Suzanne van Duikeren

Vaccine-Induced Effector-Memory CD8+ T Cell Responses Predict Therapeutic Efficacy against Tumors

CD8+ T cells have the potential to attack and eradicate cancer cells. The efficacy of therapeutic vaccines against cancer, however, lacks defined immune correlates of tumor eradication after (therapeutic) vaccination based on features of Ag-specific T cell responses. In this study, we examined CD8+ T cell responses elicited by various peptide and TLR agonist-based vaccine formulations in nontumor settings and show that the formation of CD62L−KLRG1+ effector-memory CD8+ T cells producing the effector cytokines IFN-γ and TNF predicts the degree of therapeutic efficacy of these vaccines against established s.c. tumors. Thus, characteristics of vaccine-induced CD8+ T cell responses instill a predictive determinant for the efficacy of vaccines during tumor therapy.

Vaccination during myeloid cell depletion by cancer chemotherapy fosters robust T cell responses.

Therapeutic vaccination against HPV16 is effective with chemotherapy for advanced cervical cancer patients. Vaccinating cancer away Cervical cancer, a common killer of women worldwide, is most often caused by human papillomavirus type 16 (HPV16). Although a vaccine targeting this virus is available and very effective at preventing cervical cancer, it does not work once cancer is already established, and advanced cervical cancer is very difficult to treat. Welters et al. have developed a method of therapeutic vaccination, where they synthesize long peptides mimicking key oncogenic proteins from HPV16 and use them to treat patients. Although it is too early to tell how the new vaccine will affect patient survival, combining it with chemotherapy helped strengthen patients’ immune responses against the cancer, so it is a promising candidate for further clinical development. Therapeutic vaccination with human papillomavirus type 16 synthetic long peptides (HPV16-SLPs) results in T cell–mediated regression of HPV16-induced premalignant lesions but fails to install clinically effective immunity in patients with HPV16-positive cervical cancer. We explored whether HPV16-SLP vaccination can be combined with standard carboplatin and paclitaxel chemotherapy to improve immunity and which time point would be optimal for vaccination. This was studied in the HPV16 E6/E7–positive TC-1 mouse tumor model and in patients with advanced cervical cancer. In mice and patients, the presence of a progressing tumor was associated with abnormal frequencies of circulating myeloid cells. Treatment of TC-1–bearing mice with chemotherapy and therapeutic vaccination resulted in superior survival and was directly related to a chemotherapy-mediated altered composition of the myeloid cell population in the blood and tumor. Chemotherapy had no effect on tumor-specific T cell responses. In advanced cervical cancer patients, carboplatin-paclitaxel also normalized the abnormal numbers of circulating myeloid cells, and this was associated with increased T cell reactivity to recall antigens. The effect was most pronounced starting 2 weeks after the second cycle of chemotherapy, providing an optimal immunological window for vaccination. This was validated with a single dose of HPV16-SLP vaccine given in this time window. The resulting proliferative HPV16-specific T cell responses were unusually strong and were retained after all cycles of chemotherapy. In conclusion, carboplatin-paclitaxel therapy fosters vigorous vaccine-induced T cell responses when vaccination is given after chemotherapy and has reset the tumor-induced abnormal myeloid cell composition to normal values.

Vaccine-Induced Tumor Necrosis Factor–Producing T Cells Synergize with Cisplatin to Promote Tumor Cell Death

