Synthetic Long Peptide Vaccine Research Articles

Abstract 1387: High-dimensional analysis of T cell responses to mutant neoantigens and nonmutant antigens in mouse models of melanoma

Abstract Effective cancer immunotherapy, such as immune checkpoint therapy (ICT) [e.g., anti-CTLA-4 and anti-PD-1/PD-L1], is dependent on T cell recognition of tumor antigens presented on major histocompatibility complex (MHC) and leads to durable responses in certain cancer patients. Recent advances have facilitated identification of tumor mutant neoantigens and led to efforts to develop effective personalized neoantigen cancer vaccines. However, little is known about how expression of multiple neoantigens with variable peptide-MHC (pMHC) binding or TCR affinity/avidity affects T cell responses to both discrete neoantigens and shared non-mutant antigens, and if the localization of T cells with distinctive pMHC specificity differs within the tumor. To address this, we genetically introduced multiple combinations of MHC-I and MHC-II neoantigens in the poorly immunogenic Brafv600e Pten-/- Cdkn2a-/- YUMM1.7 (Y1.7) mouse melanoma line that contains a paucity of neoantigens. We expressed neoantigens previously found in a mouse sarcoma line of the same MHC haplotype as Y1.7. The MHC-I neoantigens used were formed by G1254V mutation in Laminin alpha subunit-4 (Lama4) and A506T mutation in the Asparagine-linked glycosylation 8 (Alg8) and the MHC-II neoantigen was formed by A710T mutation in Integrin beta-1 (Itgb1). We generated Y1.7 lines expressing minigenes for mLama4, mAlg8, or mLama4 + mAlg8 MHC-I neoantigens. All three lines also expressed the mItgb1 MHC-II neoantigen. Whereas ICT-treated mice bearing the parental Y1.7 melanoma line showed rapid tumor growth, modified Y1.7 lines expressing mLama4 and/or mAlg8 plus mItgb1 were rendered sensitive to ICT. Tumors expressing mLama4 and/or mAlg8 contained mLama4- and/or mAlg8-specific CD8+ T cells, respectively. Preliminary analysis revealed expression of either MHC-I neoantigen alters the frequency of CD8+ T cells specific for non-mutant endogenous antigens in Y1.7. Interestingly, therapeutic synthetic long-peptide (SLP) vaccines incorporating mLama4 and/or mAlg8 neoantigens plus poly I:C induced tumor rejection comparable to ICT. Moreover, SLP vaccines amplified intratumoral mLama4- and mAlg8-specific CD8+ T cells, as well as in tumor-draining lymph nodes, where these neoantigen-specific CD8+ T cells were found to express IFN-γ. During either vaccination or ICT, CD8+ T cell response to mLama4 was higher than that to mAlg8 when both neoantigens were co-expressed. Altogether, these melanoma models will enable us to determine whether expression of one or more MHC-I neoantigens influences CD8+ T cell responses to both distinct neoantigens and non-mutant antigens (e.g., gp100, Trp2) during ICT and SLP vaccination. Furthermore, high-dimensional analyses approaches including scRNA-seq. and CODEX imaging are ongoing and will facilitate development of improved immunotherapies and may help uncover novel biomarkers of response. Citation Format: Sunita Keshari, Charmelle D. Williams, Elizabeth D. Sanchez, Alexander S. Shavkunov, Matthew M. Gubin. High-dimensional analysis of T cell responses to mutant neoantigens and nonmutant antigens in mouse models of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1387.

Cross-presentation of a TAP-independent signal peptide induces CD8 T immunity to escaped cancers but necessitates anchor replacement

Cancer cells frequently display defects in their antigen-processing pathway and thereby evade CD8 T cell immunity. We described a novel category of cancer antigens, named TEIPP, that emerge on cancers with functional loss of the peptide pump TAP. TEIPPs are non-mutated neoantigens despite their ‘self’ origin by virtue of their absence on normal tissues. Here, we describe the development of a synthetic long peptide (SLP) vaccine for the most immunogenic TEIPP antigen identified thus far, derived from the TAP-independent LRPAP1 signal sequence. LRPAP121–30-specific CD8 T cells were present in blood of all tested healthy donors as well as patients with non-small cell lung adenocarcinoma. SLPs with natural flanking, however, failed to be cross-presented by monocyte-derived dendritic cells. Since the C-terminus of LRPAP121–30 is an unconventional and weakly binding serine (S), we investigated if replacement of this anchor would result in efficient cross-presentation. Exchange into a valine (V) resulted in higher HLA-A2 binding affinity and enhanced T cell stimulation. Importantly, CD8 T cells isolated using the V-variant were able to bind tetramers with the natural S-variant and respond to TAP-deficient cancer cells. A functional screen with an array of N-terminal and C-terminal extended SLPs pointed at the 24-mer V-SLP, elongated at the N-terminus, as most optimal vaccine candidate. This SLP was efficiently cross-presented and consistently induced a strong polyclonal LRPAP121–30-specific CD8 T cells from the endogenous T cell repertoire. Thus, we designed a TEIPP SLP vaccine from the LRPAP1 signal sequence ready for validation in clinical trials.

