Search

Recently, an antibody which inhibits the glycoprotein A repetitions predominant (GARP)-mediated release of active transforming growth factor beta (TGFβ) from the TGFβ propeptide latency-associated peptide (LAP) showed preclinical activity in a murine model of the chronic myeloproliferative neoplasms (MPN). Consequently, we investigated the expression of the immunosuppressive molecules LAP and GARP on peripheral blood lymphocytes from 56 MPN patients and 11 healthy donors (HD). We found that lymphocytes from patients with MPN express higher levels of LAP and GARP with no strong differences found between the different MPN diagnoses. The impact of clinical parameters on the expression of LAP and GARP by lymphocytes showed that patients with calreticulin (CALR)mut MPN have increased expression compared with HD and patients with the Januskinase2 (JAK2) mutation. The fraction of lymphocytes bound to activated platelets (aPLT) strongly correlate to LAP and GARP expression suggesting that it is not the lymphocytes themselves but aPLT, which confer the increased expression of GARP and LAP on MPN patient lymphocytes. Notably, no differences in neither platelet counts nor anti-thrombotic therapy was identified between patients with JAK2- and CALRmut patients. Analysis of platelet gene expression failed to identify differences in expression of relevant genes between JAK2- and CALRmut patients., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

The origins of cancer vaccines date back to the 1800s. Since then, there have been significant efforts to generate vaccines against solid and hematologic malignancies using a variety of platforms. To date, these efforts have generally been met with minimal success. However, in the era of improved methods and technological advancements, supported by compelling preclinical and clinical data, a wave of renewed interest in the field offers the promise of discovering field-changing paradigms in the management of established and resected disease using cancer vaccines. These include novel approaches to personalized neoantigen vaccine development, as well as innovative immune-modulatory vaccines (IMVs) that facilitate activation of antiregulatory T cells to limit immunosuppression caused by regulatory immune cells. This article will introduce some of the limitations that have affected cancer vaccine development over the past several decades, followed by an introduction to the latest advancements in neoantigen vaccine and IMV therapy, and then conclude with a discussion of some of the newest technologies and progress that are occurring across the cancer vaccine space. Cancer vaccines are among the most promising frontiers for breakthrough innovations and strategies poised to make a measurable impact in the ongoing fight against cancer.

The tumor microenvironment (TME) of pancreatic cancer is highly immunosuppressive. We recently developed a transforming growth factor (TGF)β-based immune modulatory vaccine that controlled tumor growth in a murine model of pancreatic cancer by targeting immunosuppression and desmoplasia in the TME. We found that treatment with the TGFβ vaccine not only reduced the percentage of M2-like tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) in the tumor but polarized CAFs away from the myofibroblast-like phenotype. However, whether the immune modulatory properties of the TGFβ vaccine on TAM and CAF phenotypes are a direct consequence of the recognition and subsequent targeting of these subsets by TGFβ-specific T cells or an indirect consequence of the overall modulation induced within the TME remains unknown. Recognition of M2 macrophages and fibroblast by TGFβ-specific T cells was assessed by ELISpot and flow cytometry. The indirect and direct effects of the TGFβ vaccine on these cell subsets were evaluated by culturing M2 macrophages or fibroblasts with tumor-conditioned media or with T cells isolated from the spleen of mice treated with the TGFβ vaccine or a control vaccine, respectively. Changes in phenotype were assessed by flow cytometry and Bio-Plex multiplex system (Luminex). We found that TGFβ-specific T cells induced by the TGFβ vaccine can recognize M2 macrophages and fibroblasts. Furthermore, we demonstrated that the phenotype of M2 macrophages and CAFs can be directly modulated by TGFβ-specific T cells induced by the TGFβ vaccine, as well as indirectly modulated as a result of the immune-modulatory effects of the vaccine within the TME. TAMs tend to have tumor-promoting functions, harbor an immunosuppressive phenotype and are linked to decreased overall survival in pancreatic cancer when they harbor an M2-like phenotype. In