Linear doggybone DNA vaccine induces similar immunological responses to conventional plasmid DNA independently of immune recognition by TLR9 in a pre-clinical model.

Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours.

Induction of protective antitumor immunity through attenuation of ERAAP function.

The endoplasmic reticulum aminopeptidase associated with Ag processing, ERAAP, plays an important role in the trimming of antigenic peptides for presentation at the cell surface complexed with MHC class I molecules. Tumors express varying levels of ERAAP, highlighting a possible mechanism of immune-evasion through alteration of the peptide repertoire. Using the CT26 tumor model, we investigated the effects of ERAAP modulation on peptide presentation and the use of ERAAP inhibition as an antitumor therapy. We show that generation of the cross-protective tumor Ag GSW11 in the colorectal carcinoma CT26 is increased when ERAAP expression is reduced. BALB/c mice with reduced ERAAP expression challenged with CT26 induced protective immunity that was mediated by CD8(+) T cells. This antitumor immunity also protected mice when rechallenged with wild-type CT26 tumor; strong CD8(+) T cell responses to GSW11 were observed, despite its presentation being considerably lower. Furthermore, boosting the tumor immunogenicity through inhibition of ERAAP function with the small molecule inhibitor leucinethiol in vitro, or in established tumors in vivo, abrogated tumor growth and prolonged survival. Thus, our results highlight the promising possibility of using modulation of ERAAP to generate protective antitumor responses as a strategy for cancer immunotherapy.

Reactivation of low avidity tumor-specific CD8+ T cells associates with immunotherapeutic efficacy of anti-PD-1.

BACKGROUND: CD8+ T cells are a highly diverse population of cells with distinct phenotypic functions that can influence immunotherapy outcomes. Further insights on the roles of CD8+ specificities and TCR avidity of naturally arising tumor-specific T cells, where both high and low avidity T cells recognizing the same peptide-major histocompatibility complex (pMHC) coexist in the same tumor, are crucial for understanding T cell exhaustion and resistance to PD-1 immunotherapy. METHODS: CT26 models were treated with anti-PD-1 on days 3, 6 and 9 following subcutaneous tumor implantation generating variable responses during early tumor development. Tetramer staining was performed to determine the frequency and avidity of CD8+ T cells targeting the tumor-specific epitope GSW11 and confirmed with tetramer competition assays. Functional characterization of high and low avidity GSW11-specific CD8+ T cells was conducted using flow cytometry and bulk RNA-seq. In vitro cytotoxicity assays and in vivo adoptive transfer experiments were performed to determine the cytotoxicity of high and low avidity populations. RESULTS: Treatment success with anti-PD-1 was associated with the preferential expansion of low avidity (Tetlo) GSW11-specific CD8+ T cells with Vß TCR expressing clonotypes. High avidity T cells (Tethi), if present, were only found in progressing PD-1 refractory tumors. Tetlo demonstrated precursor exhausted or progenitor T cell phenotypes marked by higher expression of Tcf-1 and T-bet, and lower expression of the exhaustion markers CD39, PD-1 and Eomes compared with Tethi, whereas Tethi cells were terminally exhausted. Transcriptomics analyses showed pathways related to TCR signaling, cytotoxicity and oxidative phosphorylation were significantly enriched in Tetlo found in both regressing and progressing tumors compared with Tethi, whereas genes related to DNA damage, apoptosis and autophagy were downregulated. In vitro studies showed that Tetlo exhibits higher cytotoxicity than Tethi. Adoptive transfer of Tetlo showed more effective tumor control than Tethi, and curative responses were achieved when Tetlo was combined with two doses of anti-PD-1. CONCLUSIONS: Targeting subdominant T cell responses with lower avidity against pMHC affinity neoepitopes showed potential for improving PD-1 immunotherapy. Future interventions may consider expanding low avidity populations via vaccination or adoptive transfer.

Protective low-avidity anti-tumour CD8+ T cells are selectively attenuated by regulatory T cells.

OBJECTIVES: Regulatory T cells (Treg) play a major role in the suppression of protective anti-tumour T cell responses. In the CT26 BALB/c murine model of colorectal carcinoma, Tregs differentially suppress responses to two characterised CD8+ T epitopes, AH1 and GSW11, which results in an absence of detectable IFN-γ-producing GSW11-specific T cells in the spleen and lymph nodes of tumour challenged mice. Activation of GSW11-specific T cells correlates with protection against tumour progression. We wanted to examine the presence of non-functional GSW11-specific T cells in Treg replete and depleted mice, assess their phenotype and their affinity compared to AH1-specific T cells. METHODS: We used peptide-specific tetramers to identify tumour-specific CD8+ T cells and assessed the cell surface expression of markers associated with exhaustion (PD-1, Tim3 and Lag-3) and their function by IFN-g production using flow cytometry. We also assessed the T cell receptor (TcR) clonality of tumour-specific T cells. Tetramer competition assays were performed to determine the relative affinity of identified TcR. RESULTS: Here, we show that GSW11-specific T cells are in fact induced in Treg-replete, CT26-bearing mice, where they make up the majority of tumour-infiltrating CD8+ lymphocytes, but exhibit an 'exhausted' phenotype. This dysfunctional phenotype is induced early in the anti-tumour response in tumours. Depletion of Tregs prior to tumour challenge correlates with an altered T cell receptor (TcR) repertoire. Moreover, the avidity of GSW11-specific TcRs that expanded in the absence of Tregs was significantly lower compared with TcRs of CD8+populations that were diminished in protective anti-tumour responses. CONCLUSION: Our results indicate that Tregs suppress the induction of protective anti-tumour T cell responses and may signify that low-avidity T cells play an important role in this protection.