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Therapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all aiming at robust T cell responses. SLPs linked to the Amplivant® adjuvant (Amplivant-SLP) have shown effective delivery to dendritic cells, resulting in improved immunogenicity in mice. We have now tested virosomes as a delivery vehicle for SLPs. Virosomes are nanoparticles made from influenza virus membranes and have been used as vaccines for a variety of antigens. Amplivant-SLP virosomes induced the expansion of more antigen-specific CD8 + T memory cells in ex vivo experiments with human PBMCs than Amplivant-SLP conjugates alone. The immune response could be further improved by including the adjuvants QS-21 and 3D-PHAD in the virosomal membrane. In these experiments, the SLPs were anchored in the membrane through the hydrophobic Amplivant adjuvant. In a therapeutic mouse model of HPV16 E6/E7+ cancer, mice were vaccinated with virosomes loaded with either Amplivant-conjugated SLPs or lipid-coupled SLPs. Vaccination with both types of virosomes significantly improved the control of tumor outgrowth, leading to elimination of the tumors in about half the animals for the best combinations of adjuvants and to their survival beyond 100 days.

The combination of ISA101, a human papilloma virus (HPV) 16 peptide vaccine, and nivolumab showed a promising response rate of 33% in patients with incurable HPV-16Patients with advanced HPV-16Twenty-four patients were followed for a median of 46.5 months (95% CI, 46.0 months to not reached (NR)). The median duration of response was 11.2 months (95% CI, 8.51 months to NR); three out of eight (38%) patients with objective response were without progression at 3 years. The median and 3-year overall survival were 15.3 months (95% CI, 10.6 months to 27.2 months) and 12.5% (95% CI, 4.3% to 36%), respectively. The scores for activated T cells ((CD3Efficacy of ISA101 and nivolumab remains promising in long-term follow-up. Increased infiltration by PD-1NCT02426892.

Water-in-oil emulsion incomplete Freund’s adjuvant (IFA) has been used as an adjuvant in preventive and therapeutic vaccines since its development. New generation, highly purified modulations of the adjuvant, Montanide incomplete seppic adjuvant (ISA)-51 and Montanide ISA-720, were developed to reduce toxicity. Montanide adjuvants are generally considered to be safe, with adverse events largely consisting of antigen and adjuvant dose-dependent injection site reactions (ISRs). Peptide vaccines in Montanide ISA-51 or ISA-720 are capable of inducing both high antibody titers and durable effector T cell responses. However, an efficient T cell response depends on the affinity of the peptide to the presenting major histocompatibility complex class I molecule, CD4+T cell help and/or the level of co-stimulation. In fact, in the therapeutic cancer vaccine setting, presence of a CD4+T cell epitope seems crucial to elicit a robust and durable systemic T cell response. Additional inclusion of a Toll-like receptor ligand can further increase the magnitude and durability of the response. Use of extended peptides that need a processing step only accomplished effectively by dendritic cells (DCs) can help to avoid antigen presentation by nucleated cells other than DC. Based on recent clinical trial results, therapeutic peptide-based cancer vaccines using emulsions in adjuvant Montanide ISA-51 can elicit robust antitumor immune responses, provided that sufficient tumor-specific CD4+T cell help is given in addition to CD8+T cell epitopes. Co-treatment with PD-1 T cell checkpoint inhibitor, chemotherapy or other immunomodulatory drugs may address local and systemic immunosuppressive mechanisms, and further enhance efficacy of therapeutic cancer peptide vaccines in IFA and its modern variants. Blinded randomized placebo-controlled trials are critical to definitively prove clinical efficacy. Mineral oil-based adjuvants for preventive vaccines, to tackle spread and severity of infectious disease, induce immune responses, but require more studies to reduce toxicity.

CD8+ T-cell infiltration and effector activity in tumors are correlated with better overall survival of patients, suggesting that the ability of T cells to enter and remain in contact with tumor cells supports tumor control. CD8+ T cells express the collagen-binding integrins CD49a and CD49b, but little is known about their function or how their expression is regulated in the tumor microenvironment (TME). Here, we found that tumor-infiltrating CD8+ T cells initially expressed CD49b, gained CD49a, and then lost CD49b over the course of tumor outgrowth. This differentiation sequence was driven by antigen-independent elements in the TME, although T-cell receptor (TCR) stimulation further increased CD49a expression. Expression of exhaustion markers and CD49a associated temporally but not mechanistically. Intratumoral CD49a-expressing CD8+ T cells failed to upregulate TCR-dependent Nur77 expression, whereas CD69 was constitutively expressed, consistent with both a lack of productive antigen engagement and a tissue-resident memory-like phenotype. Imaging T cells in live tumor slices revealed that CD49a increased their motility, especially of those in close proximity to tumor cells, suggesting that it may interfere with T-cell recognition of tumor cells by distracting them from productive engagement, although we were not able to augment productive engagement by short-term CD49a blockade. CD49b also promoted relocalization of T cells at a greater distance from tumor cells. Thus, our results demonstrate that expression of these integrins affects T-cell trafficking and localization in tumors via distinct mechanisms, and suggests a new way in which the TME, and likely collagen, could promote tumor-infiltrating CD8+ T-cell dysfunction.

