Jirina Bartunkova

β ₂ ‐microglobulin (β2M) is a small protein playing a critical role in stabilizing major histocompatibility complex class I (MHC‐I) molecules on nucleated cells. Elevated levels of β2M have been observed in several cancers, inflammatory and autoimmune conditions, and renal failures. High concentrations of β2M were reported to inhibit in vitro generation of functional dendritic cells (DCs). However, our findings showed that β2M exerts a negative effect on DCs only when contaminated with endotoxins. We found that β2M preparations with a high level of endotoxin impurities matured DCs, but that this effect was not seen with functional β2M preparations with low levels of endotoxin impurities, thus showing the maturation effect was due to endotoxin stimulation. We confirmed that the high‐level endotoxin β2M compromised the in vitro differentiation of monocytes into DCs. In contrast, a low‐level endotoxin β2M had no negative impact on DC differentiation nor prevented their maturation and functionality. Moreover, regardless of the levels of endotoxin impurities, β2M stabilized the expression of MHC‐I molecules, confirming its functionality in the experimental settings. Our results show that β2M does not compromise the differentiation of DCs and indicate that elevated levels of β2M are unlikely to negatively regulate the immune system. These results have significant implications for understanding the functions of high β2M concentrations in clinical contexts and in vitro applications.

Background Prostate cancer (PCa) is one of the most common cancers in men. As a “cold tumor,” it exhibits low immunogenicity. Therefore, dendritic cells vaccines (DC) have been developed to enhance adaptive immunity. However, the efficacy of ex vivo-produced DC remains suboptimal. The antimicrobial peptide LL-37 has shown various immunomodulatory effects. Our previous studies demonstrated that LL-37 enhances the performance of DC produced from the peripheral blood of healthy volunteers. Aims In this follow-up study, we aimed to further investigate the therapeutical potential of LL-37/DC alone or in combination with anti-PD1 therapy using PCa patients’ samples and a syngeneic mouse model. Furthermore, we explored the performance of LL-37/DC under GMP conditions. Methods Human DC were prepared from leukaphereses of 10 biochemical recurrence (BR) PCa patients. The reactivity and cytotoxicity of these DC to the PCa cell line PC3 were assessed using flow cytometry and fluorescent microscopy. Mice bone marrow-derived DC from B6 TRAMP mice were inoculated subcutaneously into TRAMP tumor-bearing mice (n=9). The tumor growth and survival rates were monitored. Finally, we produced DC under GMP conditions and evaluated their performance in vitro. Results LL-37/DC-primed lymphocytes from BR PCa patients exhibited a two-fold increase in the expression of both IFN-γ and TNF-α and caused a three times higher contraction of co-cultured PC3 cells compared to DC prepared without LL-37. The increase in the LL-37/DC efficacy was not caused by enhanced DC maturation, as the expression levels of HLA-DR, CD83, CD86, and CD80 remained similar. Adding an anti-PD1 antibody did not improve either DC or LL-37/DC efficacy. Finally, we demonstrated that LL-37/DC prepared under GMP conditions was also significantly more reactive and caused two and a half times higher contraction of the co-cultured PC3 cell line compared to DC prepared without LL-37. In the TRAMP mice model, LL-37/DC exhibited significantly higher expression of CD80, extended the mice survival by 30 days, and slowed down the tumor growth as compared with the DC prepared without LL-37. The anti-PD1 therapy had no additional effect. Conclusions We demonstrated that LL-37 is a promising compound that enhances the antitumor potential of DC both in vitro and in vivo. Furthermore, this improved potential is preserved even when DC are produced under GMP conditions. On the other hand, using anti-PD1 therapy showed no additional therapeutic impact. Taken together, LL-37/DC offers potentially advantageous treatment for patients with cold tumors, who typically respond poorly to immune checkpoint inhibitor therapy.

Epithelial ovarian carcinoma (EOC) is the fifth leading cause of cancer-related death in women, largely reflecting the early dissemination of this malignant disease to the peritoneum. Due to its immunological features, EOC has poor response to immune checkpoint inhibitors (ICIs), including a limited tumor mutational burden (TMB), poor infiltration by immune cells, and active immunosuppression. Thus, novel strategies are needed to overcome the frequent lack of pre-existing immunity in patients with EOC. We developed and tested an autologous dendritic cell (DC)-based vaccine (DCVAC), which has recently been shown to be safe and to significantly improve progression-free survival (PFS) in two independent randomized phase II clinical trials enrolling patients with EOC (SOV01, NCT02107937; SOV02, NCT02107950). In addition, our exploratory data analyses suggest that the clinical benefits of the DCVAC were more pronounced in patients with EOC with lower-than-median TMBs and reduced CD8⁺ T cell infiltration. Thus, the DC-based vaccine stands out as a promising clinical tool to jumpstart anticancer immunity in patients with immunologically “cold” EOC. Our findings underscore the need for personalized immunotherapy and the clinical relevance of potential tumor-related biomarkers within the immunotherapy field. Additional clinical trials are needed to address these strategies as well as the potential value of the TMB and immune infiltration at baseline as biomarkers for guiding the clinical management of EOC.

