreting cells are enumerated after 6-h challenging of the expanded cells with the NY-ESO-1 pool of overlapping peptides, or without any peptide as control. (B) Details of longitudinal NY-ESO-1-specific CD4⁺ T-cell responses (IFNγ, TNFα, IL2, and IL-13) measured individually in each patient before and during vaccination. (C) Polyfunctionality of NY-ESO-1-specific CD4⁺ T-cell responses assessed as IFNγ⁺TNFα⁺, or IFNγ⁺TNFα⁺IL-2⁺, or IFNγ⁺TNFα⁺IL-2⁺IL-13⁺ cells, measured individually in each patient before and during vaccination. (D) Quantification of the contribution of each individual cytokine (IFNγ, TNFα, IL-13, and IL-2) to the NY-ESO-1-specific CD4⁺ T-cell responses, before and during vaccination. The mean of the response for each cytokine is shown for all patients grouped as % of the total response (that is defined as 100%). The magnitude (mean for all patients grouped) of the total response at each time point is indicated on the bottom of each pie.

Figure 4. Summary of NY-ESO-1-specific CD8⁺ and CD4⁺ T-cell responses, and antibody responses. Cellular responses were measured 14 d after IVS and humoral responses were analyzed by ELISA against the NY-ESO-1 protein in plasma collected from enrolled patients' pre- treatment and during treatment as indicated.

Figure 5. Mapping of NY-ESO-1-specific CD8⁺ and CD4⁺ T-cell responses. (A) Using individual overlapping peptides covering the entire NY-ESO-1 LSP sequence, NY-ESO-1-specific CD8⁺ T-cell responses (n = 5 patients) and CD4⁺ T-cell responses (n = 9 patients) were mapped, by monitoring IFNγ + TNFα (CD8⁺ T-cells) and IFNγ (CD4⁺ T-cells) production after 6-h peptide challenge. (B) Representative example of NY-ESO-1_94–104/B35 multimer staining directly ex vivo and after IVS of CD8⁺ T-cells from HLA-B35⁺ patients. (C) MHC class II restriction of NY-ESO-1-specific CD4⁺ T-cell responses was assessed in a 6-h peptide challenge in the absence or presence of blocking anti-DR, -DP, or -DQ antibodies. Specific responses were measured by quantification of IFNγ production. (D) Representative example of NY-ESO-1_87–99/DR7 multimer staining of IVS CD4⁺ T-cells obtained from HLA-DR7⁺ patients, before and during immunization.

Table 2. Summary of frequencies of NY-ESO-1/DR7-specific CD4⁺ T-cells, detected after one round of IVS in HLA-DR7⁺ patients.

Patient ID	Before	2vacc.	4vacc.	6vacc.	8vacc.	12vacc.
LAU 331	19.60	ND	ND	6.50	1.38	6.74
LAU 1293	2.94	2.52	5.00	ND	ND	ND
LAU 1352	1.72	ND	1.54	ND	4.91	8.62
LAU 1350	0.13	0.26	ND	6.80	ND	ND
LAU 1357	0.18	0.55	ND	1.41	1.44	ND
LAU 1397	0.08	9.67	9.94	ND	3.35	ND
LAU 466	0.10	3.69	ND	ND	ND	ND

Notes: ND, not done.

Figure 6. NY-ESO-1_87–99 peptide represents a novel MHC class II epitope. A. NY-ESO-1_87–99-specific CD4⁺ T-cell clones were generated and stained with NY-ESO-1/DR7 multimers (upper panels). Reactivity to specific peptide was tested and EC50 was calculated for both Type1 and Type2 cytokines (lower panels). (B) NY-ESO-1_87–99-specific-CD4⁺ T-cell clones were assessed for their capacity to secrete IFNγ or kill HLA-DR7⁺ target T-cells, in the presence or absence of specific peptide. (C) Representative example of direct ex vivo multimer staining of NY-ESO-1_87–99-specific-CD4⁺ T-cells in HLA-DR7⁺ patients. (D) Summary of frequencies of direct ex vivo detectable NY-ESO-1_87–99-specific-CD4⁺ T-cells in HLA-DR7⁺ patients.

Figure 7. Overall survival and progression-free survival depending on the maximal level of IFNγ⁺ NY-ESO-1-specific CD8⁺ T-cell (A) and CD4⁺ T-cell (B) frequencies reached during the study after IVS. (A). Overall survival (left panel) and progression-free survival (right panel) in patients with low frequencies of IFNγ⁺ NY-ESO-1-specific CD8⁺ T-cells (lower than the median, n = 9) and in patients with high frequencies of IFNγ⁺ NY-ESO-1-specific CD8⁺ T-cells (higher than the median, n = 9). (B) Overall survival (left panel) and progression-free survival (right panel) in patients with low frequencies of IFNγ⁺ NY-ESO-1-specific CD4⁺ T-cells (lower than the median, n = 9) and in patients with high frequencies of IFNγ⁺ NY-ESO-1-specific CD4⁺ T-cells (higher than the median, n = 9).