Therapeutic cancer vaccines have undergone a resurgence in the past decade. A better understanding of the breadth of tumour-associated antigens, the native immune response and development of novel technologies for antigen delivery has facilitated improved vaccine design. The goal of therapeutic cancer vaccines is to induce tumour regression, eradicate minimal residual disease, establish lasting antitumour memory and avoid non-specific or adverse reactions. However, tumour-induced immunosuppression and immunoresistance pose significant challenges to achieving this goal. In this Review, we deliberate on how to improve and expand the antigen repertoire for vaccines, consider developments in vaccine platforms and explore antigen-agnostic in situ vaccines. Furthermore, we summarize the reasons for failure of cancer vaccines in the past and provide an overview of various mechanisms of resistance posed by the tumour. Finally, we propose strategies for combining suitable vaccine platforms with novel immunomodulatory approaches and standard-of-care treatments for overcoming tumour resistance and enhancing clinical efficacy.This Review broadly discusses therapeutic cancer vaccines, covering resistance mechanisms and strategies to overcome these, how to improve the antigen repertoire for vaccines and vaccine platforms, and approaches for enhancing immunotherapy efficacy.

Summary During the last two decades, two main schools of modern immunotherapy have come to the forefront. The chimeric anti-CD20 antibody rituximab that was introduced for the treatment of refractory follicular lymphoma in 1998 was one of the first examples of the school of passive immunotherapy. Subsequently major and ever more costly efforts were spent on the development of blockbuster monotherapies including other monoclonal but also bispecific antibodies of highly defined specificity and subclass, antibody–drug conjugates (ADCs), as well as ex vivo expanded tumor-infiltrating lymphocytes, chimeric antigen receptor (CAR)-transduced T cells, and TCR-transduced T cells. On the other hand, there is the school that works toward active induction of patient B- or T-cell immunity against antigens of choice, or active tolerance against pathogenic allergens, auto-antigens or allo-antigens. Stradled in between these two approaches is treatment with blockers of T cell checkpoint control, which releases the brakes of T cells that have already responded to antigen. Extensive and detailed insight into the cellular and molecular interactions that regulate specific immune responses is indispensable in order to be able to optimize efficacy and rule out treatment related toxicity. This applies to all types of immunotherapy. Our knowledge of the checks and balances in the immune system is still increasing at an unprecedented pace, fostering ever more effective and specific (combination) immunotherapies and offering a rich harvest of innovative immunotherapies in the years ahead.

Autoreactive CD8+ T cells play a pivotal role in melanocyte destruction in autoimmune vitiligo. Immunotherapy for melanoma often leads to autoimmune side-effects, among which vitiligo-like depigmentation, indicating that targeting immune checkpoints can break peripheral tolerance against self-antigens in the skin. Therapeutically enhancing immune checkpoint signaling by immune cells or skin cells, making self-reactive T cells anergic, seems a promising therapeutic option for vitiligo. Here, we review the current knowledge on the PD-1/PD-L1 pathway in vitiligo as new therapeutic target for vitiligo therapy.

Many approaches to identify therapeutically relevant neoantigens couple tumor sequencing with bioinformatic algorithms and inferred rules of tumor epitope immunogenicity. However, there are no reference data to compare these approaches, and the parameters governing tumor epitope immunogenicity remain unclear. Here, we assembled a global consortium wherein each participant predicted immunogenic epitopes from shared tumor sequencing data. 608 epitopes were subsequently assessed for T cell binding in patient-matched samples. By integrating peptide features associated with presentation and recognition, we developed a model of tumor epitope immunogenicity that filtered out 98% of non-immunogenic peptides with a precision above 0.70. Pipelines prioritizing model features had superior performance, and pipeline alterations leveraging them improved prediction performance. These findings were validated in an independent cohort of 310 epitopes prioritized from tumor sequencing data and assessed for T cell binding. This data resource enables identification of parameters underlying effective anti-tumor immunity and is available to the research community.

Therapeutic cancer vaccines have effectively induced durable regressions of premalignant oncogenic human papilloma virus type 16 (HPV16)-induced anogenital lesions. However, the treatment of HPV16-induced cancers requires appropriate countermeasures to overcome cancer-induced immune suppression. We previously showed that standard-of-care carboplatin/paclitaxel chemotherapy can reduce abnormally high numbers of immunosuppressive myeloid cells in patients, allowing the development of much stronger therapeutic HPV16 vaccine (ISA101)-induced tumor immunity. We now show the clinical effects of ISA101 vaccination during chemotherapy in 77 patients with advanced, recurrent, or metastatic cervical cancer in a dose assessment study of ISA101. Tumor regressions were observed in 43% of 72 evaluable patients. The depletion of myeloid suppressive cells by carboplatin/paclitaxel was associated with detection of low frequency of spontaneous HPV16-specific immunity in 21 of 62 tested patients. Patients mounted type 1 T cell responses to the vaccine across all doses. The group of patients with higher than median vaccine-induced immune responses lived longer, with a flat tail on the survival curve. This demonstrates that chemoimmunotherapy can be exploited to the benefit of patients with advanced cancer based on a defined mode of action.

