Sjoerd H. van der Burg

Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia

BACKGROUND Vulvar intraepithelial neoplasia is a chronic disorder caused by high-risk types of human papillomavirus (HPV), most commonly HPV type 16 (HPV-16). Spontaneous regression occurs in less than 1.5% of patients, and the rate of recurrence after treatment is high. METHODS We investigated the immunogenicity and efficacy of a synthetic long-peptide vaccine in women with HPV-16-positive, high-grade vulvar intraepithelial neoplasia. Twenty women with HPV-16-positive, grade 3 vulvar intraepithelial neoplasia were vaccinated three or four times with a mix of long peptides from the HPV-16 viral oncoproteins E6 and E7 in incomplete Freunds adjuvant. The end points were clinical and HPV-16-specific T-cell responses. RESULTS The most common adverse events were local swelling in 100% of the patients and fever in 64% of the patients; none of these events exceeded grade 2 of the Common Terminology Criteria for Adverse Events of the National Cancer Institute. At 3 months after the last vaccination, 12 of 20 patients (60%; 95% confidence interval [CI], 36 to 81) had clinical responses and reported relief of symptoms. Five women had complete regression of the lesions, and HPV-16 was no longer detectable in four of them. At 12 months of follow-up, 15 of 19 patients had clinical responses (79%; 95% CI, 54 to 94), with a complete response in 9 of 19 patients (47%; 95% CI, 24 to 71). The complete-response rate was maintained at 24 months of follow-up. All patients had vaccine-induced T-cell responses, and post hoc analyses suggested that patients with a complete response at 3 months had a significantly stronger interferon-gamma-associated proliferative CD4+ T-cell response and a broad response of CD8+ interferon-gamma T cells than did patients without a complete response. CONCLUSIONS Clinical responses in women with HPV-16-positive, grade 3 vulvar intraepithelial neoplasia can be achieved by vaccination with a synthetic long-peptide vaccine against the HPV-16 oncoproteins E6 and E7. Complete responses appear to be correlated with induction of HPV-16-specific immunity.

Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines

This Review deals with recent progress in the immunotherapy of established (pre)malignant disease of viral or non-viral origin by synthetic vaccines capable of inducing robust T-cell responses. The most attractive vaccine compounds are synthetic long peptides (SLP) corresponding to the sequence of tumour viral antigens or tumour-associated non-viral antigens. Crucial to induction of therapeutic T-cell immunity is the capacity of SLP to deliver specific cargo to professional antigen-presenting cells (dendritic cells (DC)). Proper DC activation then induces the therapeutic CD4+ and CD8+ T-cell responses that are associated with regression of established (pre)malignant lesions, including those induced by high-risk human papilloma virus.

Established Human Papillomavirus Type 16-Expressing Tumors Are Effectively Eradicated Following Vaccination with Long Peptides

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E743–77, containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8+ T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4+ Th cells. The level of the induced E7-specific CD8+ T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8+ T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4 + T cells in human melanoma

Tumor-specific neo-antigens that arise as a consequence of mutations are thought to be important for the therapeutic efficacy of cancer immunotherapies. Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8+ T cells, but it is unclear whether neo-antigen–specific CD4+ T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4+ T-cell responses in tumor control, we addressed whether neo-antigen–specific CD4+ T-cell reactivity is a common property in human melanoma.

Induction of Tumor-Specific CD4+ and CD8+ T-Cell Immunity in Cervical Cancer Patients by a Human Papillomavirus Type 16 E6 and E7 Long Peptides Vaccine