Epithelial ovarian carcinoma (EOC) is the fifth-leading cause of cancer-related death in women, largely reflecting early, prediagnosis dissemination of malignant disease to the peritoneum. EOC is poorly responsive to immune check-point inhibitors due to immunologic features including limited tumor mutational burden (TMB), poor infiltration by immune cells and active immunosuppression. Thus, novel strategies are needed to overcome the frequent lack of preexisting immunity in patients with EOC. We developed and tested an autologous dendritic cell (DC)-based vaccine (DCVAC) which has recently been shown to be safe and to significantly improve progression-free survival (PFS) in two independent randomized phase II clinical trials enrolling patients with EOC (SOV01, NCT02107937; SOV02, NCT02107950). In addition, our exploratory data analyses suggest that the clinical benefits of DCVAC were more pronounced in patients with EOC with lower than-median TMB and scant CD8+ T cells infiltration. Thus, DC-based vaccine stands out as a promising clinical tool to jumpstart anticancer immunity in patients with immunologically „cold“ EOC. Our findings underscore the need for personalized immunotherapy and clinical relevance of potential tumor-related biomarkers within immunotherapy field. Additional clinical trials are needed to address these strategies as well as the potential value of TMB and immune infiltrate at baseline as biomarkers to guide the clinical management of EOC.

CD8⁺ T cells are essential for adaptive immunity against infection and tumors. Their ability to proliferate after stimulation is crucial to their functionality. Dendritic cells (DCs) are professional antigen‐presenting cells that induce their proliferation. Here, we show that thapsigargin‐induced LAD2 mast cell (MC) line‐released products can impair the ability of monocyte‐derived DCs to induce CD8⁺ T‐cell proliferation and the generation of Th1 cytokine‐producing T cells. We found that culture medium conditioned with LAD2 MCs previously stimulated with thapsigargin (thapsLAD2) induces maturation of DCs as determined by the maturation markers CD80, CD83, CD86, and HLA‐DR. However, thapsLAD2‐matured DCs produced no detectable TNFα or IL‐12 during the maturation. In addition, although their surface expression of PD‐L1 was comparable with the immature or TLR7/8‐agonist (R848)‐matured DCs, their TIM‐3 expression was significantly higher than in immature DCs and even much higher than in R848‐matured DCs. In addition, contrary to R848‐matured DCs, the thapsLAD2‐matured DCs only tended to induce enhanced proliferation of CD4⁺ T cells than immature DCs. For CD8⁺ T cells, this tendency was not even detected because thapsLAD2‐matured and immature DCs comparably induced their proliferation, which contrasted with the significantly enhanced proliferation induced by R848‐matured DCs. Furthermore, these differences were comparably recapitulated in the ability of the tested DCs to induce IFNγ‐ and IFNγ/TNFα‐producing T cells. These findings show a novel mechanism of MC‐mediated regulation of adaptive immune responses.

Supplementary Figure from An Autologous Dendritic Cell Vaccine Promotes Anticancer Immunity in Patients with Ovarian Cancer with Low Mutational Burden and Cold Tumors

Supplementary Figure from An Autologous Dendritic Cell Vaccine Promotes Anticancer Immunity in Patients with Ovarian Cancer with Low Mutational Burden and Cold Tumors

Supplementary Data from An Autologous Dendritic Cell Vaccine Promotes Anticancer Immunity in Patients with Ovarian Cancer with Low Mutational Burden and Cold Tumors

Supplementary Data from An Autologous Dendritic Cell Vaccine Promotes Anticancer Immunity in Patients with Ovarian Cancer with Low Mutational Burden and Cold Tumors

Purpose:The successful implementation of immune checkpoint inhibitors (ICI) in the clinical management of various solid tumors has raised considerable expectations for patients with epithelial ovarian carcinoma (EOC). However, EOC is poorly responsive to ICIs due to immunologic features including limited tumor mutational burden (TMB) and poor lymphocytic infiltration. An autologous dendritic cell (DC)-based vaccine (DCVAC) has recently been shown to be safe and to significantly improve progression-free survival (PFS) in a randomized phase II clinical trial enrolling patients with EOC (SOV01, NCT02107937).Patients and Methods:We harnessed sequencing, flow cytometry, multispectral immunofluorescence microscopy, and IHC to analyze (pretreatment) tumor and (pretreatment and posttreatment) peripheral blood samples from 82 patients enrolled in SOV01, with the aim of identifying immunologic biomarkers that would improve the clinical management of patients with EOC treated with DCVAC.Results:Although higher-than-median TMB and abundant CD8+ T-cell infiltration were associated with superior clinical benefits in patients with EOC receiving standard-of-care chemotherapy, the same did not hold true in women receiving DCVAC. Conversely, superior clinical responses to DCVAC were observed in patients with lower-than-median TMB and scarce CD8+ T-cell infiltration. Such responses were accompanied by signs of improved effector functions and tumor-specific cytotoxicity in the peripheral blood.Conclusions:Our findings suggest that while patients with highly infiltrated, “hot” EOCs benefit from chemotherapy, women with “cold” EOCs may instead require DC-based vaccination to jumpstart clinically relevant anticancer immune responses.