| 1. |
- Ellingsen, Espen Basmo, et al.
(författare)
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Characterization of the T cell receptor repertoire and melanoma tumor microenvironment upon combined treatment with ipilimumab and hTERT vaccination
- 2022
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Ingår i: Journal of Translational Medicine. - : Springer Nature. - 1479-5876 .- 1479-5876. ; 20
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Tidskriftsartikel (refereegranskat)abstract
- Background: This clinical trial evaluated a novel telomerase-targeting therapeutic cancer vaccine, UV1, in combination with ipilimumab, in patients with metastatic melanoma. Translational research was conducted on patient-derived blood and tissue samples with the goal of elucidating the effects of treatment on the T cell receptor repertoire and tumor microenvironment.Methods: The trial was an open-label, single-center phase I/11a study. Eligible patients had unresectable metastatic melanoma. Patients received up to 9 UV1 vaccinations and four ipilimumab infusions. Clinical responses were assessed according to RECIST 1.1. Patients were followed up for progression-free survival (PFS) and overall survival (OS). Whole-exome and RNA sequencing, and multiplex immunofluorescence were performed on the biopsies. T cell receptor (TCR) sequencing was performed on the peripheral blood and tumor tissues.Results: Twelve patients were enrolled in the study. Vaccine-specific immune responses were detected in 91% of evaluable patients. Clinical responses were observed in four patients. The mPFS was 6.7 months, and the mOS was 66.3 months. There was no association between baseline tumor mutational burden, neoantigen load, IFN-gamma gene signature, tumor-infiltrating lymphocytes, and response to therapy. Tumor telomerase expression was confirmed in all available biopsies. Vaccine-enriched TCR clones were detected in blood and biopsy, and an increase in the tumor IFN-gamma gene signature was detected in clinically responding patients.Conclusion: Clinical responses were observed irrespective of established predictive biomarkers for checkpoint inhibitor efficacy, indicating an added benefit of the vaccine-induced T cells. The clinical and immunological read-out warrants further investigation of UV1 in combination with checkpoint inhibitors.
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| 2. |
- Fotaki, Grammatiki, et al.
(författare)
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Cancer vaccine based on a combination of an infection-enhanced adenoviral vector and pro-inflammatory allogeneic DCs leads to sustained antigen-specific immune responses in three melanoma models
- 2018
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Ingår i: Oncoimmunology. - 2162-4011 .- 2162-402X. ; 7:3
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Tidskriftsartikel (refereegranskat)abstract
- Autologous patient-derived dendritic cells (DCs) modified ex vivo to present tumor-associated antigens (TAAs) are frequently used as cancer vaccines. However, apart from the stringent logistics in producing DCs on a patient basis, accumulating evidence indicate that ex vivo engineered DCs are poor in migration and in fact do not directly present TAA epitopes to naïve T cells in vivo. Instead, it is proposed that bystander host DCs take up material from vaccine-DCs, migrate and subsequently initiate antitumor T-cell responses. We used mouse models to examine the possibility of using pro-inflammatory allogeneic DCs (alloDCs) to activate host DCs and enable them to promote antigen-specific T-cell immunity. We found that alloDCs were able to initiate host DC activation and migration to draining lymph node leading to T-cell activation. The pro-inflammatory milieu created by alloDCs also led to recruitment of NK cells and neutrophils at the site of injection. Vaccination with alloDCs combined with Ad5M(gp100), an infection-enhanced adenovirus encoding the human melanoma-associated antigen gp100 resulted in generation of CD8+ T cells with a T-cell receptor (TCR) specific for the gp10025-33 epitope (gp100-TCR+). Ad5M(gp100)-alloDC vaccination in combination with transfer of gp100-specific pmel-1 T cells resulted in prolonged survival of B16-F10 melanoma-bearing mice and altered the composition of the tumor microenvironment (TME). We hereby propose that alloDCs together with TAA- or neoepitope-encoding Ad5M can become an “off-the-shelf” cancer vaccine, which can reverse the TME-induced immunosuppression and induce host cellular anti-tumor immune responses in patients without the need of a time-consuming preparation step of autologous DCs.
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| 3. |
- Fotaki, Grammatiki, et al.