Graphical abstract, Highlights • Viral infections cause oxidative stress which may be exaggerated by inflammaging. • Oxidative stress coping mechanisms can be affected by genetic/environmental factors. • SARS-CoV has been reported to modulate PARP function and thereby NAD + biosynthesis. • Cellular homeostasis and redox imbalances by SARS-CoV2 can cause stress responses. • Antioxidants such as NAC could limit ROS mediated tissue damage during COVID-19., During the current COVID-19 pandemic, a need for evaluation of already available drugs for treatment of the disease is crucial. Hereby, based on literature review from the current pandemic and previous outbreaks with corona viruses we analyze the impact of the virus infection on cell stress responses and redox balance. High levels of mortality are noticed in elderly individuals infected with SARS-CoV2 and during the previous SARS-CoV1 outbreak. Elderly individuals maintain a chronic low level of inflammation which is associated with oxidative stress and inflammatory cytokine production, a condition that increases the severity of viral infections in this population. Coronavirus infections can lead to alterations of redox balance in infected cells through modulation of NAD + biosynthesis, PARP function along with altering proteasome and mitochondrial function in the cell thereby leading to enhanced cell stress responses which further exacerbate inflammation. ROS production can increase IL-6 production and lipid peroxidation resulting in cell damage. Therefore, early treatment with anti-oxidants such as NAC during COVID-19 can be a way to bypass the excessive inflammation and cell damage that lead to severe infection, thus early NAC as intervention should be evaluated in a clinical trial setting.

Cancer immunotherapy aims to promote the activity of cytotoxic T lymphocytes (CTLs) within a tumour, assist the priming of tumour-specific CTLs in lymphoid organs and establish efficient and durable antitumour immunity. During priming, help signals are relayed from CD4+ T cells to CD8+ T cells by specific dendritic cells to optimize the magnitude and quality of the CTL response. In this Review, we highlight the cellular dynamics and membrane receptors that mediate CD4+ T cell help and the molecular mechanisms of the enhanced antitumour activity of CTLs. We outline how deficient CD4+ T cell help reduces the response of CTLs and how maximizing CD4+ T cell help can improve outcomes in cancer immunotherapy strategies. CD4+ T cells provide help to CD8+ T cells via lymph node-resident dendritic cells. In this Review, the authors discuss the molecular nature of help signals and how they can be harnessed to improve cancer immunotherapy.

Cancer cells are subjected to constant selection by the immune system, meaning that tumors that become clinically manifest have managed to subvert or hide from immunosurveillance. Immune control can be facilitated by induction of autophagy, as well as by polyploidization of cancer cells. While autophagy causes the release of ATP, a chemotactic signal for myeloid cells, polyploidization can trigger endoplasmic reticulum stress with consequent exposure of the "eat-me" signal calreticulin on the cell surface, thereby facilitating the transfer of tumor antigens into dendritic cells. Hence, both autophagy and polyploidization cause the emission of adjuvant signals that ultimately elicit immune control by CD8+ T lymphocytes. We investigated the possibility that autophagy and polyploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. Mass spectrometry led to the identification of peptides that were presented on major histocompatibility complex (MHC) class I molecules in an autophagy-dependent fashion or that were specifically exposed on the surface of polyploid cells, yet lost upon passage of such cells through immunocompetent (but not immunodeficient) mice. However, the preferential recognition of autophagy-competent and polyploid cells by the innate and cellular immune systems did not correlate with the preferential recognition of such peptides in vivo. Moreover, vaccination with such peptides was unable to elicit tumor growth-inhibitory responses in vivo. We conclude that autophagy and polyploidy increase the immunogenicity of cancer cells mostly by affecting their adjuvanticity rather than their antigenicity.

Synthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core protein derived SLP was capable of boosting CD4+ and CD8+ T cell responses in the presence of a TLR2-ligand in chronic HBV patients ex vivo. For optimal efficacy of a therapeutic vaccine in vivo, adjuvants can be conjugated to the SLP to ensure delivery of both the antigen and the co-stimulatory signal to the same antigen-presenting cell (APC). Dendritic cells (DCs) express the receptor for the adjuvant and are optimally equipped to efficiently process and present the SLP-contained epitopes to T cells. Here, we investigated TLR2-ligand conjugation of the prototype HBV-core SLP. Results indicated that TLR2-ligand conjugation reduced cross-presentation efficiency of the SLP-contained epitope by both monocyte-derived and naturally occurring DC subsets. Importantly, cross-presentation was improved after optimization of the conjugate by either shortening the SLP or by placing a valine-citrulline linker between the TLR2-ligand and the long SLP, to facilitate endosomal dissociation of SLP and TLR2-ligand after uptake. HBV-core SLP conjugates also triggered functional patient T cell responses ex vivo. These results provide an import step forward in the design of a therapeutic SLP-based vaccine to cure chronic HBV.

Importance In recurrent human papilloma virus (HPV)–driven cancer, immune checkpoint blockade with anti–programmed cell death 1 (PD-1) antibodies produces tumor regression in only a minority of patients. Therapeutic HPV vaccines have produced strong immune responses to HPV-16, but vaccination alone has been ineffective for invasive cancer. Objective To determine whether the efficacy of nivolumab, an anti–PD-1 immune checkpoint antibody, is amplified through treatment with ISA 101, a synthetic long-peptide HPV-16 vaccine inducing HPV-specific T cells, in patients with incurable HPV-16–positive cancer. Design, Setting, and Participants In this single-arm, single-center phase 2 clinical trial, 24 patients with incurable HPV-16–positive cancer were enrolled from December 23, 2015, to December 12, 2016. Duration of follow-up for censored patients was 12.2 months through August 31, 2017. Interventions The vaccine ISA101, 100 μg/peptide, was given subcutaneously on days 1, 22, and 50. Nivolumab, 3 mg/kg, was given intravenously every 2 weeks beginning day 8 for up to 1 year. Main Outcomes and Measures Assessment of efficacy reflected in the overall response rate (per Response Evaluation Criteria in Solid Tumors, version 1.1). Results Of the 24 patients (4 women and 20 men; 22 with oropharyngeal cancer; median age, 60 years [range, 36-73 years]), the overall response rate was 33% (8 patients; 90% CI, 19%-50%). Median duration of response was 10.3 months (95% CI, 10.3 months to inestimable). Five of 8 patients remain in response. Median progression-free survival was 2.7 months (95% CI, 2.5-9.4 months). Median overall survival was 17.5 months (95% CI, 17.5 months to inestimable). Grades 3 to 4 toxicity occurred in 2 patients (asymptomatic grade 3 transaminase level elevation in 1 patient and grade 4 lipase elevation in 1 patient), requiring discontinuation of nivolumab therapy. Conclusions and Relevance The overall response rate of 33% and median overall survival of 17.5 months is promising compared with PD-1 inhibition alone in similar patients. A randomized clinical trial to confirm the contribution of HPV-16 vaccination to tumoricidal effects of PD-1 inhibition is warranted for further study. Trial Registration ClinicalTrials.gov identifier:NCT02426892

