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Photochemical internalization (PCI) is a technology for inducing release of endocytosed antigens into the cell cytosol a light-induced process. Preclinical experiments have shown that PCI improves MHC class I antigen presentation, resulting in strongly enhanced CD8+ T-cell responses to polypeptide antigens. In PCI vaccination a mixture of the photosensitizing compound fimaporfin, vaccine antigens, and an adjuvant is administered intradermally followed by illumination of the vaccination site. This work describes an open label, phase I study in healthy volunteers, to assess the safety, tolerability, and immune response to PCI vaccination in combination with the adjuvant poly-ICLC (Hiltonol) (ClinicalTrials.gov Identifier: NCT02947854). The primary objective of the study was to assess the safety and local tolerance of PCI mediated vaccination, and to identify a safe fimaporfin dose for later clinical studies. A secondary objective was to analyze the immunological responses to the vaccination. Each subject received 3 doses of HPV16 E7 peptide antigens and two doses of Keyhole Limpet Hemocyanin (KLH) protein. A control group received Hiltonol and vaccine antigens only, whereas the PCI groups in addition received fimaporfin + light. Local and systemic adverse effects were assessed by standard criteria, and cellular and humoral immune responses were analyzed by ELISpot, flow cytometry, and ELISA assays. 96 healthy volunteers were vaccinated with fimaporfin doses of 0.75-50 µg. Doses below 17.5 µg were safe and tolerable, higher doses exhibited local tolerability issues in some study subjects, mainly erythema, and pain during illumination. There were few, and only mild and expected systemic adverse events. The employment of PCI increased the number of subjects exhibiting a T-cell response to the HPV peptide vaccine about 10-fold over what was achieved with the antigen/Hiltonol combination without PCI. Moreover, the use of PCI seemed to result in a more consistent and multifunctional CD8+ T-cell response. An enhancement of the humoral immune response to KLH vaccination was also observed. Using PCI in combination with Hiltonol for intradermal vaccination is safe at fimaporfin doses below 17.5 µg, and gives encouraging immune responses to peptide and protein based vaccination.

BackgroundThe presence of T cells and suppressive myeloid cells in epithelial ovarian cancer (EOC) correlate with good and bad clinical outcome, respectively. This suggests that EOC may be sensitive to adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL), provided that immunosuppression by myeloid-derived suppressor cells and M2 macrophages is reduced. Platinum-based chemotherapy can alleviate such immunosuppression, potentially creating a window of opportunity for T cell-based immunotherapy.MethodsWe initiated a phase I/II trial (NCT04072263) in patients with recurrent platinum-sensitive EOC receiving TIL during platinum-based chemotherapy. TILs were administered 2 weeks after the second, third and fourth chemotherapy course. Patients were treated in two cohorts with or without interferon-α (IFNa), as conditioning and TIL support regimen. The primary endpoint was to evaluate the feasibility and safety according to CTCAE V.4.03 criteria and the clinical response and immune modulatory effects of this treatment were evaluated as secondary endpoints.ResultsSixteen patients were enrolled. TIL could be successfully expanded for all patients. TIL treatment during chemotherapy without IFNa (n=13) was safe but the combination with IFNa added to the chemotherapy-induced toxicity with 2 out of 3 patients developing thrombocytopenia as dose-limiting toxicity. Fourteen patients completed treatment with a full TIL cycle and were further evaluated for clinical and immunological response. Platinum-based chemotherapy resulted in reduction of circulating myeloid cell numbers and IL-6 plasma levels, confirming its immunosuppression-alleviating effect. Three complete (CR), nine partial responses and two stable diseases were recorded, resulting in an objective response rate of 86% (Response Evaluation Criteria In Solid Tumors V.1.1). Interestingly, progression free survival that exceeded the previous platinum-free interval was detected in two patients, including an exceptionally long and ongoing CR in one patient that coincided with sustained alleviation of immune suppression.ConclusionTIL therapy can be safely combined with platinum-based chemotherapy but not in combination with IFNa. The chemotherapy-mediated reduction in immunosuppression and the increase in platinum-free interval for two patients warrants further exploration of properly-timed TIL infusions during platinum-based chemotherapy, possibly further benefiting from IL-2 support, as a novel treatment option for EOC patients.

