Therapeutic Vaccination against A Modified Minimal Survivin Epitope Induces Functional CD4 T Cells That Recognize Survivin‐Expressing Cells
During the last decade, cancer vaccination has received a great deal of attention; however, documentation of the clinical effect remains limited. The introduction of immune checkpoint inhibitors as a novel therapeutic modality 1-4 could by combination increase the ability of a peptide vaccination to induce clinical responses. Survivin is an anti-apoptotic protein, first discovered in 1997 5. It is overexpressed in a wide array of cancers, including malignant melanoma (MM) 5, 6, and expression is correlated with disease progression and a poor prognosis in, for example colon cancer and neuroblastoma 7, 8. Survivin is an excellent target for cancer vaccines due to its universal expression in cancer; in addition, downregulation will impair the tumour cell's ability to proliferate and expand. Many T cell epitopes from human cancer antigens have relatively low binding affinities to class I human leucocyte antigens (HLAs) 9. To increase the immunogenicity of such peptides, amino acid substitutions have been introduced at anchor positions, but not at T cell receptor contact residues, to increase the peptide binding affinity to class I HLAs. Many of these modified peptides are more immunogenic than the native epitopes and have an enhanced capacity to induce specific T cell responses. Consequently, modified minimal epitopes have been used in many clinical vaccination studies. Several natural and modified epitopes of survivin have been extensively investigated for their immunogenicity 10-12, especially a modified analogue of peptide survivin (96–104), named Sur1M2 (LMLGEFLKL), with a methionine at position 2 instead of threonine. Sur1M2 binds with high affinity to the major histocompatibility complex (MHC) class I molecule, HLA-A2 13. The ability of Sur1M2 to induce specific CD8+ T cell responses has been confirmed both in vitro and in clinical settings 13, 14. The aim of vaccination is to induce a robust T cell response, especially of cytotoxic T lymphocytes, which are able to target and eradicate tumour cells. Most clinical trials have used short epitopes consisting of 8–11 amino acids to induce CD8+ T cell responses mediated by MHC class I molecules; however, CD4+ T cells can also be induced with short epitopes, for example MHC class I epitopes from Mycobacterium tuberculosis and influenza A peptides 15, 16. Recently, in a phase I trial, Groos et al. 17 vaccinated 53 patients with a cocktail of five survivin-derived minimal epitopes (9–10 amino acids) in Montanide ISA 51VG. They found that two peptides that were modified to increase the binding to HLA-A2 and HLA-A3 13, 18 were also recognized by HLA-DP4 and HLA-DR-restricted CD4 T cells. Thus, in many patients, Sur18K10 (RISTFKNWPK) and especially the Sur1M2 epitope described above were able to activate a specific CD4 T cell repertoire, in addition to activating CD8 T cell responses. We recently performed a similar trial at our centre, where patients with confirmed MM were injected with 250 μg of Sur1M2 and 250 μg of another T cell epitope from Indoleamine 2,3-dioxygenase 19 in a Montanide ISA-51 solution (www.clinicaltrials.gov; NCT01543464). Prior to vaccination, 75 μg of granulocyte macrophage colony-stimulating factor was injected and topical Imiquimod cream was applied the day before. The patients alternated between vaccination and temozolomide treatment every second week. Based on the data from Groos et al., we examined whether a similar activation of survivin-specific T cells could be detected in our patients. Hence, we selected a HLA-A2-positive patient with a strong Sur1M2 response, measured by enzyme-linked immunospot assay (ELISPOT) after vaccination (Fig. 1A). We stimulated peripheral blood mononuclear cells (PBMCs) each week with a log10 decreasing concentration of the peptide for 3 weeks. Hereafter, we sorted the cells using a tumour necrosis factor alpha (TNF-α) secretion assay (Miltenyi Biotec). The sorted cells were then expanded in a high-dose IL-2 rapid expansion protocol to increase the number of Sur1M2 cells. As depicted in Fig. 1B, intracellular cytokine staining (ICS) with Sur1M2 stimulation revealed that 17.4% of the CD4 T cells reacted towards Sur1M2. Importantly, Sur1M2-specific T cells cross-reacted with the wild-type Sur1 peptide (Sur1 WT, LTLGEFLKL). Next, we demonstrated that the CD4+ T cell response towards Sur1M2 was HLA class II mediated, as the addition of a pan HLA class II blocking antibody (Tü39) effectively decreased the frequency of CD4+ T cells producing TNF-α from 19.2% to 0.77% (Fig. 1C). Most importantly, we showed that the Sur1M2-specific CD4+ T cells recognized cells that intracellularly expressed survivin. Hence, we stimulated the Sur1M2-specific cells with autologous PBMCs electroporated with or without mRNA encoding survivin in an ICS assay. As depicted in Fig. 1D, the reaction of CD4 T cells was significantly stronger against PBMC transfected with survivin mRNA compared with mock transfection. To summarize, we confirm the data from Groos et al. and describe that the modified survivin epitope Sur1M2, used in many preclinical and clinical studies, not only is restricted to HLA-A2, but in addition is able to induce survivin-specific CD4+ T cells. Most importantly, we show that such T cells are functionally relevant as they recognize cells intracellularly expressing survivin. These findings imply that short peptides can boost CD4+ T cells. In addition, modified peptides with amino acid substitutions at anchor residues for specific HLA molecules, for example HLA-A2, may induce immune responses restricted to additional HLA molecules, even class II HLA molecules. Thus, it is important to consider potential HLA class II-restricted responses in future studies utilizing Sur1M2, as well as other modified T cell epitopes. This study was supported by the Danish Cancer Society, the Danish Council for Independent Research, Aase og Ejnar Danielsens Fond, Toyota Foundation and Herlev Hospital. The funders did not have a role in the writing of the article or the decision to submit the article for publication.
Read more
110
