Combined Cancer Immunotherapy Against Aurora Kinase A

Abstract

Aurora kinase A (AURKA) is a centrosomal protein that is overexpressed in a number of human malignancies and can contribute to tumor progression. As we used this protein as a target of DNA immunization, we increased its immunogenicity by the addition of the PADRE helper epitope and decreased its potential oncogenicity by mutagenesis of the kinase domain. For in vitro analysis of induced immune responses in mice, we identified the Aurka(220-228) nonapeptide representing an H-2Kb epitope. As DNA vaccination against the Aurka self-antigen by a gene gun did not show any antitumor effect, we combined DNA immunization with anti-CD25 treatment that depletes mainly regulatory T cells. Whereas 1 anti-CD25 dose injected before DNA vaccination did not enhance the activation of Aurka-specific splenocytes, 3 doses administered on days of immunizations augmented about 10-fold immunity against Aurka. However, an opposite effect was found for antitumor immunity-only 1 anti-CD25 dose combined with DNA vaccination reduced tumor growth. Moreover, the administration of 3 doses of anti-CD25 antibody alone accelerated tumor growth. Analysis of tumor-infiltrating cells showed that 3 anti-CD25 doses not only efficiently depleted regulatory T cells but also activated helper T cells and CD3(-)CD25(+) cells. Next, we found that blockade of the PD-1 receptor initiated 1 week after the first immunization was necessary for significant inhibition of tumor growth with therapeutic DNA vaccination against Aurka combined with depletion of CD25 cells. Our results suggest that combined cancer immunotherapy should be carefully evaluated to achieve the optimal antitumor effect.

Experimental Combined Immunotherapy of Tumours with Major Histocompatibility Complex Class I Downregulation

“…The delayed delivery of adjuvants probably enables activation of adaptive immunity induced by DNA vaccination that subsequently cooperates with innate immunity stimulated by adjuvants [ 38 ]. This enhancement is similar to the impact of delayed PD-1/PD-L1 blockade [ 36 , 39 ].…”

“…Since, in our previous studies, we had demonstrated that TC-1/A9- induced tumors were poorly sensitive to DNA vaccination against the E7 oncoprotein [ 34 ] and in tumors developed from parental TC-1 tumor cells, the effect of DNA immunization was markedly enhanced by systemic delivery of ODN1826 or LMS [ 35 ], we tested these immunostimulatory drugs against MHC-I-deficient TC-1/A9 cells in this study. Moreover, as we identified a substantial proportion of NK and NKT cells in immune cells infiltrating TC-1-induced tumors [ 36 ], we also evaluated the effect of ODN1585 used for the activation of NK cells and α-GalCer inducing NKT cells.…”

“…An IFN-γ ELISPOT assay was performed on mononuclear cells isolated from pools of splenocytes (3 mice per group) according to the protocol described previously [ 36 ]. Cells were incubated with either 0.1 μg/mL of the E7 49–57 peptide (RAHYNIVTF, > 96% pure; Clonestar Biotech, Brno, Czech Republic) derived from the HPV16 E7 oncoprotein or 1 μg/mL of the PADRE peptide (AKFVAAWTLKAAA, >81% pure; GenScript, Piscataway, NJ, USA) at 37 °C in 5% CO2 for 24 h.…”

“…The preparation of a single cell suspension from a tumor tissue was performed as described previously [ 36 ] (see also isolation of TAMs). Four panels of fluorescent-labeled antibodies were used to identify the main cell populations infiltrating the tumor ( Table 1 ).…”

Implementation of Mass Cytometry for Immunoprofiling of Patients with Solid Tumors

“…Collagenase and DNase I are enzymes commonly used for the dissociation of tumor tissues, and their applicability for mass cytometry has recently been demonstrated [9]. Based on our prior experiences with the usage of Col NB8 (SERVA) for preparation of tumor-infiltrating cells from mouse tumors [10] and Col D (Roche) for HNSCC [11], we compared these two types of collagenases. Tumor samples were divided into two parts of the same weight and processed in the gentleMACS Octo Dissociator using predefined programs with the enzymatic treatment at 37°C for 40 min.…”