Integrating Cancer Vaccines in the Standard-of-Care of Ovarian Cancer: Translating Preclinical Models to Human.

As the majority of ovarian cancer (OC) patients are diagnosed with metastatic disease, less than 40% will survive past 5 years after diagnosis. OC is characterized by a succession of remissions and recurrences. The most promising time point for immunotherapeutic interventions in OC is following debulking surgery. Accumulating evidence shows that T cells are important in OC; thus, cancer vaccines capable of eliciting antitumor T cells will be effective in OC treatment. In this review, we discuss different cancer vaccines and propose strategies for their incorporation into the OC standard-of-care regimens. Using the murine ID8 ovarian tumor model, we provide evidence that a cancer vaccine can be effectively combined with OC standard-of-care to achieve greater overall efficacy. We demonstrate several important similarities between the ID8 model and OC patients, in terms of response to immunotherapies, and the ID8 model can be an important tool for evaluating combinatorial regimens and clinical trial designs in OC. Other emerging models, including patient-derived xenograft and genetically engineered mouse models, are continuing to improve and can be useful for evaluating cancer vaccination therapies in the near future. Here, we provide a comprehensive review of the completed and current clinical trials evaluating cancer vaccines in OC

Rate of Freeze Impacts the Survival and Immune Responses Post Cryoablation of Melanoma.

The emergence of ablative therapies has revolutionized the treatment of inoperable solid tumors. Cryoablation stands out for its uniqueness of operation based on hypothermia, and for its ability to unleash the native tumor antigens, resulting in the generation of anti-tumor immune responses. It is not clearly understood how alterations in the rate of freeze impact the immune response outcomes. In this study, we tested fast freeze and slow freeze rates for their locoregional effectiveness and their ability to elicit immune responses in a B16F10 mouse model of melanoma. Tumor bearing mice treated with fast freeze protocol survived better than the ones treated with slow freeze protocol. Fast freeze resulted in a higher magnitude of CD4 <sup>+</sup> and CD8 <sup>+</sup> T-cell responses, and a significantly extended survival post re-challenge. Thus, fast freeze rate should be applied in any future studies employing cryoablation as an in vivo vaccination tool in conjunction with targeted immunotherapies

Longitudinal analysis of DC subsets in patients with ovarian cancer: Implications for immunotherapy.

The use of circulating cDC1 to generate anti-cancer vaccines is among the most promising approaches to overcome the limited immunogenicity and clinical efficacy of monocyte-derived DC. However, the recurrent lymphopenia and the reduction of DC numbers and functionality in patients with cancer may represent an important limitation of such approach. In patients with ovarian cancer (OvC) that had received chemotherapy, we previously showed that cDC1 frequency and function were reduced. We recruited healthy donors (HD, n=7) and patients with OvC at diagnosis and undergoing interval debulking surgery (IDS, n=6), primary debulking surgery (PDS, n=6) or at relapse (n=8). We characterized longitudinally phenotypic and functional properties of peripheral DC subsets by multiparametric flow cytometry. We show that the frequency of cDC1 and the total CD141+ DC capacity to take up antigen are not reduced at the diagnosis, while their TLR3 responsiveness is partially impaired in comparison with HD. Chemotherapy causes cDC1 depletion and increase in cDC2 frequency, but mainly in patients belonging to the PDS group, while in the IDS group both total lymphocytes and cDC1 are preserved. The capacity of total CD141 <sup>+</sup> DC and cDC2 to take up antigen is not impacted by chemotherapy, while the activation capacity upon Poly(I:C) (TLR3L) stimulation is further decreased. Our study provides new information about the impact of chemotherapy on the immune system of patients with OvC and sheds a new light on the importance of considering timing with respect to chemotherapy when designing new vaccination strategies that aim at withdrawing or targeting specific DC subsets

Development and Optimization of a GMP-Compliant Manufacturing Process for a Personalized Tumor Lysate Dendritic Cell Vaccine.

With the emergence of immune checkpoint inhibitors and adoptive T-cell therapies, there is a considerable interest in using personalized autologous dendritic cell (DC) vaccines in combination with T cell-targeting immunotherapies to potentially maximize the therapeutic impact of DC vaccines. Here, we describe the development and optimization of a Good Manufacturing Practice (GMP)-compliant manufacturing process based on tumor lysate as a tumor antigen source for the production of an oxidized tumor cell lysate loaded DC (OC-DC) vaccine. The manufacturing process required one day for lysate preparation and six days for OC-DC vaccine production. Tumor lysate production was standardized based on an optimal tumor digestion protocol and the immunogenicity was improved through oxidation using hypochloric acid prior to freeze-thaw cycles resulting in the oxidized tumor cell lysate (OC-L). Next, monocytes were selected using the CliniMACS prodigy closed system and were placed in culture in cell factories in the presence of IL-4 and GM-CSF. Immature DCs were loaded with OC-L and matured using MPLA-IFNγ. After assessing the functionality of the OC-DC cells (IL12p70 secretion and COSTIM assay), the OC-DC vaccine was cryopreserved in multiple doses for single use. Finally, the stability of the formulated doses was tested and validated. We believe this GMP-compliant DC vaccine manufacturing process will facilitate access of patients to personalized DC vaccines, and allow for multi-center clinical trials

A Phase I/II trial comparing autologous dendritic cell vaccine pulsed either with personalized peptides (PEP-DC) or with tumor lysate (OC-DC) in patients with advanced high-grade ovarian serous carcinoma.

