Booster vaccination of cancer patients with MAGE-A3 protein reveals long-term immunological memory or tolerance depending on priming

Abstract

We previously reported results of a phase II trial in which recombinant MAGE-A3 protein was administered with or without adjuvant AS02B to 18 non-small-cell lung cancer (NSCLC) patients after tumor resection. We found that the presence of adjuvant was essential for the development of humoral and cellular responses against selected MAGE-A3 epitopes. In our current study, 14 patients that still had no evidence of disease up to 3 years after vaccination with MAGE-A3 protein with or without adjuvant received an additional four doses of MAGE-A3 protein with adjuvant AS02B. After just one boost injection, six of seven patients originally vaccinated with MAGE-A3 protein plus adjuvant reached again their peak antibody titers against MAGE-A3 attained during the first vaccination. All seven patients subsequently developed even stronger antibody responses. Furthermore, booster vaccination widened the spectrum of CD4(+) and CD8(+) T cells against various new and known MAGE-A3 epitopes. In contrast, only two of seven patients originally vaccinated with MAGE-A3 protein alone developed high-titer antibodies to MAGE-A3, and all these patients showed very limited CD4(+) and no CD8(+) T cell reactivity, despite now receiving antigen in the presence of adjuvant. Our results underscore the importance of appropriate antigen priming using an adjuvant for generating persistent B and T cell memory and allowing typical booster responses with reimmunization. In contrast, absence of adjuvant at priming compromises further immunization attempts. These data provide an immunological rationale for vaccine design in light of recently reported favorable clinical responses in NSCLC patients after vaccination with MAGE-A3 protein plus adjuvant AS02B.

Fig. 1.

Antibody responses to MAGE-A3 in vaccinated patients. Reciprocal antibody titers against baculovirus-derived MAGE-A3 protein were measured by ELISA and are shown for each time point from prestudy (pre) to day (d) 85 for both vaccine cycles in the presence (yellow arrows) or absence (gray arrows) of adjuvant. Each box represents one patient, with cohort 1 patients on the left, cohort 2 patients on the right, and SPP patients at the bottom. None of the patients developed any significant reactivity against control proteins NY-ESO-1, LAGE-1, or p53. Results are representative of at least three independent experiments.

Fig. 2.

CD4⁺ and CD8⁺ T cell response to MAGE-A3 in vaccinated patients. Mean numbers of IFN-γ producing cells of 50,000 presensitized CD4⁺ (A) or CD8⁺ (B) T cells were measured by ELISPOT against indicated individual or pooled MAGE-A3 long overlapping peptide(s) as indicated by color symbols and are shown for each time point from prestudy (pre) to day (d) 85 for both vaccine cycles in the presence (yellow arrows) or absence (gray arrows) of adjuvant. If detectable, responses to irrelevant peptide-pulsed targets are shown as gray bars. Each box represents one patient, with cohort 1 patients on the left, cohort 2 patients on the right, and SPP patients at the bottom. Results are representative of at least two independent experiments, and error bars represent SD of replicates.

Fig. 3.

Functional analyses of vaccine-induced MAGE-A3-specific CD4⁺ T cells. (A) Intracellular cytokine staining of CD4⁺ T cells of representative patient LK-19 on day 43b responding to vaccination with MAGE-A3 protein. CD4⁺ T cells were presensitized for 14 days by using pooled MAGE-A3 peptides, and intracellular cytokine staining of Th1 (Upper) and Th2 (Lower) cytokines was performed after reexposure to single MAGE-A3 peptides. To differentiate effector from target cells, T-APC were stained beforehand by using the intracellular dye CFSE and gated out. Percentages of CD4⁺ effector T cells expressing the given cytokine in response to MAGE-A3 peptide 141–160 are indicated. Background levels were determined by using T-APC pulsed with irrelevant peptide (irrelev pept) and are shown in parentheses. (B) Perforin ELISPOT assays for cytolytic activity of MAGE-A3-specific CD4⁺ T cells. Representative results are shown for CD4⁺ T cells obtained on day 85 from patient WG-13 responding against pooled MAGE-A3 peptides (#16–30) and single MAGE-A3 peptide 151–170 (#16). (C) After in vitro sensitization with pooled peptides, MAGE-A3-specific CD4⁺ T cells did not coexpress Treg marker FOXP3. Representative results of intracellular costaining of IFN-γ and FOXP3 are shown for MAGE-A3 141–160-specific CD4⁺ T cells of patient LK-19 obtained on day 43b.

Fig. 4.

Summary of T cell epitopes from MAGE-A3 previously described and found in this study. (Left) Epitopes in the context of HLA class I (left, blue bars) or class II (right, red bars) are shown aligned to scale along the amino acid sequence of full-length MAGE-A3 protein (black graduated bar). Whenever defined, a selection of potential HLA restriction alleles is indicated. (Right) Summary of HLA class I- or class II-restricted epitopes of MAGE-A3 defined by analyzing T cell responses after vaccination with full-length MAGE-A3 protein.