(författare)
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Pro-inflammatory allogeneic DCs promote activation of bystander immune cells and thereby license antigen-specific T-cell responses
- 2018
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Ingår i: Oncoimmunology. - 2162-4011 .- 2162-402X. ; 7:3
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Tidskriftsartikel (refereegranskat)abstract
- Accumulating evidence support an important role for endogenous bystander dendritic cells (DCs) in the efficiency of autologous patient-derived DC-vaccines, as bystander DCs take up material from vaccine-DCs, migrate to draining lymph node and initiate antitumor T-cell responses. We examined the possibility of using allogeneic DCs as vaccine-DCs to activate bystander immune cells and promote antigen-specific T-cell responses. We demonstrate that human DCs matured with polyI:C, R848 and IFN-γ (denoted COMBIG) in combination with an infection-enhanced adenovirus vector (denoted Ad5M) exhibit a pro-inflammatory state. COMBIG/Ad5M-matured allogeneic DCs (alloDCs) efficiently activated T-cells and NK-cells in allogeneic co-culture experiments. The secretion of immunostimulatory factors during the co-culture promoted the maturation of bystander-DCs, which efficiently cross-presented a model-antigen to activate antigen-specific CD8+ T-cells in vitro. We propose that alloDCs, in combination with Ad5M as loading vehicle, may be a cost-effective and logistically simplified DC vaccination strategy to induce anti-tumor immune responses in cancer patients.
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| 4. |
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| 5. |
- Kerzeli, Iliana Kyriaki
(författare)
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Dissecting the Microenvironment of Urothelial Bladder Cancer : Therapy, Modelling and Biomarkers
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The complexity of the tumor microenvironment (TME) impacts therapy responses and the survival of cancer patients. The scope of this thesis is to study the effects of immune modulation on tumor-infiltrating leukocytes, and to explore the TME of urothelial cancer to provide the research society with new knowledge and potential therapeutic targets.In paper I, a small molecule MALT1 inhibitor demonstrated in vitro selective inhibition of immune suppressive T regulatory cells (Tregs). However, the inhibitor failed to demonstrate anti-tumor therapeutic effects in vivo. The effect of the MALT1 inhibitor was assessed on immune cell subsets in tumors, as well as in secondary lymphoid organs. Ultimately, the inhibitor was found to have a depleting effect on antigen-specific T cells.Paper II describes the development and characterization of a novel murine urothelial cancer model in both sexes by using a low-dose carcinogen exposure via drinking water combined with a unique transgenic strain. The model recapitulates the profile of basal histology subtype urothelial cancer with a progression outcome, presents transcriptomic tumor heterogeneity, and displays early immune activation signatures despite its failure to respond to checkpoint blockade immunotherapy. Moreover, the study demonstrates that the female sex displayed accelerated tumor development, but also benefited from therapeutic TLR9 stimulation in contrast to males.Therefore, in paper III, an in-depth transcriptomic analysis of the immune cell compartment of the TME was performed in both sexes by using this novel model of urothelial cancer. Infiltrating immune cell subsets varied between stages, while cell communication analysis pinpointed distinct interactions among immune and tumor cells and pathways of immune regulation suitable for therapeutic targeting. Also, differentially expressed genes in immune cell subsets of the TME between sexes revealed higher immune responsiveness in some female immune cell subsets and higher metabolic activity in several male immune cell subsets.In paper IV, proximity extension assay (PEA) analysis of urine and plasma was assessed for liquid biopsy biomarker discovery. Samples from patients at first diagnosis revealed that high MMP12 plasma levels were associated with poor survival. This finding was reproduced in serum PEA analysis of an independent urothelial cancer patient cohort. Single cell transcriptomic analysis of public datasets demonstrated that the production of MMP12 in urothelial cancer could be attributed to tumor-infiltrating macrophages.In conclusion, this thesis illustrates the multi-faceted opportunities and challenges of targeting and studying the microenvironment of urothelial cancer in murine models and human patients.
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| 6. |
- Kerzeli, Iliana Kyriaki, et al.