Purpose: Cancer immunotherapy, such as vaccination, is an increasingly successful treatment modality, but its interaction with chemotherapy remains largely undefined. Therefore, we explored the mechanism of synergy between vaccination with synthetic long peptides (SLP) of human papillomavirus type 16 (HPV16) and cisplatin in a preclinical tumor model for HPV16. Experimental Design: SLP vaccination in this preclinical tumor model allowed the elucidation of novel mechanisms of synergy between chemo- and immunotherapy. By analyzing the tumor immune infiltrate, we focused on the local intratumoral effects of chemotherapy, vaccination, or the combination. Results: Of several chemotherapeutic agents, cisplatin synergized best with SLP vaccination in tumor eradication, without requirement for the maximum-tolerated dose (MTD). Upon SLP vaccination, tumors were highly infiltrated with HPV-specific, tumor necrosis factor-α (TNFα)- and interferon-γ (IFNγ)–producing T cells. Upon combined treatment, tumor cell proliferation was significantly decreased compared with single treated and untreated tumors. Furthermore, we showed that TNFα strongly enhanced cisplatin-induced apoptotic tumor cell death in a JNK-dependent manner. This is consistent with upregulation of proapoptotic molecules and with enhanced cell death in vivo upon combined SLP vaccination and cisplatin treatment. In vivo neutralization of TNFα significantly reduced the antitumor responses induced by the combined treatment. Conclusion: Taken together, our data show that peptide vaccination with cisplatin treatment leads to decreased tumor cell proliferation and TNFα-induced enhanced cisplatin-mediated killing of tumor cells, together resulting in superior tumor eradication. Clin Cancer Res; 21(4); 781–94. ©2014 AACR.

Therapeutic Peptide Vaccine-Induced CD8 T Cells Strongly Modulate Intratumoral Macrophages Required for Tumor Regression

Intratumor macrophages were found to be functionally malleable and can support, or be inhospitable to, tumors. Vaccine-induced cytokine-producing CD8 T cells modified intratumoral macrophage subsets, and both T cells and macrophages were indispensable for tumor regressions. Abundant macrophage infiltration of solid cancers commonly correlates with poor prognosis. Tumor-promoting functions of macrophages include angiogenesis, metastasis formation, and suppression of Th1-type immune responses. Here, we show that successful treatment of cervical carcinoma in mouse models with synthetic long peptide (SLP) vaccines induced influx of cytokine-producing CD8 T cells that strongly altered the numbers and phenotype of intratumoral macrophages. On the basis of the expression of CD11b, CD11c, F4/80, Ly6C, Ly6G, and MHC II, we identified four myeloid subpopulations that increased in numbers from 2.0-fold to 8.7-fold in regressing tumors. These changes of the intratumoral myeloid composition coincided with macrophage recruitment by chemokines, including CCL2 and CCL5, and were completely dependent on a vaccine-induced influx of tumor-specific CD8 T cells. CD4 T cells were dispensable. Incubation of tumor cells with T cell–derived IFNγ and TNFα recapitulated the chemokine profile observed in vivo, confirming the capacity of antitumor CD8 T cells to mediate macrophage infiltration of tumors. Strikingly, complete regressions of large established tumors depended on the tumor-infiltrating macrophages that were induced by this immunotherapy, because a small-molecule drug inhibitor targeting CSF-1R diminished the number of intratumoral macrophages and abrogated the complete remissions. Survival rates after therapeutic SLP vaccination deteriorated in the presence of CSF-1R blockers. Together, these results show that therapeutic peptide vaccination could induce cytokine-producing T cells with strong macrophage-skewing capacity necessary for tumor shrinkage, and suggest that the development of macrophage-polarizing, rather than macrophage-depleting, agents is warranted. Cancer Immunol Res; 3(9); 1042–51. ©2015 AACR.

Prediction of the immunogenic potential of frameshift-mutated antigens in microsatellite instable cancer

Microsatellite instable (MSI) cancers express frameshift‐mutated antigens, the C‐terminal polypeptides of which are foreign to the immune system. Consequently, these antigens constitute a unique pool of tumor‐specific antigens that can be exploited for patient diagnosis and selective, immune‐mediated targeting of cancers. However, other than their sequence, very little is known about the characteristics of the majority of these proteins. We therefore developed a methodology for predicting their immunogenic behavior that is based on a gene‐expression system, in which each of the proteins was fused to a short C‐terminal polypeptide comprising two epitopes that can be readily detected by T‐cells and antibodies, respectively. In this manner, accumulation of the antigens and processing of peptides derived thereof into MHC can be monitored systematically. The antigens, which accumulate in the cells in which they are synthesized, are of primary interest for cancer immunotherapy, because peptide epitopes derived thereof can be presented by dendritic cells in addition to the tumor cells themselves. As a result, these antigens constitute the best targets for a coordinated immune response by both CD8+ and CD4+ T‐cells, which increases the likelihood that tumor‐induced immunity would be detectable against these antigens in cancer patients, as well as the potential value of these antigens as components of anticancer vaccines. Our data indicate that, of 15 frameshift‐mutated antigens examined in our study, 4 (TGFβR2‐1, MARCKS‐1, MARCKS‐2 and CDX2‐2) are of primary interest, and 4 additional antigens (TAF1B‐1, PCNXL2‐2, TCF7L2‐2 and Baxα+1) are of moderate interest for further tumor immunological research.