BackgroundExpression of killer cell lectin-like receptor B1 (KLRB1), the gene encoding the cell surface molecule CD161, is associated with favorable prognosis in many cancers. CD161 is expressed by several lymphocyte populations, but its role and regulation on tumor-specific CD4+ T cells is unknown.MethodsWe examined the clinical impact of CD4+CD161+ T cells in human papillomavirus (HPV)16+ oropharyngeal squamous cell carcinoma (OPSCC), analyzed their contribution in a cohort of therapeutically vaccinated patients and used HPV16-specific CD4+CD161+ tumor-infiltrating lymphocytes and T cell clones for in-depth mechanistic studies.ResultsCentral and effector memory CD4+ T cells express CD161, but only CD4+CD161+ effector memory T cells (Tem) are associated with improved survival in OPSCC. Therapeutic vaccination activates and expands type 1 cytokine-producing CD4+CD161+ effector T cells. The expression of CD161 is dynamic and follows a pattern opposite of the checkpoint molecules PD1 and CD39. CD161 did not function as an immune checkpoint molecule as demonstrated using multiple experimental approaches using antibodies to block CD161 and gene editing to knockout CD161 expression. Single-cell transcriptomics revealed KLRB1 expression in many T cell clusters suggesting differences in their activation. Indeed, CD4+CD161+ effector cells specifically expressed the transcriptional transactivator SOX4, known to enhance T cell receptor (TCR) signaling via CD3ε. Consistent with this observation, CD4+CD161+ cells respond more vigorously to limiting amounts of cognate antigen in presence of interleukin (IL)-12 and IL-18 compared to their CD161- counterparts. The expression of CD161/KLRB1 and SOX4 was downregulated upon TCR stimulation and this effect was boosted by transforming growth factor (TGF)β1.ConclusionHigh levels of CD4+CD161+ Tem are associated with improved survival and our data show that CD161 is dynamically regulated by cell intrinsic and extrinsic factors. CD161 expressing CD4+ T cells rapidly respond to suboptimal antigen stimulation suggesting that CD161, similar to SOX4, is involved in the amplification of TCR signals in CD4+ T cells.

BackgroundAmplivant 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.MethodsA 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.ResultsToxicity 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.ConclusionsAmplivant-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 numbersNCT02821494 and 2014-000658-12.

BackgroundIn melanoma, cancer germline antigen (CGA)-directed vaccination has shown to induce objective clinical responses accompanied by strong anti-tumor immune responses.1 As CGAs are immunogenic and highly expressed by hepatocellular carcinoma (HCC) tumor cells, these have demonstrated to be attractive targets to be implemented in therapeutic anti-liver cancer vaccination as well.2Synthetic long peptide (SLP) vaccination has proven to elicit efficient anti-tumor CD4+ and CD8+ T cell responses and to have promising clinical effects.3 We aimed to develop an SLP vaccine targeting HCC-restricted CGA-epitopes covering at least five different HLA super types that are highly prevalent globally.MethodsWe applied an integrative pre-clinical approach of in silico epitope prediction, immunopeptidomics, and in vitro tools to select GSAs and validate CGA-SLPs in HCC patient-derived tumor infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs).ResultsOut of a set of 13 CGAs, previously shown to be expressed in primary human HCC tissues, two CGAs (i.e., CGA-A and -B) demonstrated no healthy tissue expression and covered >75% of HCC patients collectively (N = 55). Immunopeptidome analysis of human HCC-derived hepatocytes (N = 12), together with in silico CGA-related epitope predictions according to epitope immunogenicity, enabled identification of 196 and 220 potential epitopes for CGA-A and -B, respectively. HLA-A*02:01 binding of these epitopes was validated in vitro using a HLA-A2 stabilization assay and ranked accordingly. Six SLPs were designed incorporating 54 HLA-A*02:01, 25 HLA-A*01:01, 24 HLA-A*03:01, 27 HLA-A*24:01, and 15 HLA-B*07:02 predicted and/or validated CGA-A- and -B-related epitopes. Top three-ranked epitopes were selected to validate ex vivo intra-tumor immune reactivity using corresponding peptide-HLA-A*02:01 dextramers in human HCC-derived TILs. Tumors of 8/11 patients contained CGA-A- and CGA-B-specific TILs that were characterized by a tumor reactive phenotype. Upon in vitro enrichment, SLP immunogenicity was demonstrated through Interferon gamma ELISPOT in 2/3 of human HCC-derived PBMCs using an in vitro co-culture system with autologous antigen presenting cells.ConclusionsHere, we describe the intelligent design of a set of immunogenic SLPs comprising CGA-related epitopes for the global population that can be further exploited for the development of an off-the shelf anti-cancer vaccine to treat HCC.ReferencesAn RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature 2020;585(7823):107–112.Expression of cancer testis antigens in tumor-adjacent normal liver is associated with post-resection recurrence of hepatocellular carcinoma. Cancers (Basel) 2021;13(10):2499.Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med. 2009;361(19):1838.Ethics ApprovalAll study procedures were approved by the local ethics committee (Medische Ethische Toetsings Commissie Erasmus MC Rotterdam; NL58534,078.16). Patients had given informed consent for tissue and blood donation as well as usage of personal data.ConsentPatients had given informed consent for tissue and blood donation as well as usage of personal data.