Most ovarian cancer patients are diagnosed at a late stage with 85% of them relapsing after surgery and standard chemotherapy; for this reason, new treatments are urgently needed. Ovarian cancer has become a candidate for immunotherapy by reason of their expression of shared tumor-associated antigens (TAAs) and private mutated neoantigens (NeoAgs) and the recognition of the tumor by the immune system. Additionally, the presence of intraepithelial tumor infiltrating lymphocytes (TILs) is associated with improved progression-free and overall survival of patients with ovarian cancer. The aim of active immunotherapy, including vaccination, is to generate a new anti-tumor response and amplify an existing immune response. Recently developed NeoAgs-based cancer vaccines have the advantage of being more tumor specific, reducing the potential for immunological tolerance, and inducing robust immunogenicity. We propose a randomized phase I/II study in patients with advanced ovarian cancer to compare the immunogenicity and to assess safety and feasibility of two personalized DC vaccines. After standard of care surgery and chemotherapy, patients will receive either a novel vaccine consisting of autologous DCs pulsed with up to ten peptides (PEP-DC), selected using an agnostic, yet personalized, epitope discovery algorithm, or a sequential combination of a DC vaccine loaded with autologous oxidized tumor lysate (OC-DC) prior to an equivalent PEP-DC vaccine. All vaccines will be administered in combination with low-dose cyclophosphamide. This study is the first attempt to compare the two approaches and to use NeoAgs-based vaccines in ovarian cancer in the adjuvant setting. The proposed treatment takes advantage of the beneficial effects of pre-treatment with OC-DC prior to PEP-DC vaccination, prompting immune response induction against a wide range of patient-specific antigens, and amplification of pre-existing NeoAgs-specific T cell clones. Trial registration This trial is already approved by Swissmedic (Ref.: 2019TpP1004) and will be registered at http://www.clinicaltrials.gov before enrollment opens

The Ovarian Cancer Chemokine Landscape Is Conducive to Homing of Vaccine-Primed and CD3/CD28-Costimulated T Cells Prepared for Adoptive Therapy.

PURPOSE: Chemokines are implicated in T-cell trafficking. We mapped the chemokine landscape in advanced stage ovarian cancer and characterized the expression of cognate receptors in autologous dendritic cell (DC)-vaccine primed T cells in the context of cell-based immunotherapy. EXPERIMENTAL DESIGN: The expression of all known human chemokines in patients with primary ovarian cancer was analyzed on two independent microarray datasets and validated on tissue microarray. Peripheral blood T cells from five HLA-A2 patients with recurrent ovarian cancer, who previously received autologous tumor DC vaccine, underwent CD3/CD28 costimulation and expansion ex vivo. Tumor-specific T cells were identified by HER2/neu pentamer staining and were evaluated for the expression and functionality of chemokine receptors important for homing to ovarian cancer. RESULTS: The chemokine landscape of ovarian cancer is heterogeneous with high expression of known lymphocyte-recruiting chemokines (CCL2, CCL4, and CCL5) in tumors with intraepithelial T cells, whereas CXCL10, CXCL12, and CXCL16 are expressed quasi-universally, including in tumors lacking tumor-infiltrating T cells. DC-vaccine primed T cells were found to express the cognate receptors for the above chemokines. Ex vivo CD3/CD28 costimulation and expansion of vaccine-primed Tcells upregulated CXCR3 and CXCR4, and enhanced their migration toward universally expressed chemokines in ovarian cancer. CONCLUSIONS: DC-primed tumor-specific T cells are armed with the appropriate receptors to migrate toward universal ovarian cancer chemokines, and these receptors are further upregulated by ex vivo CD3/CD28 costimulation, which render T cells more fit for migrating toward these chemokines. Clin Cancer Res; 21(12); 2840-50. ©2015 AACR

Quantitative and qualitative impairments in dendritic cell subsets of patients with ovarian or prostate cancer