Abstract 1582: A pre-existing inflammatory immune microenvironment predicts the clinical and immunological response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 peptide vaccination

Abstract Background: Vulvar High-grade Squamous Intraepithelial Lesion (vHSIL) is predominantly induced by high-risk Human Papilloma Virus type 16 (HPV16). In two independent trials, therapeutic vaccination against the oncoproteins of HPV16 with synthetic long peptides (SLP) resulted in vHSIL regression in about half of the patients after 12 months. Several studies have shown that the immune microenvironment influences therapy outcome. Therefore, a thorough investigation of the vHSIL immune microenvironment before and after SLP vaccination was performed, and its impact on clinical response was studied. Methods: Two novel multiplex immunofluorescence panels were designed and fully optimized for formalin-fixed paraffin-embedded tissue, one for T cells (CD3, CD8, Foxp3, Tim3, Tbet, PD-1, DAPI) and one for myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI). Pre- and post-vaccination biopsies of 29 vHSIL patients, as well as 27 healthy vulva excisions, were stained, scanned with the Vectra multispectral imaging system, and automatically phenotyped and counted with inForm advanced image analysis software. Results: A pre-existing pro-inflammatory tumor microenvironment, marked by high numbers of CD4+ and CD8+ T cells expressing Tbet and/or PD-1 as well as CD14+ inflammatory macrophages, is a strong predictor for good clinical response to therapeutic HPV16 SLP vaccination. A clear stepwise increase in pre-vaccination infiltrating Tbet+, CD4+, CD8+ T cells and CD14+ macrophages, and decrease in Foxp3+ regulatory T cells was observed as response increased from non to partial to complete response. Moreover, the pre-vaccination immune microenvironment of complete responders resembled healthy vulva. Vaccination further increased infiltrating CD4+ and Tbet+ T cells and CD14+ macrophages, and decreased Foxp3+ regulatory T cells and CD68+CD163+ M2 macrophages in the complete and partial responders, but not in the non responders. Conclusion: Clinical responsiveness to therapeutic HPV16 SLP vaccination requires a pre-existing inflamed type 1 immune contexture in vHSIL. Hence, only patients with an inflamed microenvironment should be selected for monotherapy by therapeutic peptide vaccination, since this strategy is incapable of overcoming an immunologically cold tumor microenvironment. Citation Format: Ziena Abdulrahman, Noel F. de Miranda, Mariette I. van Poelgeest, Sjoerd H. van der Burg. A pre-existing inflammatory immune microenvironment predicts the clinical and immunological response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 peptide vaccination [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1582.

3O A pre-existing inflammatory immune microenvironment predicts the clinical and immunological response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 peptide vaccination

Abstract Background Vulvar High-grade Squamous Intraepithelial Lesion (vHSIL) is predominantly induced by high-risk Human Papilloma Virus type 16 (HPV16). In two independent trials, therapeutic vaccination against the oncoproteins of HPV16 with synthetic long peptides (SLP) resulted in vHSIL regression in about half of the patients after 12 months. Several studies have shown that the immune microenvironment influences therapy outcome. Therefore, a thorough investigation of the vHSIL immune microenvironment before and after SLP vaccination was performed, and its impact on clinical response was studied. Methods Two novel multiplex immunofluorescence panels were designed for formalin-fixed paraffin-embedded tissue, one for T cells (CD3, CD8, FoxP3, Tim3, Tbet, PD-1, DAPI) and one for myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI). Pre- and 3 months post-vaccination biopsies of 29 patients and 27 healthy vulva excisions were stained, scanned with the Vectra multispectral imaging system, and automatically phenotyped and counted with inForm advanced image analysis software. Results A pre-existing pro-inflammatory TME, marked by high numbers of CD4 and CD8 T cells expressing Tbet and/or PD-1 as well as CD14+ inflammatory macrophages, is a strong predictor for good clinical response. A clear stepwise increase in pre-vaccination infiltrating Tbet+, CD4+, CD8+ T cells and CD14+ macrophages, and decrease in Foxp3+ Tregs was observed as response increased from non to partial to complete response. Moreover, the pre-vaccination immune microenvironment of complete responders resembled healthy vulva. Vaccination further increased infiltrating CD4+ and Tbet+ T cells and CD14+ macrophages and decreased FoxP3+ Tregs in the complete and partial responders, but not in the non responders. Conclusion Clinical responsiveness to therapeutic HPV16 SLP vaccination requires a pre-existing inflamed type 1 immune contexture in vHSIL. Hence, only patients with an inflamed TME should be selected for monotherapy by therapeutic vaccination, since this strategy is incapable of creating an inflamed TME in patients where this is absent. Legal entity responsible for the study The authors. Funding Leiden University Medical Center. Disclosure S.H. van der Burg: Advisory / Consultancy: ISA Pharmaceuticals. All other authors have declared no conflicts of interest.