Tumor Eradication by Cisplatin Is Sustained by CD80/86-Mediated Costimulation of CD8+ T Cells

Certain cytotoxic chemotherapeutic drugs are immunogenic, stimulating tumor immunity through mechanisms that are not completely understood. Here we show how the DNA-damaging drug cisplatin modulates tumor immunity. At the maximum tolerated dose (MTD), cisplatin cured 50% of mice with established murine TC-1 or C3 tumors, which are preclinical models of human papillomavirus (HPV)-associated cancer. Notably, the curative benefit of cisplatin relied entirely upon induction of tumor-specific CD8+ T cells. Mechanistic investigations showed that cisplatin stimulated tumor infiltration of inflammatory antigen-presenting cells (APC) expressing relatively higher levels of the T-cell costimulatory ligands CD70, CD80, and CD86. Cell death triggered by cisplatin was associated with the release of at least 19 proteins in the tumor environment that could act as damage-associated molecular patterns and upregulate costimulatory molecules, either alone or in concert, but the responsible proteins remain unknown. Essentially, the curative effect of cisplatin was abrogated in mice lacking expression of CD80 and CD86 on APCs. Furthermore, cisplatin treatment was improved by CTLA-4 blockade, which increases the availability of CD80/86 to bind to CD28. In contrast, there was no effect of CD27 stimulation, which replaces CD70 interaction. At the cisplatin MTD, cure rates could also be increased by vaccination with synthetic long peptides, whereas cures could also be achieved at similar rates at 80% of the MTD with reduced side effects. Our findings reveal an essential basis for the immunogenic properties of cisplatin, which are mediated by the induction of costimulatory signals for CD8+ T-cell-dependent tumor destruction. Cancer Res; 76(20); 6017-29. ©2016 AACR.

Enhanced Cross-Presentation and Improved CD8+ T Cell Responses after Mannosylation of Synthetic Long Peptides in Mice

The use of synthetic long peptides (SLP) has been proven to be a promising approach to induce adaptive immune responses in vaccination strategies. Here, we analyzed whether the efficiency to activate cytotoxic T cells by SLP-based vaccinations can be increased by conjugating SLPs to mannose residues. We could demonstrate that mannosylation of SLPs results in increased internalization by the mannose receptor (MR) on murine antigen-presenting cells. MR-mediated internalization targeted the mannosylated SLPs into early endosomes, from where they were cross-presented very efficiently compared to non-mannosylated SLPs. The influence of SLP mannosylation was specific for cross-presentation, as no influence on MHC II-restricted presentation was observed. Additionally, we showed that vaccination of mice with mannosylated SLPs containing epitopes from either ovalbumin or HPV E7 resulted in enhanced proliferation and activation of antigen-specific CD8+ T cells. These findings demonstrate that mannosylation of SLPs augments the induction of a cytotoxic T cell response in vitro and in vivo and might be a promising approach to induce cytotoxic T cell responses in e.g. cancer therapy and anti-viral immunity.

The Breadth of Synthetic Long Peptide Vaccine-Induced CD8+ T Cell Responses Determines the Efficacy against Mouse Cytomegalovirus Infection.