Purpose: The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides vaccine on the antigen-specific T-cell response in cervical cancer patients. Experimental Design: Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. HPV16-specific T-cell immune responses were analyzed by evaluating the magnitude, breadth, type, and polarization by proliferation assays, IFNγ-ELISPOT, and cytokine production and phenotyped by the T-cell markers CD4, CD8, CD25, and Foxp3. Results: Vaccine-induced T-cell responses against HPV16 E6 and E7 were detected in six of six and five of six patients, respectively. These responses were broad, involved both CD4+ and CD8+ T cells, and could be detected up to 12 months after the last vaccination. The vaccine-induced responses were dominated by effector type CD4+CD25+Foxp3− type 1 cytokine IFNγ-producing T cells but also included the expansion of T cells with a CD4+CD25+Foxp3+ phenotype. Conclusions: The HPV16 E6 and E7 synthetic long peptides vaccine is highly immunogenic, in that it increases the number and activity of HPV16-specific CD4+ and CD8+ T cells to a broad array of epitopes in all patients. The expansion of CD4+ and CD8+ tumor-specific T cells, both considered to be important in the antitumor response, indicates the immunotherapeutic potential of this vaccine. Notably, part of the vaccine-induced T cells display a CD4+CD25+Foxp3+ phenotype that is frequently associated with regulatory T-cell function, suggesting that strategies to disarm this subset of T cells should be considered as components of immunotherapeutic modalities against HPV-induced cancers.

A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence

In response to tenacious stress signals, such as the unscheduled activation of oncogenes, cells can mobilize tumour suppressor networks to avert the hazard of malignant transformation. A large body of evidence indicates that oncogene-induced senescence (OIS) acts as such a break, withdrawing cells from the proliferative pool almost irreversibly, thus crafting a vital pathophysiological mechanism that protects against cancer. Despite the widespread contribution of OIS to the cessation of tumorigenic expansion in animal models and humans, we have only just begun to define the underlying mechanism and identify key players. Although deregulation of metabolism is intimately linked to the proliferative capacity of cells, and senescent cells are thought to remain metabolically active, little has been investigated in detail about the role of cellular metabolism in OIS. Here we show, by metabolic profiling and functional perturbations, that the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) is a crucial mediator of senescence induced by BRAFV600E, an oncogene commonly mutated in melanoma and other cancers. BRAFV600E-induced senescence was accompanied by simultaneous suppression of the PDH-inhibitory enzyme pyruvate dehydrogenase kinase 1 (PDK1) and induction of the PDH-activating enzyme pyruvate dehydrogenase phosphatase 2 (PDP2). The resulting combined activation of PDH enhanced the use of pyruvate in the tricarboxylic acid cycle, causing increased respiration and redox stress. Abrogation of OIS, a rate-limiting step towards oncogenic transformation, coincided with reversion of these processes. Further supporting a crucial role of PDH in OIS, enforced normalization of either PDK1 or PDP2 expression levels inhibited PDH and abrogated OIS, thereby licensing BRAFV600E-driven melanoma development. Finally, depletion of PDK1 eradicated melanoma subpopulations resistant to targeted BRAF inhibition, and caused regression of established melanomas. These results reveal a mechanistic relationship between OIS and a key metabolic signalling axis, which may be exploited therapeutically.

Identification and manipulation of tumor associated macrophages in human cancers

Evading immune destruction and tumor promoting inflammation are important hallmarks in the development of cancer. Macrophages are present in most human tumors and are often associated with bad prognosis. Tumor associated macrophages come in many functional flavors ranging from what is known as classically activated macrophages (M1) associated with acute inflammation and T-cell immunity to immune suppressive macrophages (M2) associated with the promotion of tumor growth. The role of these functionally different myeloid cells is extensively studied in mice tumor models but dissimilarities in markers and receptors make the direct translation to human cancer difficult. This review focuses on recent reports discriminating the type of infiltrating macrophages in human tumors and the environmental cues present that steer their differentiation. Finally, immunotherapeutic approaches to interfere in this process are discussed.

Human Papillomavirus Type 16-Positive Cervical Cancer Is Associated with Impaired CD4+ T-Cell Immunity against Early Antigens E2 and E6