Combining Immune Checkpoint Blockade and Tumor-Specific Vaccine for Patients With Incurable Human Papillomavirus 16–Related Cancer

ImportanceIn 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.ObjectiveTo 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 ParticipantsIn 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.InterventionsThe 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 MeasuresAssessment of efficacy reflected in the overall response rate (per Response Evaluation Criteria in Solid Tumors, version 1.1).ResultsOf 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 RelevanceThe 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 RegistrationClinicalTrials.gov identifier: NCT02426892

Development of a novel immunoproteasome digestion assay for synthetic long peptide vaccine design.

Recently, many autologous tumor antigens have been examined for their potential use in cancer immunotherapy. However, the success of cancer vaccines in clinical trials has been limited, partly because of the limitations of using single, short peptides in most attempts. With this in mind, we aimed to develop multivalent synthetic long peptide (SLP) vaccines containing multiple cytotoxic T-lymphocyte (CTL) epitopes. However, to confirm whether a multivalent vaccine can induce an individual epitope-specific CTL, the only viable screening strategies currently available are interferon-gamma (IFN-μ enzyme-linked immunospot (ELISPOT) assays using human peripheral blood mononuclear cells, or expensive human leukocyte antigen (HLA)-expressing mice. In this report, we evaluated the use of our developed murine-20S immunoproteasome (i20S) digestion assay, and found that it could predict the results of IFN-μ ELISPOT assays. Importantly, the murine-i20S digestion assay not only predicted CTL induction, but also antitumor activity in an HLA-expressing mouse model. We conclude that the murine-i20S digestion assay is an extremely useful tool for the development of “all functional” multivalent SLP vaccines.

Preclinical development of peptide vaccination combined with oncolytic MG1-E6E7 for HPV-associated cancer

Human papilloma virus (HPV)-associated cancer is a significant global health burden and despite the presence of viral transforming antigens within neoplastic cells, therapeutic vaccinations are ineffective for advanced disease. HPV positive TC1 cells are susceptible to viral oncolysis by MG1-E6E7, a custom designed oncolytic Maraba virus. Epitope mapping of mice vaccinated with MG1-E6E7 enabled the rational design of synthetic long peptide (SLP) vaccines against HPV16 and HPV18 antigens. SLPs were able to induce specific CD8+ immune responses and the magnitude of these responses significantly increased when boosted by MG1-E6E7. Logically designed vaccination induced multi-functional CD8+ T cells and provided complete sterilising immunity of mice challenged with TC1 cells. In mice bearing large HPV-positive tumours, SLP vaccination combined with MG1-E6E7 was able to clear tumours in 60% of mice and these mice were completely protected against a long term aggressive re-challenge with the TC1 tumour model. Combining conventional SLPs with the multi-functional oncolytic MG1-E6E7 represents a promising approach against advanced HPV positive neoplasia.

Efficient Eradication of Established Tumors in Mice with Cationic Liposome-Based Synthetic Long-Peptide Vaccines.

Therapeutic vaccination with synthetic long peptides (SLP) can be clinically effective against HPV-induced premalignant lesions; however, their efficiency in established malignant lesions leaves room for improvement. Here, we report the high therapeutic potency of cationic liposomes loaded with well-defined tumor-specific SLPs and a TLR3 ligand as adjuvant. The cationic particles, with an average size of 160 nm, could strongly activate functional, antigen-specific CD8+ and CD4+ T cells and induced in vivo cytotoxicity against target cells after intradermal vaccination. At a low dose (1 nmol) of SLP, our liposomal formulations significantly controlled tumor outgrowth in two independent models (melanoma and HPV-induced tumors) and even cured 75%-100% of mice of their large established tumors. Cured mice were fully protected from a second challenge with an otherwise lethal dose of tumor cells, indicating the potential of liposomal SLP in the formulation of powerful vaccines for cancer immunotherapy. Cancer Immunol Res; 5(3); 222-33. ©2017 AACR.