There is an ultimate need for efficacious vaccines against human cytomegalovirus (HCMV), which causes severe morbidity and mortality among neonates and immunocompromised individuals. In this study we explored synthetic long peptide (SLP) vaccination as a platform modality to protect against mouse CMV (MCMV) infection in preclinical mouse models. In both C57BL/6 and BALB/c mouse strains, prime-booster vaccination with SLPs containing MHC class I restricted epitopes of MCMV resulted in the induction of strong and polyfunctional (i.e., IFN-γ+, TNF+, IL-2+) CD8+ T cell responses, equivalent in magnitude to those induced by the virus itself. SLP vaccination initially led to the formation of effector CD8+ T cells (KLRG1hi, CD44hi, CD127lo, CD62Llo), which eventually converted to a mixed central and effector-memory T cell phenotype. Markedly, the magnitude of the SLP vaccine-induced CD8+ T cell response was unrelated to the T cell functional avidity but correlated to the naive CD8+ T cell precursor frequency of each epitope. Vaccination with single SLPs displayed various levels of long-term protection against acute MCMV infection, but superior protection occurred after vaccination with a combination of SLPs. This finding underlines the importance of the breadth of the vaccine-induced CD8+ T cell response. Thus, SLP-based vaccines could be a potential strategy to prevent CMV-associated disease.

Predicting the efficacy of cancer vaccines by evaluating T-cell responses

In a preclinical experimental model of human papilloma virus (HPV)-induced cervical carcinoma, we have shown that the efficacy of cancer vaccines can be predicted by the evaluation of vaccine-induced T-cell responses in healthy subjects. We argue that such knowledge can be used to screen candidates for vaccination, which in turn may accelerate the development and increase the overall efficacy of cancer vaccines.

Synergy of therapeutic vaccination against HPV16 oncogenic proteins and standard chemotherapeutics

We previously developed a synthetic long peptide (SLP) vaccine against HPV16 oncoproteins that induced lesion regression in patients with HPV16+ high-grade vulvar intraepithelial neoplasia, correlated with strong vaccine-prompted HPV16-specific T cell responses. In patients with HPV16-induced metastatic cervical cancer, vaccine-induced T cell responses were weaker and did not result in improved clinical outcome. In a preclinical HPV16 E6/E7+ mouse tumor model we studied the efficacy of SLP vaccination combined with chemotherapy. Mice that received either peptide vaccination or chemotherapy showed only temporary tumor regression. Importantly, combined chemo-immunotherapy induced complete tumor eradication in 75% of the mice, which was associated with a strong tumor influx of vaccine specific tumor necrosis factor alpha (TNFα) and interferon gamma (IFN-γ) producing CD8+ CTLs. Tumor cells incubated with TNFα and IFN-γ, together with cisplatin, enhanced their chemokine expression and SLP vaccine-induced CTLs appeared to migrate earlier into the tumor beds. Combination treatment in vitro caused a decrease in proliferation of tumor cells and TNFα-induced enhancement of cisplatin- mediated tumor cell death, accompanied by increased expression of pro-apoptotic molecules. SLP vaccination together with carboplatin and paclitaxel, a standard combined chemotherapy, caused marked decline in the abnormally high numbers of myeloid cells in blood and tumor in the mouse model, again associated with synergy in tumor eradication. Hence, standard chemotherapy promotes the effects of SLP vaccination by better attraction of T cells into tumors, greater sensitivity of tumors to TNFα-mediated apoptosis, and better expansion of T cells through depletion of myeloid derived suppressor cells without suppression of T cells, allowing synergy in tumor eradication. A clinical pilot study on the composition of blood leukocytes in late stage cervical cancer patients also revealed high numbers of myeloid cells, associated with low T cell responses, indicating an immunosuppressed status. When these patients were treated with carboplatin-paclitaxel chemotherapy their immune profile was normalized to that of healthy subjects. Therefore a clinical trial was performed in which late-stage cervical cancer patients were treated with standard chemotherapy in combination with HPV16 SLP vaccination. Immunomonitoring confirmed the beneficial effect of myeloid cell depletion associated with a robust induction of HPV16-specific T cell responses that were sustained throughout several cycles of chemotherapy.