Background: Therapeutic vaccines based on synthetic long peptides (SLPs) have a great potential for immunotherapy of cancer patients as these SLPs include both human leukocyte antigen (HLA) class I and II epitopes and no patient selection for HLA types is required. The antigen-induced immune response can be strengthened with immune stimulating additives. Amplivant (AV) is a synthetic Toll-like receptor 2 ligand which can be directly conjugated to tumor peptide antigens. In preclinical studies, AV-conjugation to antigens led to both enhanced antigen presentation by dendritic cells and T-cell priming and caused superior induction of effective anti-tumor responses. Moreover, AV-conjugated SLPs showed a 100 times higher immune response compared to unconjugated SLP. The current study is a first-in-human trial to investigate safety and immunogenicity of AV-conjugated human papillomavirus (HPV)16-SLPs. Methods: A dose escalation phase I trial was performed in 24 patients with HPV16 positive (pre-) malignant lesions. AV was conjugated to two SLPs derived from the most immunodominant regions of the HPV16 E6 oncoprotein. Four dose groups (1, 5, 20 or 50 μg of each peptide) in 6 patients each were studied. The vaccine was injected three times intradermally in DMSO / water with a three-week interval. Adverse events (AE) were collected according to CTCAE v4.0 up to 26 weeks. Peptide-specific T-cell immune responses were determined in blood samples taken before and after vaccination using complementary immunological assays (proliferation assay, IFNγ-ELISPOT and cytokine bead array). Results: Toxicity after three AV-conjugated HPV16-SLP vaccinations was limited to CTCAE grade 1 or 2, with predominantly inflammation at the vaccination site and sometimes flu-like symptoms, which generally resolved within one day. Dose increase resulted from no AE in the lowest dose group to mild/moderate AE in all vaccinated persons in the highest dose group. In the lowest dose group, minor vaccine-induced T-cell responses were observed in three of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T-cell response after vaccination. The induced T-cell response against HPV16 lasted until the end of the trial. Conclusions: This first-in-human study showed that AV conjugated to SLPs can safely be used as an intradermal therapeutic vaccine. AV-conjugated HPV16-SLP was able to induce robust HPV16-specific T-cell immunity in patients treated for HPV16 positive (pre-) malignancies without any other vaccine adjuvant or formulation. Increase in dose resulted in both a higher number of mild adverse events as well as stronger T-cell immunity. Clinical trial information: NCT02821494.

Purpose: Therapeutic vaccination with human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides (SLP) is effective against HPV16-induced high-grade vulvar and vaginal intraepithelial neoplasia (VIN/VaIN). However, clinical nonresponders displayed weak CD8+ T-cell reactivity. Here, we studied if imiquimod applied at the vaccine site could improve CD8+ T-cell reactivity, clinical efficacy, and safety of HPV16-SLP (ISA101). Experimental Design: A multicenter open-label, randomized controlled trial was conducted in patients with HPV16+ high-grade VIN/VaIN. Patients received ISA101 vaccination with or without application of 5% imiquimod at the vaccine site. The primary objective was the induction of a directly ex vivo detectable HPV16-specific CD8+ T-cell response. The secondary objectives were clinical responses (lesion size, histology, and virology) and their relation with the strength of vaccination-induced immune responses. Results: Forty-three patients were assigned to either ISA101 with imiquimod (n = 21) or ISA101 only (n = 22). Imiquimod did not improve the outcomes of vaccination. However, vaccine-induced clinical responses were observed in 18 of 34 (53%; 95% CI, 35.1–70.2) patients at 3 months and in 15 of 29 (52%; 95% CI, 32.5–70.6) patients, 8 of whom displayed a complete histologic response, at 12 months after the last vaccination. All patients displayed vaccine-induced T-cell responses, which were significantly stronger in patients with complete responses. Importantly, viral clearance occurred in all but one of the patients with complete histologic clearance. Conclusions: This new study confirms that clinical efficacy of ISA101 vaccination is related to the strength of vaccine-induced HPV16-specific T-cell immunity and is an effective therapy for HPV16-induced high-grade VIN/VaIN. Clin Cancer Res; 22(10); 2342–50. ©2016 AACR. See related commentary by Karaki et al., p. 2317