Background Dendritic cells (DCs) are the most efficient antigen-presenting cells, hence initiating a potent and cancer-specific immune response. This ability (mainly using monocyte-derived DCs) has been exploited in vaccination strategies for decades with limited clinical efficacy. Another alternative would be the use of conventional DCs (cDCs) of which at least three subsets circulate in human blood: cDC1s (CD141bright), cDC2s (CD1c+) and plasmacytoid DCs. Despite their paucity, technical advances may allow for their selection and clinical use. However, many assumptions concerning the DC subset biology depend on observations from mouse models, hindering their translational potential. In this study, we characterise human DCs in patients with ovarian cancer (OvC) or prostate cancer (PrC). Patients and methods Whole blood samples from patients with OvC or PrC and healthy donors (HDs) were evaluated by flow cytometry for the phenotypic and functional characterisation of DC subsets. Results In both patient groups, the frequency of total CD141+ DCs was lower than that in HDs, but the cDC1 subset was only reduced in patients with OvC. CD141+ DCs showed a reduced response to the TLR3 agonist poly (I:C) in both groups of patients. An inverse correlation between the frequency of cDC1s and CA125, the OvC tumour burden marker, was observed. Consistently, high expression of CLEC9A in OvC tissue (The Cancer Genome Atlas data set) indicated a better overall survival. Conclusions cDC1s are reduced in patients with OvC, and CD141+ DCs are quantitatively and qualitatively impaired in patients with OvC or PrC. CD141+ DC activation may predict functional impairment. The loss of cDC1s may be a bad prognostic factor for patients with OvC

Adenosine mediates functional and metabolic suppression of peripheral and tumor-infiltrating CD8⁺ T cells.

Several mechanisms are present in the tumor microenvironment (TME) to impair cytotoxic T cell responses potentially able to control tumor growth. Among these, the accumulation of adenosine (Ado) contributes to tumor progression and represents a promising immunotherapeutic target. Ado has been shown to impair T cell effector function, but the role and mechanisms employed by Ado/Ado receptors (AdoRs) in modulating human peripheral and tumor-infiltrating lymphocyte (TIL) function are still puzzling. CD8 <sup>+</sup> T cell cytokine production following stimulation was quantified by intracellular staining and flow cytometry. The cytotoxic capacity of tumor infiltrating lymphocytes (TILs) was quantified by the chromium release assay following co-culture with autologous or anti-CD3-loaded tumor cell lines. The CD8 <sup>+</sup> T cell metabolic fitness was evaluated by the seahorse assay and by the quantification of 2-NBDG uptake and CD71/CD98 upregulation upon stimulation. The expression of AdoRs was assessed by RNA flow cytometry, a recently developed technology that we validated by semiquantitative RT-PCR (qRT-PCR), while the impact on T cell function was evaluated by the use of selective antagonists and agonists. The influence of Ado/AdoR on the PKA and mTOR pathways was evaluated by phosphoflow staining of p-CREB and p-S6, respectively, and validated by western blot. Here, we demonstrate that Ado signaling through the A2A receptor (A2AR) in human peripheral CD8 <sup>+</sup> T cells and TILs is responsible for the higher sensitivity to Ado-mediated suppression of T central memory cells. We confirmed that Ado is able to impair peripheral and tumor-expanded T cell effector functions, and we show for the first time its impact on metabolic fitness. The Ado-mediated immunosuppressive effects are mediated by increased PKA activation that results in impairment of the mTORC1 pathway. Our findings unveil A2AR/PKA/mTORC1 as the main Ado signaling pathway impairing the immune competence of peripheral T cells and TILs. Thus, p-CREB and p-S6 may represent useful pharmacodynamic and efficacy biomarkers of immunotherapies targeting Ado. The effect of Ado on T cell metabolic fitness reinforces the importance of the adenosinergic pathway as a target for next-generation immunotherapy

PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rβ-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential

The clinical application of cancer immunotherapy based on naturally circulating dendritic cells

Dendritic cells (DCs) can initiate and direct adaptive immune responses. This ability is exploitable in DC vaccination strategies, in which DCs are educated ex vivo to present tumor antigens and are administered into the patient with the aim to induce a tumor-specific immune response. DC vaccination remains a promising approach with the potential to further improve cancer immunotherapy with little or no evidence of treatment-limiting toxicity. However, evidence for objective clinical antitumor activity of DC vaccination is currently limited, hampering the clinical implementation. One possible explanation for this is that the most commonly used monocyte-derived DCs may not be the best source for DC-based immunotherapy. The novel approach to use naturally circulating DCs may be an attractive alternative. In contrast to monocyte-derived DCs, naturally circulating DCs are relatively scarce but do not require extensive culture periods. Thereby, their functional capabilities are preserved, the reproducibility of clinical applications is increased, and the cells are not dysfunctional before injection. In human blood, at least three DC subsets can be distinguished, plasmacytoid DCs, CD141+ and CD1c+ myeloid/conventional DCs, each with distinct functional characteristics. In completed clinical trials, either CD1c+ myeloid DCs or plasmacytoid DCs were administered and showed encouraging immunological and clinical outcomes. Currently, also the combination of CD1c+ myeloid and plasmacytoid DCs as well as the intratumoral use of CD1c+ myeloid DCs is under investigation in the clinic. Isolation and culture strategies for CD141+ myeloid DCs are being developed. Here, we summarize and discuss recent clinical developments and future prospects of natural DC-based immunotherapy