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Background The survival of patients with cervical cancer who are treated with cisplatin in conjunction with the topoisomerase I inhibitor topotecan is enhanced when compared with patients treated with only one of these chemotherapeutics. Moreover, cisplatin-based and T cell-based immunotherapy have been shown to synergize, resulting in stronger antitumor responses. Here, we interrogated whether topotecan could further enhance the synergy of cisplatin with T cell-based cancer immunotherapy.Methods Mice bearing human papilloma virus 16 (HPV16) E6/E7-expressing TC-1 tumors were vaccinated with HPV16 E7 long peptides and additionally received chemotherapy consisting of cisplatin and topotecan. We performed an in-depth study of this combinatorial chemoimmunotherapy on the effector function and expansion/contraction kinetics of vaccine-induced CD8+ T cells in the peripheral blood and tumor microenvironment (TME). In addition, we interrogated the particular role of chemotherapy-induced upregulation of costimulatory ligands by tumor-infiltrated myeloid cells on T cell proliferation and survival.Results We show that E7 long peptide vaccination combined with cisplatin and topotecan, results in CD8+ T cell-dependent durable rejection of established tumors and 94% long-term survival. Although topotecan initially repressed the expansion of vaccine-induced CD8+ T cells, these cells eventually expanded vigorously, which was followed by delayed contraction. These effects associated with the induction of the proliferation marker Ki-67 and the antiapoptosis molecule Bcl-2 by intratumoral tumor-specific CD8+ T cells, which was regulated by topotecan-mediated upregulation of the costimulatory ligand CD70 on myeloid cells in the TME.Conclusions Taken together, our data show that although treatment with cisplatin, topotecan and vaccination initially delays T cell expansion, this combinatorial therapy results eventually in a more robust T cell-mediated tumor eradication due to enhancement of costimulatory molecules in the TME.

Background Amplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long peptides (SLPs) strongly enhanced antigen presentation by dendritic cells, T cell priming and induction of effective antitumor responses. The current study is a first-in-human trial to investigate safety and immunogenicity of amplivant conjugated to human papillomavirus (HPV) 16-SLP.Methods A dose escalation phase I vaccination trial was performed in 25 patients treated for HPV16 positive (pre-)malignant lesions. Amplivant was conjugated to two SLPs derived from the two most immunodominant regions of the HPV16 E6 oncoprotein. The vaccine, containing a mix of these two conjugates in watery solution without any other formulation, was injected intradermally three times with a 3-week interval in four dose groups (1, 5, 20 or 50 µg per conjugated peptide). Safety data were collected during the study. Peptide-specific T cell immune responses were determined in blood samples taken before, during and after vaccination using complementary immunological assays.Results Toxicity after three amplivant-conjugated HPV16-SLP vaccinations was limited to grade 1 or 2, observed as predominantly mild skin inflammation at the vaccination site and sometimes mild flu-like symptoms. Adverse events varied from none in the lowest dose group to mild/moderate vaccine-related inflammation in all patients and flu-like symptoms in three out of seven patients in the highest dose group, after at least one injection. In the lowest dose group, vaccine-induced T cell responses were observed in the blood of three out of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T cell response after vaccination. These HPV16-specific T cell responses lasted until the end of the trial.Conclusions Amplivant-conjugated SLPs can safely be used as an intradermal therapeutic vaccine to induce robust HPV16-specific T cell immunity in patients previously treated for HPV16 positive (pre-) malignancies. Increased vaccine dose was associated with a higher number of mild adverse events and with stronger systemic T cell immunity.Trial registration numbers NCT02821494 and 2014-000658-12.

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.

Background 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-16+ cancer. Here we report long-term clinical outcomes and immune correlates of response.Methods Patients with advanced HPV-16+ cancer and less than two prior regimens for recurrence were enrolled to receive ISA101 (100 µg/peptide) on days 1, 22, and 50 and nivolumab 3 mg/kg every 2 weeks beginning day 8 for up to 1 year. Baseline tumor samples were stained with multiplex immunofluorescence for programmed death-ligand 1 (PD-L1), programmed cell death protein-1 (PD-1), CD3, CD8, CD68, and pan-cytokeratin in a single panel and scanned with the Vectra 3.0 multispectral microscope. Whole transcriptome analysis of baseline tumors was performed with Affymetrix Clariom D arrays. Differential gene expression analysis was performed on responders versus non-responders.Results Twenty-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 ((CD3+PD-1+)+(CD3+CD8+PD-1+)), activated cytotoxic T cells (CD3+CD8+PD-1+), and total macrophage ((CD68+PD-L1−)+(CD68+PD-L1+)) in tumor were directly correlated with clinical response (p

Background Stress-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.Methods We 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.Results The 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.Conclusions Modi-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.

Abstract Background Chemoradiotherapy (CRT) remains one of the most common cancer treatment modalities, and recent data suggest that CRT is maximally effective when there is generation of an anti-tumoral immune response. However, CRT has also been shown to promote immunosuppressive mechanisms which must be blocked or reversed to maximize its immune stimulating effects. Methods Therefore, using a preclinical model of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), we developed a clinically relevant therapy combining CRT and two existing immunomodulatory drugs: cyclophosphamide (CTX) and the small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). In this model, we treated the syngeneic HPV-HNSCC mEER tumor-bearing mice with fractionated (10 fractions of 3 Gy) tumor-directed radiation and weekly cisplatin administration. We compared the immune responses induced by CRT and those induced by combinatory treatment (CRT + CTX/L-NIL) with flow cytometry, quantitative multiplex immunofluorescence and by profiling immune-related gene expression changes. Results We show that combination treatment favorably remodels the tumor myeloid immune microenvironment including an increase in anti-tumor immune cell types (inflammatory monocytes and M1-like macrophages) and a decrease in immunosuppressive granulocytic myeloid-derived suppressor cells (MDSCs). Intratumoral T cell infiltration and tumor antigen specificity of T cells were also improved, including a 31.8-fold increase in the CD8+ T cell/ regulatory T cell ratio and a significant increase in tumor antigen-specific CD8+ T cells compared to CRT alone. CTX/LNIL immunomodulation was also shown to significantly improve CRT efficacy, leading to rejection of 21% established tumors in a CD8-dependent manner. Conclusions Overall, these data show that modulation of the tumor immune microenvironment with CTX/L-NIL enhances susceptibility of treatment-refractory tumors to CRT. The combination of tumor immune microenvironment modulation with CRT constitutes a translationally relevant approach to enhance CRT efficacy through enhanced immune activation.

Abstract 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.