addition, myofibroblast-like CAFs create a stiff extracellular matrix that restricts T cell infiltration, impeding the effectiveness of immune therapies in desmoplastic tumors, such as pancreatic ductal adenocarcinoma. Reducing immunosuppression and immune exclusion in pancreatic tumors by targeting TAMs and CAFs with the TGFβ-based immune modulatory vaccine emerges as an innovative strategy for the generation of a more favorable environment for immune-based therapies, such as immune checkpoint inhibitors., Competing Interests: Competing interests: MHA has developed an invention based on the use of transforming growth factor-β-derived peptides for vaccinations. A patent application directed to the invention is owned by the company IO Biotech ApS and lists MHA as the sole inventor. MHA is advisor and shareholder at IO Biotech. IL and EM are employees at IO Biotech. The additional authors declare no competing financial interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) arise due to acquired somatic driver mutations in stem cells and develop over 10-30 years from the earliest cancer stages (essential thrombocythemia, polycythemia vera) towards the advanced myelofibrosis stage with bone marrow failure. The JAK2V617F mutation is the most prevalent driver mutation. Chronic inflammation is considered to be a major pathogenetic player, both as a trigger of MPN development and as a driver of disease progression. Chronic inflammation in MPNs is characterized by persistent connective tissue remodeling, which leads to organ dysfunction and ultimately, organ failure, due to excessive accumulation of extracellular matrix (ECM). Considering that MPNs are acquired clonal stem cell diseases developing in an inflammatory microenvironment in which the hematopoietic cell populations are progressively replaced by stromal proliferation-"a wound that never heals"-we herein aim to provide a comprehensive review of previous promising research in the field of circulating ECM fragments in the diagnosis, treatment and monitoring of MPNs. We address the rationales and highlight new perspectives for the use of circulating ECM protein fragments as biologically plausible, noninvasive disease markers in the management of MPNs.

Background: Therapeutic cancer vaccination against mutant calreticulin ( CALR ) in patients with CALR -mutant ( CALR mut) myeloproliferative neoplasms (MPN) induces strong T-cell responses against mutant CALR yet fails to demonstrate clinical activity. Infiltration of tumor specific T cells into the tumor microenvironment is needed to attain a clinical response to therapeutic cancer vaccination., Aim: Determine if CALRmut specific T cells isolated from vaccinated patients enrich in the bone marrow upon completion of vaccination and explore possible explanations for the lack of enrichment., Methods: CALRmut specific T cells from four of ten vaccinated patients were expanded, enriched, and analyzed by T-cell receptor sequencing (TCRSeq). The TCRs identified were used as fingerprints of CALRmut specific T cells. Bone marrow aspirations from the four patients were acquired at baseline and at the end of trial. T cells were enriched from the bone marrow aspirations and analyzed by TCRSeq to identify the presence and fraction of CALRmut specific T cells at the two different time points. In silico calculations were performed to calculate the ratio between transformed cells and effector cells in patients with CALR mut MPN., Results: The fraction of CALRmut specific T cells in the bone marrow did not increase upon completion of the vaccination trial. In general, the T cell repertoire in the bone marrow remains relatively constant through the vaccination trial. The enriched and expanded CALRmut specific T cells recognize peripheral blood autologous CALR mut cells. In silico analyses demonstrate a high imbalance in the fraction of CALR mut cells and CALRmut specific effector T-cells in peripheral blood., Conclusion: CALRmut specific T cells do not enrich in the bone marrow after therapeutic cancer peptide vaccination against mutant CALR. The specific T cells recognize autologous peripheral blood derived CALR mut cells. In silico analyses demonstrate a high imbalance between the number of transformed cells and CALRmut specific effector T-cells in the periphery. We suggest that the high burden of transformed cells in the periphery compared to the number of effector cells could impact the ability of specific T cells to enrich in the bone marrow., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Holmström, Andersen, Traynor, Ahmad, Lisle, Handlos Grauslund, Skov, Kjær, Ottesen, Gjerstorff, Hasselbalch and Andersen.)