Enforced OX40 Stimulation Empowers Booster Vaccines to Induce Effective CD4+ and CD8+ T Cell Responses against Mouse Cytomegalovirus Infection.

There is an imperative need for effective preventive vaccines against human cytomegalovirus as it poses a significant threat to the immunologically immature, causing congenital disease, and to the immune compromised including transplant recipients. In this study, we examined the efficacy of synthetic long peptides (SLPs) as a CD4+ and CD8+ T cell-eliciting preventive vaccine approach against mouse CMV (MCMV) infection. In addition, the use of agonistic OX40 antibodies to enhance vaccine efficacy was explored. Immunocompetent C57BL/6 mice were vaccinated in a prime-boost vaccination regiment with SLPs comprising various MHC class I- and II-restricted peptide epitopes of MCMV-encoded antigens. Enforced OX40 stimulation resulted in superior MCMV-specific CD4+ as CD8+ T cell responses when applied during booster SLP vaccination. Vaccination with a mixture of SLPs containing MHC class II epitopes and OX40 agonistic antibodies resulted in a moderate reduction of the viral titers after challenge with lytic MCMV infection. Markedly, the combination of SLP vaccines containing both MHC class I and II epitopes plus OX40 activation during booster vaccination resulted in polyfunctional (i.e., IFN-γ+, TNF+, IL-2+) CD4+ and CD8+ T cell responses that were even higher in magnitude when compared to those induced by the virus, and this resulted in the best containment of virus dissemination. Our results show that the induction of strong T cell responses can be a fundamental component in the design of vaccines against persistent viral infections.

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.

Combination of Photodynamic Therapy and Specific Immunotherapy Efficiently Eradicates Established Tumors.

The efficacy of immunotherapy against advanced cancer may be improved by combination strategies. Photodynamic therapy (PDT) is a local tumor ablation method based on localized activation of a photosensitizer, leading to oxygen radical-induced tumor cell death. PDT can enhance antitumor immune responses by release of antigen and danger signals, supporting combination protocols of PDT with immunotherapy. We investigated the local and systemic immune effects of PDT after treatment of established tumors. In two independent aggressive mouse tumor models, TC-1 and RMA, we combined PDT with therapeutic vaccination using synthetic long peptides (SLP) containing epitopes from tumor antigens. PDT of established tumors using the photosensitizer Bremachlorin resulted in significant delay of tumor outgrowth. Combination treatment of PDT with therapeutic SLP vaccination cured one third of mice. Importantly, all cured mice were fully protected against subsequent tumor rechallenge, and combination treatment of primary tumors led to eradication of distant secondary tumors, indicating the induction of a systemic antitumor immune response. Indeed, PDT by itself induced a significant CD8(+) T-cell response against the tumor, which was increased when combined with SLP vaccination and essential for the therapeutic effect of combination therapy. We show that immunotherapy can be efficiently combined with PDT to eradicate established tumors, based on strong local tumor ablation and the induction of a robust systemic immune response. These results suggest combination of active immunotherapy with tumor ablation by PDT as a feasible novel treatment strategy for advanced cancer.

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

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.

A phase 1/2 study combining gemcitabine, Pegintron and p53 SLP vaccine in patients with platinum-resistant ovarian cancer

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). This is a phase 1/2 trial in which patients sequential 6 cycles of gemcitabine (1000 mg/kg2 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 2nd and 6th cycle, blood was collected for immunomonitoring. Toxicity, CA-125, and radiologic response were evaluated after 3 and 6 cycles of chemotherapy. 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. 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.

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

Abstract 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

Vaccine-induced tumor necrosis factor-producing T cells synergize with cisplatin to promote tumor cell death.

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. 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. 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. 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.

Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

The immune system plays key roles in determining the fate of developing cancers by not only functioning as a tumour promoter facilitating cellular transformation, promoting tumour growth and sculpting tumour cell immunogenicity1–6, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion1,2,7. Yet clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer induced immunosuppression. In many individuals, immunosuppression is mediated by Cytotoxic T-Lymphocyte Associated Antigen-4 (CTLA-4) and Programmed Death-1 (PD-1), two immunomodulatory receptors expressed on T cells8,9. Monoclonal antibody (mAb) based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies10–13. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Herein, we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T cell rejection antigens following αPD-1 and/or αCTLA-4 therapy of mice bearing progressively growing sarcomas and show that therapeutic synthetic long peptide (SLP) vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Whereas, mutant tumour antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with αPD-1- and/or αCTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens (TSMA) are not only important targets of checkpoint blockade therapy but also can be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

Cationic liposomes loaded with a synthetic long peptide and poly(I:C): a defined adjuvanted vaccine for induction of antigen-specific T cell cytotoxicity.

For effective cancer immunotherapy by vaccination, co-delivery of tumour antigens and adjuvants to dendritic cells and subsequent activation of antigen-specific cytotoxic T cells (CTLs) is crucial. In this study, a synthetic long peptide (SLP) harbouring the model CTL epitope SIINFEKL was encapsulated with the TLR3 ligand poly(inosinic-polycytidylic acid) (poly(I:C)) in cationic liposomes consisting of DOTAP and DOPC. The obtained particles were down-sized to about 140 nm (measured by dynamic light scattering) and had a positive zeta-potential of about 26 mV (according to laser Doppler electrophoresis). SLP loading efficiency was about 40% as determined by HPLC. Poly(I:C) loading efficiency was about 50%, as assessed from the fluorescence intensity of fluorescently labelled poly(I:C). Immunogenicity of the liposomal SLP vaccine was evaluated in vitro by its capacity to activate dendritic cells (DCs) and present the processed SLP to SIINFEKL-specific T cells. The effectiveness of the vaccine to activate CD8(+) T cells was analysed in vivo after intradermal and subcutaneous immunisation in mice, by measuring antigen-specific T cells in blood and spleens and assessing their functionality by cytokine production and in vivo cytotoxicity. The liposomal formulation efficiently delivered the SLP to DCs in vitro and induced a functional CD8(+) T cell immune response in vivo to the CTL epitope present in the SLP. The SLP-specific CD8(+) T cell frequency induced by the poly(I:C)-adjuvanted liposomal SLP formulation showed an at least 25 fold increase over the T cell frequency induced by the poly(I:C)-adjuvanted soluble SLP. In conclusion, cationic liposomes loaded with SLP and poly(I:C) have potential as a powerful therapeutic cancer vaccine formulation.

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.

Cancer/testis antigen HCA587-derived long peptide vaccine generates potent immunologic responses and antitumor effects in mouse model.

The cancer/testis antigen HCA587 (also known as MAGE-C2), one of the most immunogenic tumor antigens, is overexpressed in a wide spectrum of malignant tumors and can serve as a target for immunotherapy. In this study, we synthesized 14 overlapping (25-35 amino acids) long peptides representing the sequence of the most immunogenic part of the HCA587 protein and evaluated the antigen-specific immune responses and antitumor effects generated by immunization with the synthetic long peptide (SLP) vaccine in a mouse model. HCA587 SLPs in combination with adjuvants CFA and CpG ODN induced potent T-cell responses, which were dominated by type 1 cytokine IFN-γ-producing CD4(+) T cells as measured by ELISPOT and intracellular cytokine staining assay. Moreover, HCA587 SLP vaccination conferred protection against challenge with HCA587-expressing B16 melanoma in a therapeutic setting. Our findings may provide a scientific basis for the use of HCA587-derived long overlapping peptide vaccine for the treatment of patients with cancer in future clinical trials.

A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions

The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping long-peptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16+ high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre- and post-vaccination by ELISPOT, proliferation assay and cytokine assays in PBMC and HSIL-infiltrating lymphocytes, and delayed-type hypersensitivity skin tests. Motivational problems of this patient group to postpone treatment of their premalignant lesions affected the inclusion rates and caused the study to stop prematurely. Of the accrued patients, 4 received a placebo and 5 received 1–2 vaccinations. Side effects mainly were flu-like symptoms and injection site reactions. A strong HPV-specific IFNγ-associated T-cell response was detected by ELISPOT in all vaccinated patients. The outcome of the skin tests correlated well with the ELISPOT analysis. The cytokine profile associated with HPV16-specific proliferation varied from robust type 1 to dominant type 2 responses. No conclusions could be drawn on vaccine-enhanced T-cell infiltration of the lesion, and there was no HPV clearance at the time of LEEP excision. Thus, vaccination of HSIL patients results in increased HPV16-specific T-cell immunity. Further development of this type of treatment relies on the ability to motivate patients and in the reduction in the side effects.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-012-1292-7) contains supplementary material, which is available to authorized users.