Purpose: Tumor-infiltrating lymphocytes (TIL) are associated with a better prognosis in high-grade serous ovarian cancer (HGSC). However, it is largely unknown how this prognostic benefit of TIL relates to current standard treatment of surgical resection and (neo-)adjuvant chemotherapy. To address this outstanding issue, we compared TIL infiltration in a unique cohort of patients with advanced-stage HGSC primarily treated with either surgery or neoadjuvant chemotherapy. Experimental Design: Tissue microarray slides containing samples of 171 patients were analyzed for CD8+ TIL by IHC. Freshly isolated CD8+ TIL subsets were characterized by flow cytometry based on differentiation, activation, and exhaustion markers. Relevant T-cell subsets (CD27+) were validated using IHC and immunofluorescence. Results: A prognostic benefit for patients with high intratumoral CD8+ TIL was observed if primary surgery had resulted in a complete cytoreduction (no residual tissue). By contrast, optimal ( Conclusions: Our findings indicate that treatment regimen, surgical result, and the differentiation of TIL should all be taken into account when studying immune factors in HGSC or, by extension, selecting patients for immunotherapy trials. Clin Cancer Res; 22(3); 714–24. ©2015 AACR.

Summary Monobenzone is a pro-hapten that is exclusively metabolized by melanocytes, thereby haptenizing melanocyte-specific antigens, which results in cytotoxic autoimmunity specifically against pigmented cells. Studying monobenzone in a setting of contact hypersensitivity (CHS), we observed that monobenzone induced a long-lasting, melanocyte-specific immune response that was dependent on NK cells, yet fully intact in the absence of T- and B cells. Consistent with the concept of "memory NK cells," monobenzone-induced NK cells resided in the liver and transfer of these cells conferred melanocyte-specific immunity to naive animals. Monobenzone-exposed skin displayed macrophage infiltration and cutaneous lymph nodes showed an inflammasome-dependent influx of macrophages with a tissue-resident phenotype, coinciding with local NK cell activation. Indeed, macrophage depletion or the absence of the NLRP3 inflammasome, the adaptor protein ASC or interleukin-18 (IL-18) abolished monobenzone CHS, thereby establishing a non-redundant role for the NLRP3 inflammasome as a critical proinflammatory checkpoint in the induction of hapten-dependent memory NK cells.

The T cells of the immune system can destroy tumours, but their activation can be inefficient. Vaccines that exploit tumour mutations elicit robust T-cell responses to tumours, with potential clinical benefits. See Letters p.217 & p.222 Neoantigens have long been considered optimal targets for anti-tumour vaccines, and recent mutation coding and prediction techniques have aimed to streamline their identification and selection. Two papers in this issue report results from personalized neoantigen vaccine trials in patients with cancer. Catherine Wu and colleagues report the results of a phase I trial of a personalized cancer vaccine that targets up to 20 patient neoantigens. The vaccine was safe and induced tumour-antigen-specific immune responses. Four out of six patients treated showed no recurrence at 25 months, and progressing patients responded to further therapy with checkpoint inhibitor. Ugur Sahin and colleagues report the first-in-human application of a personalized neoantigen vaccine in patients with melanoma. Their vaccination strategy includes sequencing and computational identification of neoantigens from patients, and design and manufacture of a poly-antigen RNA vaccine for treatment. In 13 patients, the vaccine boosted immunity against some of the selected tumour antigens from the individual patients, and two patients showed infiltration of tumour-reactive T cells. These results suggest that personalized vaccines could be refined and tailored to provide clinical benefit as cancer immunotherapies.

Editorial summary Advances in mass spectrometry have allowed the high-throughput quantitative identification of human leukocyte antigen (HLA) class I ligands, and recent studies have reported on the breadth and diversity of the HLA class I immunopeptidome. These findings have far-reaching implications for immunosurveillance by T cells and translational value for immunotherapy.

BackgroundStress-induced post-translational modifications occur during autophagy and can result in generation of new epitopes and immune recognition. One such modification is the conversion of arginine to citrulline by peptidylarginine deiminase enzymes.MethodsWe used Human leukocyte antigen (HLA) transgenic mouse models to assess the immunogenicity of citrullinated peptide vaccine by cytokine Enzyme linked immunosorbant spot (ELISpot) assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma and ID8 ovarian models expressing either constitutive or interferon-gamma (IFNγ) inducible Major Histocompatibility Complex (MHC) class II (MHC-II) as represented by most human tumors. To determine the importance of CD4 T cells in tumor therapy, we analyzed the immune cell infiltrate into murine tumors using flow cytometry and performed therapy studies in the presence of CD4 and CD8 T cell depletion. We assessed the T cell repertoire to citrullinated peptides in ovarian cancer patients and healthy donors using flow cytometry.ResultsThe combination of citrullinated vimentin and enolase peptides (Modi-1) stimulated strong CD4 T cell responses in mice. Responses resulted in a potent anti-tumor therapy against established tumors and generated immunological memory which protected against tumor rechallenge. Depletion of CD4, but not CD8 T cells, abrogated the primary anti-tumor response as well as the memory response to tumor rechallenge. This was further reinforced by successful tumor regression being associated with an increase in tumor-infiltrating CD4 T cells and a reduction in tumor-associated myeloid suppressor cells. The anti-tumor response also relied on direct CD4 T cell recognition as only tumors expressing MHC-II were rejected. A comparison of different Toll-like receptor (TLR)-stimulating adjuvants showed that Modi-1 induced strong Th1 responses when combined with granulocyte-macrophage colony-stimulating factor (GMCSF), TLR9/TLR4, TLR9, TLR3, TLR1/2 and TLR7 agonists. Direct linkage of the TLR1/2 agonist to the peptides allowed the vaccine dose to be reduced by 10-fold to 100-fold without loss of anti-tumor activity. Furthermore, a CD4 Th1 response to the citrullinated peptides was seen in ovarian cancer patients.ConclusionsModi-1 citrullinated peptide vaccine induces potent CD4-mediated anti-tumor responses in mouse models and a CD4 T cell repertoire is present in ovarian cancer patients to the citrullinated peptides suggesting that Modi-1 could be an effective vaccine for ovarian cancer patients.