(författare)
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Elevated levels of MMP12 sourced from macrophages are associated with poor prognosis in urothelial bladder cancer
- 2023
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Ingår i: BMC Cancer. - : BioMed Central (BMC). - 1471-2407 .- 1471-2407. ; 23:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Urothelial bladder cancer is most frequently diagnosed at the non-muscle-invasive stage (NMIBC). However, recurrences and interventions for intermediate and high-risk NMIBC patients impact the quality of life. Biomarkers for patient stratification could help to avoid unnecessary interventions whilst indicating aggressive measures when required.Methods In this study, immuno-oncology focused, multiplexed proximity extension assays were utilised to analyse plasma (n = 90) and urine (n = 40) samples from 90 newly-diagnosed and treatment-naive bladder cancer patients. Public single-cell RNA-sequencing and microarray data from patient tumour tissues and murine OH-BBN-induced urothelial carcinomas were also explored to further corroborate the proteomic findings.Results Plasma from muscle-invasive, urothelial bladder cancer patients displayed higher levels of MMP7 (p = 0.028) and CCL23 (p = 0.03) compared to NMIBC patients, whereas urine displayed higher levels of CD27 (p = 0.044) and CD40 (p = 0.04) in the NMIBC group by two-sided Wilcoxon rank-sum tests. Random forest survival and multivariable regression analyses identified increased MMP12 plasma levels as an independent marker (p < 0.001) associated with shorter overall survival (HR = 1.8, p < 0.001, 95% CI:1.3-2.5); this finding was validated in an independent patient OLINK cohort, but could not be established using a transcriptomic microarray dataset. Single-cell transcriptomics analyses indicated tumour-infiltrating macrophages as a putative source of MMP12.Conclusions The measurable levels of tumour-localised, immune-cell-derived MMP12 in blood suggest MMP12 as an important biomarker that could complement histopathology-based risk stratification.As MMP12 stems from infiltrating immune cells rather than the tumor cells themselves, analyses performed on tissue biopsy material risk a biased selection of biomarkers produced by the tumour, while ignoring the surrounding microenvironment.
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| 7. |
- Kerzeli, Iliana Kyriaki, et al.
(författare)
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MALT1 inhibition suppresses T-cell dependent immune surveillance
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- MALT1 supports the development of natural regulatory T cells (Tregs), while protease-dead MALT1 (MALT1-PD) mice develop autoimmunity and an intrinsic capacity to reject syngeneic tumor transplantation. Herein, a small molecule inhibitor targeting MALT1 was developed and evaluated for potential use in Treg inhibition as part of a cancer immunotherapy strategy.In vitro, MALT1 inhibitor treatment inhibited the proteolytic cleavage of the MALT1 substrate HOIL1 in Jurkat cells and blocked IL-2 secretion by immune cells. Moreover, orally administrated MV088428 inhibited anti-CD3 induced IL-2 release in vivo. In vitro MALT1 inhibition selectively suppressed the proliferation of PBMC derived CD25+ FoxP3+ CD4+ T cells, while no direct effect was noted on the proliferation and viability of CD25- CD4+ T cells. In vivo, no evident anti-tumor effect as a monotherapy in the MB49 bladder cancer model was achieved and despite selective decrease of Treg frequencies in lymph nodes of tumor bearing animals, intratumoral Treg depletion was not observed. No synergistic anti-tumor effects were noted when MALT1 inhibitor was combined with anti-PD1 therapy, and concomitant treatment with MALT1 inhibitor abrogated the efficacy of anti-CTLA4 therapy. MALT1 inhibition had no impact on the frequencies of viable NK, lymphocyte and myeloid cells or on proliferation of conventional CD4 and CD8 T cells. However, there was a significant decrease of antigen-specific T cells in vivo upon adoptive T cell transfer and peptide vaccination. Thus, while MALT1 inhibition substantially reduced Treg populations in lymph nodes, but less so in tumors, off-target effects on antigen-experienced T cells along with the lack of impact on tumor Tregs likely abolish the compound’s efficacy. The off-target effect on antigen-experienced T cells could present implications for the use of MALT1 inhibitors for cancer indications where tumor control is likely to be mediated via T cell driven immune surveillance. Thus, dosing length and combination therapy strategies should be carefully designed and evaluated further.
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| 8. |
- Kerzeli, Iliana Kyriaki, et al.