Checkpoint inhibition (CPI) therapy and adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL-based ACT) are the two most effective immunotherapies for the treatment of metastatic melanoma. While CPI has been the dominating therapy in the past decade, TIL-based ACT is beneficial for individuals even after progression on previous immunotherapies. Given that notable differences in response have been made when used as a subsequent treatment, we investigated how the qualities of TILs changed when the ex vivo microenvironment of intact tumor fragments were modulated with checkpoint inhibitors targeting programmed death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Initially, we show that unmodified TILs from CPI-resistant individuals can be produced, are overwhelmingly terminally differentiated, and are capable of responding to tumor. We then investigate these properties in ex vivo checkpoint modulated TILs finding that that they retain these qualities. Lastly, we confirmed the specificity of the TILs to the highest responding tumor antigens, and identified this reactivity resides largely in CD39 + CD69 + terminally differentiated populations. Overall, we found that anti-PD-1 will alter the proliferative capacity while anti-CTLA4 will influence breadth of antigen specificity., Competing Interests: IMS has lectured for or had advisory board relationships with MSD, Sanofi Aventis, BMS, Pierre Fabre, Novartis, TILT Biotherapeutics, IO Biotech, and Novo Nordisk. IS has received research grants from Lytix biopharma, IO Biotech, BMS, Adaptimmune, and TILT Biotherapeutics. IS is a co-founder and shareholder for the company IO Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor NO declared a shared parent affiliation with the authors TH, CF, JG, TB, MP, MD, MA, IMS and OM at the time of review., (Copyright © 2023 Hulen, Friese, Kristensen, Granhøj, Borch, Peeters, Donia, Andersen, Hadrup, Svane and Met.)

Background: We have previously published initial efficacy of the indoleamine 2,3-dioxygenase (IDO)/anti-programmed death ligand 1 (PD-L1) vaccine in combination with nivolumab in 30 anti-PD-1 therapy naïve patients with metastatic melanoma (cohort A). We now report long-term follow-up of patients in cohort A. Further, we report results from cohort B, where the peptide vaccine was added to anti-PD-1 therapy for patients with progressive disease during anti-PD-1 treatment., Methods: All patients were treated with a therapeutic peptide vaccine in Montanide targeting IDO and PD-L1 combined with nivolumab (NCT03047928). A long-term follow-up of safety, response rates, and survival rates were performed in cohort A including patient subgroup analyses. Safety and clinical responses were analyzed for cohort B., Results: Cohort A: At data cut-off, January 5, 2023, the overall response rate (ORR) was 80%, and 50% of the 30 patients obtained a complete response (CR). The median progression-free survival (mPFS) was 25.5 months (95% CI 8.8 to 39), and median overall survival (mOS) was not reached (NR) (95% CI 36.4 to NR). The minimum follow-up time was 29.8 months, and the median follow-up was 45.3 months (IQR 34.8-59.2). A subgroup evaluation further revealed that cohort A patients with unfavorable baseline characteristics, including either PD-L1 negative tumors (n=13), elevated lactate dehydrogenase (LDH) levels (n=11), or M1c (n=17) obtained both favorable response rates and durable responses. The ORR was 61.5%, 79%, and 88% for patients with PD-L1 - tumors, elevated LDH, and M1c, respectively. The mPFS was 7.1 months for patients with PD-L1 - tumors, 30.9 months for patients with elevated LDH, and 27.9 months for M1c patients. Cohort B: At data cut-off, the best overall response was stable disease for 2 of the 10 evaluable patients. The mPFS was 2.4 months (95% CI 1.38 to 2.52), and the mOS was 16.7 months (95% CI 4.13 to NR)., Conclusion: This long-term follow-up confirms the promising and durable responses in cohort A. Subgroup analyses of patients with unfavorable baseline characteristics revealed that high response rates and survival rates were also found in patients with either PD-L1 negative tumors, elevated LDH levels, or M1c. No meaningful clinical effect was demonstrated in cohort B patients., Trial Registration Number: NCT03047928., Competing Interests: Competing interests: MHA has patent