Integrins α1β1 (CD49a), α2β1 (CD49b) and αEβ7 (CD103) mediate retention of lymphocytes in peripheral tissues, and their expression is upregulated on tumor infiltrating lymphocytes (TIL) compared to circulating lymphocytes. Little is known about what induces expression of these retention integrins (RI) nor whether RI define subsets in the tumor microenvironment (TME) with a specific phenotype. Human metastatic melanoma-derived CD8 TIL could be grouped into five subpopulations based on RI expression patterns: RI(neg), CD49a(+) only, CD49a(+)CD49b(+), CD49a(+)CD103(+), or positive for all three RI. A significantly larger fraction of the CD49a(+) only subpopulation expressed multiple effector cytokines, whereas CD49a(+)CD103(+) and CD49a(+)CD49b(+) cells expressed IFNγ only. RI(neg) and CD49a(+)CD49b(+)CD103(+) CD8 TIL subsets expressed significantly less effector cytokines overall. Interestingly, however, CD49a(+)CD49b(+)CD103(+) CD8 expressed lowest CD127, and highest levels of perforin and exhaustion markers PD-1 and Tim3, suggesting selective exhaustion rather than conversion to memory. To gain insight into RI expression induction, normal donor PBMC were cultured with T cell receptor (TCR) stimulation and/or cytokines. TCR stimulation alone induced two RI(+) cell populations: CD49a single positive and CD49a(+)CD49b(+) cells. TNFα and IL-2 each were capable of inducing these populations. Addition of TGFβ to TCR stimulation generated two additional populations; CD49a(+)CD49b(neg)CD103(+) and CD49a(+)CD49b(+)CD103(+.) Taken together, our findings identify opportunities to modulate RI expression in the TME by cytokine therapies and to generate subsets with a specific RI repertoire in the interest of augmenting immune therapies for cancer or for modulating other immune-related diseases such as autoimmune diseases.

Background Ligands for the Toll-like receptor (TLR) family can induce activation of cells of the innate immune system and are widely studied for their potential to enhance adaptive immunity. Conjugation of TLR2-ligand Pam3CSK4 to synthetic long peptides (SLPs) was shown to strongly enhance the induction of antitumor immunity. To further improve cancer vaccination, we have previously shown that the novel TLR2-L Amplivant (AV), a modified Pam3CSK4, potentiates the maturation effects on murine DCs. In the current study, we further assessed the immunological properties of AV. Methods Naïve mice were vaccinated with a conjugate of either Pam3CSK4 or AV and an SLP to assess specific T cell priming efficiency in vivo. The potency of AV and Pam3CSK4, either as free compounds or conjugated to different SLPs, to mature murine DCs was compared by stimulating murine dendritic cells overnight followed by ELISA and flow cytometry analysis. Murine tumor experiments were carried out by vaccinating mice carrying established HPV16 E6 and E7-expressing tumors and subsequently analyzing myeloid and lymphoid cells infiltrating the tumor microenvironment. Furthermore, tumor outgrowth after vaccination was monitored to enable comparison of the efficiency to induce antitumor immunity by Pam3CSK-SLP and AV-SLP conjugates. To enhance therapeutic efficacy, AV-SLP conjugate vaccination was combined with ablative therapies to assess whether synergism between such therapies would occur. Results SLPs conjugated to AV induce stronger DC maturation, in vivo T cell priming and antitumor immunity compared to conjugates with Pam3CSK4. Interestingly, AV-SLP conjugates modulate the macrophage populations in the tumor microenvironment, correlating with a therapeutic effect in an aggressive murine tumor model. The potency of AV-SLP conjugates in cancer vaccination operates optimally in combination with chemotherapy or photodynamic therapy. Conclusion These data allow further optimization of vaccination-based immunotherapy of cancer by use of the improved TLR2-ligand Amplivant. Electronic supplementary material The online version of this article (10.1186/s40425-018-0455-2) contains supplementary material, which is available to authorized users.

Cancer immunotherapy aims to promote the activity of cytotoxic T lymphocytes (CTLs) within a tumour, assist the priming of tumour-specific CTLs in lymphoid organs and establish efficient and durable antitumour immunity. During priming, help signals are relayed from CD4

Enhancing T cell responses against both viral and tumor Ags requires efficient costimulation and directed delivery of peptide Ags into APCs. Long peptide vaccines are considered favorable vaccine moieties from a clinical perspective, as they can harbor more than one immunogenic epitope enabling treatment of a broader target population. In addition, longer peptides are not extracellularly loaded on MHC class I; rather, they require intracellular processing and will thereby be presented to T cells mainly by professional APCs, thereby avoiding the risk of tolerance induction. The drawback of peptide vaccines regardless of peptide length is that naked peptides are not actively targeted to and taken up by APCs, and the standard nonconjugated adjuvant-peptide mixtures do not ensure cotargeting of the two to the same APC. We have identified a tetanus toxin–derived B cell epitope that can mediate the formation of immune complexes in the presence of circulating Abs. In this study, we show that these immune complexes improve both Ag uptake by APCs (blood monocytes and CD1c+ dendritic cells) and consequently improve CD8+ T cell recall responses in a human ex vivo blood loop system. The uptake of the peptide conjugate by blood monocytes is dependent on Abs and the complement component C1q. We envision that this strategy can be used to facilitate active uptake of Ags into APCs to improve T cell responses against pathogens or cancer.

