Immunization with a HER-2/neu helper peptide vaccine generates HER-2/neu CD8 T-cell immunity in cancer patients

Abstract

CD4 T-cell help is required during the generation and maintenance of effective antitumor CD8 T cell-mediated immunity. The goal of this study was to determine whether HER-2/neu-specific CD8 T-cell immunity could be elicited using HER-2/neu-derived MHC class II "helper" peptides, which contain encompassed HLA-A2-binding motifs. Nineteen HLA-A2 patients with HER-2/neu-overexpressing cancers received a vaccine preparation consisting of putative HER-2/neu helper peptides p369-384, p688-703, and p971-984. Contained within these sequences are the HLA-A2-binding motifs p369-377, p689-697, and p971-979. After vaccination, the mean peptide-specific T-cell precursor frequency to the HLA-A2 peptides increased in the majority of patients. In addition, the peptide-specific T cells were able to lyse tumors. The responses were long-lived and detectable for more than 1 year after the final vaccination in select patients. These results demonstrate that HER-2/neu MHC class II epitopes containing encompassed MHC class I epitopes are able to induce long-lasting HER-2-specific IFN-gamma-producing CD8 T cells.

Figure 1

Patients immunized with a 15-aa HER-2/neu peptide–based vaccine develop HER-2/neu peptide–specific and protein–specific T-cell proliferation responses. Shown are the preimmunization (open circles) and maximal postimmunization (filled circles) proliferative responses (SI) for the HER-2/neu peptides, p369–384, p688–703, p971–984, and the HER-2/neu protein domains, ECD and ICD. For comparison, the maximal responses to tetanus toxoid (TT) are shown. Each symbol represents a measurement from a single unique subject, calculated from 24 replicates. The solid lines indicate the mean SI for the group.

Figure 2

The majority of patients could be immunized to HER-2/neu. Data are shown as the percentage of the population before immunization and after immunization that had a positive proliferation response (SI > 2) to each of the peptides in the vaccine, p369–384 (light gray bar), p688–703 (gray bar), p971–984 (filled bar), as well as to the HER-2 protein domains, ECD (open bar) and ICD (dark gray bar). The mean (range) preimmunization SI of patients considered as having a positive response to p369–384 was 2.4 (one patient only), to p688–703 was 2.5 (2.4–2.5), to p971–984 was 2.5 (one patient only), to ECD (no patients), and to ICD was 2.2 (one patient only). The mean (range) postimmunization SI of patients considered as having a positive response to p369–384 was 7.5 (2.9–35.6), to p688–703 was 3.5 (2.1–6.4), to p971–984 was 6.2 (2.4–26.1), to ECD was 3.3 (2.5–5.1), and to ICD was 6.2 (2.7–18).

Figure 3

Patients immunized with a 15-aa HER-2/neu vaccine increase T-cell precursors to the encompassed HLA-A2 9-aa peptides. (a) Combined ELIspot responses (precursors/10⁶ PBMCs) to HLA-A2–binding epitopes of influenza and CMV virus are shown. Data are from normal HLA-A2 volunteers (triangles) and study subjects (squares), with the mean delineated by a bar. (b) Preimmunization and maximal postimmunization ELIspot responses (precursors/10⁶ PBMCs) to the HLA-A2 HER-2/neu peptides, p369–377, p689–697, p971–979, in subjects are shown. For comparison, the maximal responses to TT are shown. Each symbol represents a measurement from a single unique subject, calculated on six replicates.

Figure 4

The majority of patients could be immunized to HLA-A2 HER-2/neu peptides. Data are shown as the percentage of the population before immunization and after immunization that had a detectable ELIspot response to each of the HER-2/neu, HLA-A2 peptides, p369–377 (light gray bar), p689–697 (dark gray bar), p971–979 (filled bar). The mean (range) preimmunization HER-2/neu–specific precursor frequency of patients considered as having a positive response to p369–377 was 125 (32–217, two patients only), to p689–697 (no patients), and to p971–979 was 74 (13–135, two patients only). The mean (range) maximal postimmunization HER-2/neu–specific precursor frequency of patients considered as having a positive response to p369–377 was 111 (11–417), to p689–697 was 60 (25–143), and to p971–979 was 97 (13–185).

Figure 5

Peptide-specific T cells isolated from a breast cancer patient after immunization can lyse HLA-A2 cells overexpressing HER-2/neu protein. PBMCs from a representative patient, 0107, were examined for cytolytic activity against BLCL-A2 alone (open circles), peptide-loaded A2-BLCLs (filled symbols), or HER-2/neu–expressing BLCL-A2 (open squares) at three different E/T ratios. The peptides (p369–377, p689–697, p971–979) used to pulse the BLCL-A2 were the HLA-A2–binding peptides encompassed in the 15-aa HER-2/neu vaccine. Each point represents the mean of three replicates.

Figure 6

Peptide-specific T-cell clones isolated from an ovarian cancer patient after immunization can lyse HLA-A2⁺ tumor cells overexpressing HER-2/neu protein. A p369–377–specific clone was examined for cytolytic activity against BLCL-A2 alone (open circles), p369–377-loaded BLCL-A2 (filled circles), or the HER-2/neu–overexpressing tumor cells, SKOV3 (open squares) and SKOV3-A2 (filled squares). SKOV3-A2 are SKOV3 cells stably expressing HLA-A2. Each point represents the mean of three replicates (± SEM). The absence of errors indicates a standard of the mean less than 1%.