applications allocated to the biotech company IO Biotech ApS concerning IDO and PD-L1 peptide therapy. MHA is a shareholder, founder, and advisor for the company IO Biotech. EE is employed at IO Biotech. IMS has either lectured for or had relationships regarding advisory board with Novo Nordisk, MSD, Novartis, Pierre Fabre Sanofi Aventis, BMS, IO Biotech, and TILT Biotherapeutics. IMS received research grants from BMS, IO Biotech, Adaptimmune, Lytix biopharma, and TILT Biotherapeutics. IMS is the shareholder and cofounder of the biotech company IO Biotech ApS. CLL and JWK do not have conflicts of interest., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The B-cell lymphoma-extra-large (Bcl-XL) protein plays an important role in cancer cells' resistance to apoptosis. Pre-clinical studies have shown that vaccination with Bcl-XL-derived peptides can induce tumor-specific T cell responses that may lead to the elimination of cancer cells. Furthermore, pre-clinical studies of the novel adjuvant CAF ® 09b have shown that intraperitoneal (IP) injections of this adjuvant can improve the activation of the immune system. In this study, patients with hormone-sensitive prostate cancer (PC) received a vaccine consisting of Bcl-XL-peptide with CAF ® 09b as an adjuvant. The primary aim was to evaluate the tolerability and safety of IP and intramuscular (IM) administration, determine the optimal route of administration, and characterize vaccine immunogenicity., Patients and Methods: Twenty patients were included. A total of six vaccinations were scheduled: in Group A (IM to IP injections), ten patients received three vaccines IM biweekly; after a three-week pause, patients then received three vaccines IP biweekly. In Group B (IP to IM injections), ten patients received IP vaccines first, followed by IM under a similar vaccination schedule. Safety was assessed by logging and evaluating adverse events (AE) according to Common Terminology Criteria for Adverse Events (CTCAE v. 4.0). Vaccines-induced immune responses were analyzed by Enzyme-Linked Immunospot and flow cytometry., Results: No serious AEs were reported. Although an increase in T cell response against the Bcl-XL-peptide was found in all patients, a larger proportion of patients in group B demonstrated earlier and stronger immune responses to the vaccine compared to patients in group A. Further, we demonstrated vaccine-induced immunity towards patient-specific CD4, and CD8 T cell epitopes embedded in Bcl-XL-peptide and an increase in CD4 and CD8 T cell activation markers CD107a and CD137 following vaccination. At a median follow-up of 21 months, no patients had experienced clinically significant disease progression., Conclusion: The Bcl-XL-peptide-CAF ® 09b vaccination was feasible and safe in patients with l hormone-sensitive PC. In addition, the vaccine was immunogenic and able to elicit CD4 and CD8 T cell responses with initial IP administration eliciting early and high levels of vaccine-specific responses in a higher number og patients., Clinical Trial Registration: https://clinicaltrials.gov, identifier NCT03412786., Competing Interests: MD has the past two years received proprietary data access from Bristol-Myers Squibb and Genentech, is advisor of Achilles Therapeutics, and has received honoraria for lectures from Roche and Novartis. IS has received for consultancies honoraria as well as lectures from Roche, MSD, Novartis, Pierre Fabre, and Bristol-Myers Squibb; and financial support to attend conferences from Bristol-Myers Squibb, MSD, and Novartis. GW, LA, and DC are co-inventors on patents covering CAF®09b. All the rights have been assigned to Statens Serum Institut SSI, a Danish not-for-profit governmental institute.Within the last two years, PK has received honoraria for lectures from Jannsen Cilag and financial support to attend conferences from Pfizer. Author EM was employed by company IO Biotech Aps, Copenhagen, Denmark. Author MK was employed by company EVAXION BIOTECH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SEBM declared a past co-authorship with the author DC to the handling editor., (Copyright © 2023 Mørk, Kongsted, Westergaard, Albieri, Granhøj, Donia, Martinenaite, Holmström, Madsen, Kverneland, Kjeldsen, Holmstroem, Lorentzen, Nørgaard, Andreasen, Wood, Christensen, Klausen, Hadrup, thor Straten, Andersen and Svane.)