Vitiligo development in melanoma patients during immunotherapy is a favorable prognostic sign and indicates breakage of tolerance against melanocytic/melanoma antigens. We investigated a novel immunotherapeutic approach of the skin-depigmenting compound monobenzone synergizing with imiquimod in inducing antimelanoma immunity and melanoma regression. Stage III-IV melanoma patients with non-resectable cutaneous melanoma metastases were treated with monobenzone and imiquimod (MI) therapy applied locally to cutaneous metastases and adjacent skin during 12 weeks, or longer. Twenty-one of 25 enrolled patients were evaluable for clinical assessment at 12 weeks. MI therapy was well-tolerated. Partial regression of cutaneous metastases was observed in 8 patients and stable disease in 1 patient, reaching the statistical endpoint of treatment efficacy. Continued treatment induced clinical response in 11 patients, including complete responses in three patients. Seven patients developed vitiligo-like depigmentation on areas of skin that were not treated with MI therapy, indicating a systemic effect of MI therapy. Melanoma-specific antibody responses were induced in 7 of 17 patients tested and melanoma-specific CD8+T-cell responses in 11 of 15 patients tested. These systemic immune responses were significantly increased during therapy as compared to baseline in responding patients. This study shows that MI therapy induces local and systemic anti-melanoma immunity and local regression of cutaneous metastases in 38% of patients, or 52% during prolonged therapy. This study provides proof-of-concept of MI therapy, a low-cost, broadly applicable and well-tolerated treatment for cutaneous melanoma metastases, attractive for further clinical investigation.

Immune complexes are potent mediators of cellular immunity and have been extensively studied for their disease mediating properties in humans and for their role in anti-cancer immunity. However, a viable approach to use antibody-complexed antigen as vehicle for specific immunotherapy has not yet reached clinical use. Since virtually all people have endogenous antibodies against tetanus toxoid (TTd), such commonly occurring antibodies are promising candidates to utilize for immune modulation. As an initial proof-of-concept we investigated if anti-tetanus IgG could induce potent cross-presentation of a conjugate with SIINFEKL, a MHC class I presented epitope of ovalbumin (OVA), to TTd. This protein conjugate enhanced OVA-specific CD8+ T cell responses when administrated to seropositive mice. Since TTd is poorly defined, we next investigated whether a synthetic peptide-peptide conjugate, with a chemically defined linear B cell epitope of tetanus toxin (TTx) origin, could improve cellular immune responses. Herein we identify one linear B cell epitope, here after named MTTE thru a screening of overlapping peptides from the alpha and beta region of TTx, and by assessment of the binding of pooled IgG, or individual human IgG from high-titer TTd vaccinated donors, to these peptides. Subsequently, we developed a chemical protocol to synthesize defined conjugates containing multiple copies of MTTE covalently attached to one or more T cell epitopes of choice. To demonstrate the potential of the above approach we showed that immune complexes of anti-MTTE antibodies with MTTE-containing conjugates are able to induce DC and T cell activation using model antigens.

Prototype hepatitis B virus (HBV)-derived synthetic long peptide (SLP) cross-presented by autologous dendritic cells boosted HBV-specific (CD4,CD8) T-cell responses in chronic HBV (CHB) patients ex vivo. Often, PD-L1 blockade improved SLP-responses. This supports therapeutic SLPbased vaccine development for CHB treatment., Background Vaccination with synthetic long peptides (SLP) is a promising new treatment strategy for chronic hepatitis B virus (CHB). SLP can induce broad T-cell responses for all HLA types. Here we investigated the ability of a prototype HBV-core (HBc)-sequence-derived SLP to boost HBV-specific T cells in CHB patients ex vivo. Methods HBc-SLP was used to assess cross-presentation by monocyte-derived dendritic cells (moDC) and BDCA1+ blood myeloid DC (mDC) to engineered HBV-specific CD8+ T cells. Autologous SLP-loaded and toll-like receptor (TLR)-stimulated DC were used to activate patient HBc-specific CD8+ and CD4+ T cells. Results HBV-SLP was cross-presented by moDC, which was further enhanced by adjuvants. Patient-derived SLP-loaded moDC significantly increased autologous HBcAg18-27-specific CD8+ T cells and CD4+ T cells ex vivo. HBV-specific T cells were functional as they synthesized tumor necrosis factor-alpha and interferon-gamma. In 6/7 of patients blockade of PD-L1 further increased SLP effects. Also, importantly, patient-derived BDCA1+ mDC cross-presented and activated autologous T-cell responses ex vivo. Conclusions As a proof of concept, we showed a prototype HBc-SLP can boost T-cell responses in patients ex vivo. These results pave the way for the development of a therapeutic SLP-based vaccine to induce effective HBV-specific adaptive immune responses in CHB patients.

