Vaccination with an NY-ESO-1 peptide of HLA class I/II specificities induces integrated humoral and T cell responses in ovarian cancer

Abstract

NY-ESO-1 is a "cancer-testis" antigen expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. The NY-ESO-1 peptide epitope, ESO(157-170), is recognized by HLA-DP4-restricted CD4+ T cells and HLA-A2- and A24-restricted CD8+ T cells. To test whether providing cognate helper CD4+ T cells would enhance the antitumor immune response, we conducted a phase I clinical trial of immunization with ESO(157-170) mixed with incomplete Freund's adjuvant (Montanide ISA51) in 18 HLA-DP4+ EOC patients with minimal disease burden. NY-ESO-1-specific Ab responses and/or specific HLA-A2-restricted CD8+ and HLA-DP4-restricted CD4+ T cell responses were induced by a course of at least five vaccinations at three weekly intervals in a high proportion of patients. There were no serious vaccine-related adverse events. Vaccine-induced CD8+ and CD4+ T cell clones were shown to recognize NY-ESO-1-expressing tumor targets. T cell receptor analysis indicated that tumor-recognizing CD4+ T cell clones were structurally distinct from non-tumor-recognizing clones. Long-lived and functional vaccine-elicited CD8+ and CD4+ T cells were detectable in some patients up to 12 months after immunization. These results confirm the paradigm that the provision of cognate CD4+ T cell help is important for cancer vaccine design and provides the rationale for a phase II study design using ESO(157-170) epitope or the full-length NY-ESO-1 protein for immunotherapy in patients with EOC.

Fig. 1.

NY-ESO-1 serum Ab was assessed by ELISA before and after vaccination. Two baseline seronegative patients (10 and 13) converted to seropositive during the course of immunization.

Fig. 2.

CD4⁺ T cell response to ESO_157–170 vaccine. (A) There is a significant relationship between the number of vaccines and the maximal number of detectable ESO_157–170-reactive CD4⁺ T cells (P = 0.002) when data from all patients are examined cumulatively. (B) CD4⁺ T cells were stimulated with ESO_157–170 peptide and tested against target cells (T-APC loaded with ESO_157–170 peptide or irrelevant peptide Flu NP_206–229, Flu NP) by IFN-γ ICS.

Fig. 3.

Tumor recognition by vaccine-induced CD4⁺ T cells. (A) ESO_157–170-specific CD4⁺ T cells were assessed for recognition of ESO⁺DP4⁺ SK-Mel-37 or ESO^−veDP4⁺ SK-Mel-23 by intracellular TNF-α staining. (B) Reactivity of CD4⁺ T cell clones to ESO_157–170 peptide with concentration ranging from 10 μM to 1 nM. (C) Only clone 5-B-8 recognized SK-Mel-37, and SK-Mel-23 was only recognized by all clones when pulsed with ESO_157–170. (D and E) Assessment of tumor recognition by IFN-γ ICS.

Fig. 4.

CD8⁺ T cell response to ESO_157–170 vaccine. (A) CD8⁺ T cells were stimulated with ESO_157–170 peptide and stained with HLA-A2/ESO_157–165 multimer or HLA-A2/Flu HA_58–66 multimer. (B) IFN-γ production by ESO_157–170 specific CD8⁺ T cells was analyzed by ELISPOT. (C and D) Confirmation by IFN-γ ICS.

Fig. 5.

Tumor recognition by vaccine-induced CD8⁺ T cells. (A) After vaccine, ESO_157–170-specific CD8⁺ T cells from patient 18 were tested against ESO-1b or irrelevant peptide Flu-HA in a CD107a/b surface and IFN-γ ICS assay. (B) HLA-A2/ESO_157–165 multimer-reactive CD8⁺ T cells recognized HLA2⁺ESO⁺ cell line MZ-MEL-19 but not HLA-A2⁺ESO⁻ line SK-MEL-23. (C) Reactivity of CD8⁺ T cell clones to ESO_157–165 peptide (range 10 nM to 1 pM). (D–F) All four CD8⁺ T cell clones recognized HLA2⁺ESO⁺ MZ-Mel-19 but not HLA2⁺ESO⁻ SK-Mel-23.

Fig. 6.

Long-lived T cell immunity. (A) ESO_157–170-specific CD4⁺ T cell response at 6 months for patient 2. (B) A2/ESO_157–165 multimer-reactive CD8⁺ T cells recognized SK-Mel-37 and MZ-Mel-19, but not SK-Mel-23, when tested for CD107a/b and IFN-γ by ICS.