(författare)
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Proximity Extension Assay reveals MMP12 in plasma as a predictor of outcome in urothelial bladder cancer
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background Urothelial bladder cancer (UBC) is most-frequently diagnosed at the non-muscle-invasive stage (NMIBC). However, recurrences and interventions for intermediate and high-risk NMIBC patients impact quality of life. Biomarkers for patient stratification can help to avoid unnecessary interventions whilst indicating aggressive measures when required.Methods Proximity Extension Assay (PEA) was utilised to analyse plasma (n=90) and urine (n=40) samples from 90 newly-diagnosed and treatment-naïve UBC patients with an immuno-oncology protein panel. Public single-cell and bulk RNA sequencing data from patient tumour tissues and a murine OH-BBN induced UBC model were also explored. Results Plasma samples from muscle-invasive UBC patients displayed higher levels of MMP7 and CCL23 compared to NMIBC patients, whereas urine samples displayed higher levels of CD27 and CD40 in the NMIBC group. Increased MMP12 plasma levels were identified as an independent marker of poor survival outcome and this finding was further validated in an independent cohort. Moreover, scRNA-seq data analysis indicated tumour infiltrating macrophages as a putative source of MMP12. Conclusion The local production, but systemic diffusion of MMP12, suggests that MMP12 could be an important biomarker to complement histopathology-based risk stratification. Additionally, it may represent a pharmacological target in bladder cancer.
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| 9. |
- Kerzeli, Iliana Kyriaki, et al.
(författare)
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Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations
- 2021
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 16:7
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Tidskriftsartikel (refereegranskat)abstract
- Bladder cancer, one of the most prevalent malignancies worldwide, remains hard to classify due to a staggering molecular complexity. Despite a plethora of diagnostic tools and therapies, it is hard to outline the key steps leading up to the transition from high-risk non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). Carcinogen-induced murine models can recapitulate urothelial carcinogenesis and natural anti-tumor immunity. Herein, we have developed and profiled a novel model of progressive NMIBC based on 10 weeks of OH-BBN exposure in hepatocyte growth factor/cyclin dependent kinase 4 (R24C) (Hgf-Cdk4(R24C)) mice. The profiling of the model was performed by histology grading, single cell transcriptomic and proteomic analysis, while the derivation of a tumorigenic cell line was validated and used to assess in vivo anti-tumor effects in response to immunotherapy. Established NMIBC was present in females at 10 weeks post OH-BBN exposure while neoplasia was not as advanced in male mice, however all mice progressed to MIBC. Single cell RNA sequencing analysis revealed an intratumoral heterogeneity also described in the human disease trajectory. Moreover, although immune activation biomarkers were elevated in urine during carcinogen exposure, anti-programmed cell death protein 1 (anti-PD1) monotherapy did not prevent tumor progression. Furthermore, anti-PD1 immunotherapy did not control the growth of subcutaneous tumors formed by the newly derived urothelial cancer cell line. However, treatment with CpG-oligodeoxynucleotides (ODN) significantly decreased tumor volume, but only in females. In conclusion, the molecular map of this novel preclinical model of bladder cancer provides an opportunity to further investigate pharmacological therapies ahead with regards to both targeted drugs and immunotherapies to improve the strategies of how we should tackle the heterogeneous tumor microenvironment in urothelial bladder cancer to improve responses rates in the clinic.
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| 10. |
- Kerzeli, Iliana Kyriaki, et al.
(författare)
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The immunological landscape in bladder cancer: a single cell RNA seq analysis of tumor bearing mouse bladders with a focus on sex differences
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In urothelial bladder cancer differences in clinical outcomes and responses to immunotherapy associate with tumor stage and sex. However, the tumor microenvironment landscape in urothelial cancer and its contexture in different stages and sexes is not fully deciphered. We utilized an autochthonous urothelial cancer model sampled at the non-muscle and muscle invasive stage from both sexes. Single cell RNA sequencing was applied to analyze intercellular interactions and sex differences in the two stages with focus on immune cell subsets. We discerned the Cd6-Alcam interaction to occur between lymphoid, tumor and myeloid cells and that female tumor bearing bladders were enriched in cells of lymphoid lineage compared to males, although male lymphoid cells were metabolically more active. Furthermore, we identified two subsets of Macrophages, Ccl8+ and Spp1+ that were characteristic for NMIBC and MIBC respectively, however both were found to be more active in immune cell recruitment, activation and cytokine signaling in females than males. Analysis of intercellular interactions of immune cells subsets revealed the role of Lyc6c2+ and Ace+ Monocytes as antigen presenting cells equipped with co-stimulatory receptors, and the Anxa1-Fpr2 immune suppressive axis to be active among tumor cells and subsets of granulocytes, macrophages and monocytes. Collectively our results demonstrate the complexity and sex specific differences of the tumor microenvironment in urothelial cancer using a clinically relevant murine model of progressive disease, and distinguish the Cd6-Alcam and Anxa1-Fpr2 interaction pathways of lymphoid, myeloid and tumor cells as potential targets of immune modulation for cancer therapy.
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