// Eveline M. Dijkgraaf 1 , Saskia J.A.M. Santegoets 1 , An K.L. Reyners 2 , Renske Goedemans 1 , Hans W. Nijman 3 , Mariette I.E. van Poelgeest 4 , Arien R. van Erkel 5 , Vincent T.H.B.M. Smit 6 , Toos A.H.H. Daemen 7 , Jacobus J.M. van der Hoeven 1 , Cornelis J.M. Melief 8 , Marij J.P. Welters 1 , Judith R. Kroep 1, * , Sjoerd H. van der Burg 1, * 1 Department of Medical Oncology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands 2 Department of Clinical Oncology, University Medical Center Groningen, University of Groningen, 9713 GZ, Groningen, The Netherlands 3 Department of Gynecologic Oncology, University Medical Center Groningen, University of Groningen, 9713 GZ, Groningen, The Netherlands 4 Department of Gynecology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands 5 Department of Radiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands 6 Department of Pathology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands 7 Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, 9713 GZ, Groningen, The Netherlands 8 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands * These authors have contributed equally to this work Correspondence to: Sjoerd H. van der Burg, e-mail: shvdburg@lumc.nl Judith R. Kroep, e-mail: j.r.kroep@lumc.nl Keywords: ovarian cancer, p53, immunotherapy, chemoresistance, tumor immunity Received: May 27, 2015 Accepted: August 03, 2015 Published: August 14, 2015 ABSTRACT Purpose : Preclinical tumor models show that chemotherapy has immune modulatory properties which can be exploited in the context of immunotherapy. The purpose of this study was to determine the feasibility and immunogenicity of combinations of such an immunomodulatory chemotherapeutic agent with immunotherapy, p53 synthetic long peptide (SLP) vaccine and Pegintron (IFN-α) in patients with platinum-resistant p53-positive epithelial ovarian cancer (EOC). Experimental design : This is a phase 1/2 trial in which patients sequential 6 cycles of gemcitabine (1000 mg/kg 2 iv; n = 3), gemcitabine with Pegintron before and after the first gemcitabine cycle (Pegintron 1 μg/kg sc; n = 6), and gemcitabine and Pegintron combined with p53 SLP vaccine (0.3 mg/peptide, 9 peptides; n = 6). At baseline, 22 days after the 2 nd and 6 th cycle, blood was collected for immunomonitoring. Toxicity, CA-125, and radiologic response were evaluated after 3 and 6 cycles of chemotherapy. Results : None of the patients enrolled experienced dose-limiting toxicity. Predominant grade 3/4 toxicities were nausea/vomiting and dyspnea. Grade 1/2 toxicities consisted of fatigue (78%) and Pegintron-related flu-like symptoms (72%). Gemcitabine reduced myeloid-derived suppressor cells ( p = 0.0005) and increased immune-supportive M1 macrophages ( p = 0.04). Combination of gemcitabine and Pegintron stimulated higher frequencies of circulating proliferating CD4+ and CD8+ T-cells but not regulatory T-cells. All vaccinated patients showed strong vaccine-induced p53-specific T-cell responses. Conclusion : Combination of gemcitabine, the immune modulator Pegintron and therapeutic peptide vaccination is a viable approach in the development of combined chemo-immunotherapeutic regimens to treat cancer.

The clinical benefit of therapeutic cancer vaccines has been established. Whereas regression of lesions was shown for premalignant lesions caused by HPV, clinical benefit in cancer patients was mostly noted as prolonged survival. Suboptimal vaccine design and an immunosuppressive cancer microenvironment are the root causes of the lack of cancer eradication. Effective cancer vaccines deliver concentrated antigen to both HLA class I and II molecules of DCs, promoting both CD4 and CD8 T cell responses. Optimal vaccine platforms include DNA and RNA vaccines and synthetic long peptides. Antigens of choice include mutant sequences, selected cancer testis antigens, and viral antigens. Drugs or physical treatments can mitigate the immunosuppressive cancer microenvironment and include chemotherapeutics, radiation, indoleamine 2,3-dioxygenase (IDO) inhibitors, inhibitors of T cell checkpoints, agonists of selected TNF receptor family members, and inhibitors of undesirable cytokines. The specificity of therapeutic vaccination combined with such immunomodulation offers an attractive avenue for the development of future cancer therapies.

Peptides have attracted much interest as a platform for the induction of therapeutic T cell responses, ever since the discovery of MHC-presented protein fragments (peptides) as the antigenic basis for T cell recognition. CD8+ cytotoxic T lymphocytes (Tc) recognize short peptides of 8–11 amino acids in length, presented by MHC class I molecules (HLA class I in human beings), expressed on all nucleated cell types. CD4+ T helper cells (Th) recognize the combination of slightly longer peptides of 12–15 amino acids in length in the context of HLA class II molecules, expressed mainly on cells of the immune system such as dendritic cells (DC), macrophages, and B cells. Since T cells recognize these antigen fragments (epitopes) only when expressed on cell surfaces by HLA molecules tethered to cell membranes, T cells are not distracted by free antigen in body fluids, but are specialized in the recognition and destruction of cells presenting abnormal (microbial, nonself) fragments by T cell receptors on T cells that have not been deleted in the thymus by central tolerance.

High-risk human papillomaviruses infect the basal cells of human epithelia. There it deploys several mechanisms to suppress pathogen receptor recognition signalling, impeding the immune system to control viral infection. Furthermore, infected cells become more resistant to type I and II interferon, tumour necrosis factor-α and CD40 activation, via interference with downstream programs halting viral replication or regulating the proliferation and cell death. Consequently, some infected individuals fail to raise early protein-specific T-cell responses that are strong enough to protect against virus-induced premalignant disease and ultimately cancer. Therapeutic vaccines triggering a strong T-cell response against the early proteins can successfully be used to treat patients at the premalignant stage but combinations of different treatment modalities are required for cancer therapy.