Cervical cancer is the possible outcome of genital infection with high-risk human papillomavirus (HPV) and is preceded by a phase of persistent HPV infection during which the host immune system fails to eliminate the virus. Fortunately, the majority of genital HPV infections are cleared before the development of (pre)malignant lesions. Analysis of CD4+ T-helper (Th) immunity against the E2, E6, and E7 antigens of HPV16 in healthy women revealed strong proliferative E2- and E6-specific responses associated with prominent IFN-γ and interleukin 5 secretion. This indicates that the naturally arising virus-induced immune response displays a mixed Th1/Th2 cytokine profile. Of all HPV16+ cervical cancer patients, approximately half failed to mount a detectable immune response against the HPV16-derived peptides. The other half of the patients showed impaired HPV16-specific proliferative responses, which generally lacked both IFN-γ and interleukin 5. This indicates that the HPV16-specific CD4+ T-cell response in cervical cancer patients is either absent or severely impaired, despite a relatively good immune status of the patients, as indicated by intact responses against recall antigens. It is highly conceivable that proper CD4+ T-cell help is important for launching an effective immune attack against HPV because infection of cervical epithelia by this virus is, at least initially, not accompanied by gross disturbance of this tissue and/or strong proinflammatory stimuli. Therefore, our observations concerning the lack of functional HPV16-specific CD4+ T-cell immunity in patients with cervical cancer offer a possible explanation for the development of this disease.

High Number of Intraepithelial CD8+ Tumor-Infiltrating Lymphocytes Is Associated with the Absence of Lymph Node Metastases in Patients with Large Early-Stage Cervical Cancer

In a prospective study, we have examined the tumor-specific immune response in a group of 59 patients with human papillomavirus (HPV) 16-positive (HPV16(+))-induced or HPV18(+)-induced cervical cancer. Local antitumor immunity was analyzed by the enumeration of tumor-infiltrating dendritic cells and CD4+, CD8+, and regulatory T cells as well as by calculation of the ratio of CD8+/CD4+ T cells and CD8+/regulatory T cells. Systemic tumor-specific immunity was assessed by determination of the HPV E6- and/or E7-specific T-cell response in the blood of these patients. Finally, these variables were evaluated with respect to known histopathologic prognostic variables, including the absence (LN-) or presence (LN+) of lymph node metastases. Stratification according to the lymph node status of patients revealed a significantly stronger CD8+ T-cell tumor infiltration, a higher CD8+/CD4+ T-cell ratio, and higher CD8+/regulatory T-cell ratio in the group of patients in which the tumor failed to metastasize to the tumor-draining lymph node. Subdivision according to the presence (IR+) or absence (IR-) of circulating HPV-specific T cells disclosed that the highest number of tumor-infiltrating CD8+ T cells was found in the group of LN- patients displaying a concomitant systemic tumor-specific immune response (LN-IR+). CD8+ T-cell infiltration in LN-IR- patients was comparable with that of LN+ patients. In cervical cancer, the absence of lymph node metastases is strongly associated with a better prognosis. Our data indicate that, especially in a subgroup of LN- patients, a strong and effective interaction between immune system and tumor exists. This subgroup of cervical cancer patients may have the best prognosis.

Phase I Immunotherapeutic Trial with Long Peptides Spanning the E6 and E7 Sequences of High-Risk Human Papillomavirus 16 in End-Stage Cervical Cancer Patients Shows Low Toxicity and Robust Immunogenicity

Purpose: To determine the toxicity, safety, and immunogenicity of a human papillomavirus 16 (HPV16) E6 and E7 long peptide vaccine administered to end-stage cervical cancer patients. Experimental Design: Three groups of end-stage cervical cancer patients (in total n = 35) were s.c. vaccinated with HPV16 E6 combined with or separated from HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant, four times at 3-week intervals. Group 1 received 300 μg/peptide at a single site and group 2 received 100 μg/peptide of the E6 peptides in one limb and 300 μg/peptide of the E7 peptides in a second limb. Group 3 received separate injections of E6 and E7 peptides, each at a dose of 50 μg/peptide. The primary end point was to determine safety and toxicity of the HPV16 long peptides vaccine. In addition, the vaccine-induced T-cell response was assessed by IFNγ enzyme-linked immunospot. Results: No toxicity beyond grade 2 was observed during and after four vaccinations. In a few patients, transient flu-like symptoms were observed. Enzyme-linked immunospot analysis of the vaccine-induced immune response revealed that coinjection of the E6 and E7 peptides resulted in a strong and broad T-cell response dominated by immunity against E6. Injection of the E6 and E7 peptides at two different sites increased the E7 response but did not affect the magnitude of the E6-induced immune response. Conclusions: The HPV16 E6 and E7 long peptide-based vaccine is well tolerated and capable of inducing a broad IFNγ-associated T-cell response even in end-stage cervical cancer patients.