Tetje C. van der Sluis

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.

Inhibition of CSF-1R Supports T-Cell Mediated Melanoma Therapy

Tumor associated macrophages (TAM) can promote angiogenesis, invasiveness and immunosuppression. The cytokine CSF-1 (or M-CSF) is an important factor of TAM recruitment and differentiation and several pharmacological agents targeting the CSF-1 receptor (CSF-1R) have been developed to regulate TAM in solid cancers. We show that the kinase inhibitor PLX3397 strongly dampened the systemic and local accumulation of macrophages driven by B16F10 melanomas, without affecting Gr-1+ myeloid derived suppressor cells. Removal of intratumoral macrophages was remarkably efficient and a modest, but statistically significant, delay in melanoma outgrowth was observed. Importantly, CSF-1R inhibition strongly enhanced tumor control by immunotherapy using tumor-specific CD8 T cells. Elevated IFNγ production by T cells was observed in mice treated with the combination of PLX3397 and immunotherapy. These results support the combined use of CSF-1R inhibition with CD8 T cell immunotherapy, especially for macrophage-stimulating tumors.

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.

New approaches in vaccine-based immunotherapy for human papillomavirus-induced cancer

The identification of human papillomavirus as the etiological factor for cervical cancer provides an opportunity to treat these malignancies by vaccination. Although therapeutic vaccination against viral oncogenes regularly induces a specific T cell response, clinical effectivity remains low. Three factors are particularly important for clinical outcome: the balance between cytotoxic T cells and regulatory immune subsets, the balance between cytotoxic T cells and tumor cells and finally the killing efficiency of cytotoxic T cells within the tumor. To improve these three factors, therapeutic vaccination is combined with other treatments. Here, we review those studies that are based on understanding the inhibitory mechanisms that prevent unleashing the full power of therapeutic vaccine-induced T cells and utilize combinatorial interventions based on these insights.

Abstract 2494: Vaccine-induced TNF alpha producing T cells synergize with cisplatin in tumor eradication

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Previously we have shown that Synthetic Long Peptide (SLP) vaccination against Human Papilloma Virus (HPV) oncogenic proteins is safe and induces functional T-cell responses in mice and humans. Although vaccination induces potent clinical responses in mice and in patients with premalignant lesions, no clinical responses were observed in patients with cervical cancer. Here we show in a preclinical mouse model for HPV induced malignancies that SLP vaccination can be safely combined with clinically relevant chemotherapeutics to eradicate tumors. In mice that received either peptide vaccination or chemotherapy, only a temporary regression in tumor size was observed. Importantly, combined chemo-immunotherapy induced complete and sustained tumor eradication in nearly all mice. The chemotherapeutic agent cisplatin displayed the strongest synergy with SLP vaccination. Long term regression of tumors occurred at both the maximal tolerated dose of cisplatin and at 40% of that dose when combined with SLP vaccination, but at the later dose side effects such as body weight loss were absent. Upon vaccination tumors were highly infiltrated with Tumor Necrosis Factor alpha (TNFα) and Interferon gamma (IFN-γ) producing CD8+ CTLs. Tumor cells incubated with these cytokines and cisplatin in vitro strongly enhance their chemokine expression, compatible with the abundant leukocyte infiltration into the tumor upon combined chemo-immunotherapy. Accordingly, when combined with cisplatin treatment, SLP vaccine-induced CTLs appeared to migrate earlier from the tumor rim into the tumor beds. Moreover, analysis of the tumor cells in vivo showed that combined treatment significantly decreased the proliferative capacity of tumor cells compared to single treated or untreated tumors. Furthermore, we showed that TNFα enhanced cisplatin- induced, JNK dependent tumor cell apoptosis. This cell death was accompanied by an increased expression of pro-apoptotic molecules. The synergy between cisplatin and SLP vaccination was largely abolished by in vivo treatment with monoclonal antibody against TNFα. Together, our data show that combined peptide treatment with cisplatin leads to superior tumor eradication in the absence of T cell immunosuppression. Citation Format: Cornelis J. Melief, Tetje C. van der Sluis, Sjoerd H. van der Burg. Vaccine-induced TNF alpha producing T cells synergize with cisplatin in tumor eradication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2494. doi:10.1158/1538-7445.AM2015-2494

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.

Abstract 2938: Synergistic effects of properly timed HPV16 synthetic long peptide vaccination during standard carboplatin-paclitaxel chemotherapy in animals and in patients with metastatic cervical carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We previously developed a synthetic long overlapping HPV16 E6/E7 peptides (HPV16-SLP) vaccine that was clinically active in patients with HPV16+ high-grade vulvar intraepithelial neoplasia. Complete lesion regression was related to a strong vaccine-prompted HPV16-specific effector T-cell response. However, in patients with HPV16-induced metastatic cervical cancer the vaccine-induced T-cell responses were weaker and did not result in clear clinical benefit. A new study on the immune constitution of PBMC of such patients revealed increased numbers of myeloid cells, low T-cell reactivity against common microbial recall antigens and a lower stimulatory capacity of antigen presenting cells, indicating an immunosuppressive status. When these patients were subsequently treated with standard carboplatin-paclitaxel chemotherapy we observed that at a specific time point during chemotherapy this tumor-biased immune profile was normalized to a profile similar to that found in healthy subjects. Experiments in an HPV16+ mouse tumor model showed that the tumor-induced abnormally high myeloid cell level was also normalized by this chemotherapy, not only among PBMC but also locally in the tumor. Residual tumor-present myeloid cells phenotypically mirrored activated non-suppressive antigen presenting cells. Moreover, combined HPV16 SLP vaccination and chemotherapy effectively led to a higher cure rate of mice with established tumors. Together these data indicated that standard chemotherapy has an immune stimulatory effect by deletion of suppressive myeloid cells in the mouse tumor model and in patients with metastatic cervical cancer . Therefore, a clinical trial was started in which such patients were treated with standard chemotherapy in combination with HPV16 SLP (ISA101) vaccination. Comprehensive immune monitoring 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. The data of these studies will be reported. Currently, we have started a multicenter clinical trial (ISA-HPV-01-12) in which vaccination with the HPV16 SLP ISA101 vaccine is combined with carboplatin-paclitaxel and type I interferon to assess clinical and immunological outcome in a group of HPV16+ metastatic cervical cancer patients. Citation Format: Sjoerd H. Van Der Burg, Tetje C. van der Sluis, Helene van Meir, Judith R. Kroep, Gemma G. Kenter, Mariette I.E. van Poelgeest, Koos Burggraaf, Cornelis J.M. Melief, Marij J.P. Welters. Synergistic effects of properly timed HPV16 synthetic long peptide vaccination during standard carboplatin-paclitaxel chemotherapy in animals and in patients with metastatic cervical carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2938. doi:10.1158/1538-7445.AM2014-2938