Background: Circulating transforming growth factor-β (TGF-β)-specific T cells that recognize TGF-β-expressing immune regulatory cells have been described in patients with cancer. TGF-β-derived peptide vaccination modulates the tumor microenvironment and has shown clinical effects in animal models of pancreatic cancer (PC). TGF-β-expressing regulatory cells are especially elevated in PC and may prevent the clinical response to immune checkpoint inhibitors (ICIs). Thus, in the present study we investigated the significance of TGF-β-specific T-cell immunity in patients with PC treated with ICI combined with radiotherapy in a randomized phase 2 study (CheckPAC)., Methods: Immune responses to a TGF-β-derived epitope entitled TGF-β-15 as well as epitopes from Clostridium tetani (tetanus) and influenza were measured in peripheral blood mononuclear cells (PBMCs) with interferon-ɣ enzyme-linked immunospot assays. PBMCs were isolated before and after treatment. Correlations between immune response data and clinical data were evaluated with parametric and non-parametric statistical methods. Survival was analyzed with univariate and multivariate Cox-regression. TGF-β-15 specific T cells were isolated and expanded and examined for recognition of autologous regulatory immune cells by flow cytometry., Results: PBMCs from 32 patients were analyzed for immune responses to the TGF-β-derived epitope entitled TGF-β-15. Patients with a strong TGF-β-specific immune response at treatment initiation had longer progression-free and overall survival, compared with patients with a weak or no TGF-β-specific immune response. This remained significant in multivariate analysis. Patients with weak and strong TGF-β-specific responses displayed similar responses towards viral antigens. Furthermore, we show that TGF-β-specific T cells from a clinical responder specifically reacted to and lysed autologous, regulatory immune cells. Finally, mimicking a TGF-β-15 vaccination, we showed that repeated stimulations with the TGF-β-15 epitope in vitro enhanced the immune response to TGF-β-15., Conclusion: A strong TGF-β-15 specific immune response was associated with clinical benefit and improved survival after ICI/radiotherapy for patients with PC. Importantly, the lack of TGF-β-specific T cells in some patients was not caused by a general immune dysfunction. TGF-β-specific T cells recognized regulatory immune cells and could be introduced in vitro in patients without spontaneous responses. Taken together, our data suggest that combining TGF-β-based vaccination with ICI/radiotherapy will be beneficial for patients with PC., Competing Interests: Competing interests: MHA has made an invention based on the use of TGF-β for vaccinations. The rights of the invention have been transferred to Copenhagen University Hospital Herlev, according to the Danish Law of Public Inventions at Public Research Institutions. The capital region has licensed the rights to the company IO Biotech ApS. The patent application was filed by IO Biotech ApS. MHA is founder, advisor and shareholder in IO Biotech. IMS is co-founder, advisor and shareholder in IO Biotech. The additional authors declare no competing financial interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The identification and characterization of tumor antigens are central objectives in developing anti-cancer immunotherapy. Traditionally, tumor-associated antigens (TAAs) are considered relatively restricted to tumor cells (i.e., overexpressed proteins in tumor cells), whereas tumor-specific antigens (TSAs) are considered unique to tumor cells. Recent studies have focused on identifying patient-specific neoantigens, which might be highly immunogenic because they are not expressed in normal tissues. The opposite strategy has emerged with the discovery of anti-regulatory T cells (anti-Tregs) that recognize and attack many cell types in the tumor microenvironment, such as regulatory immune cells, in addition to tumor cells. The term proposed in this review is "tumor microenvironment antigens" (TMAs) to describe the antigens that draw this attack. As therapeutic targets, TMAs offer several advantages that differentiate them from more traditional tumor antigens. Targeting TMAs leads not only to a direct attack on tumor cells but also to modulation of the tumor microenvironment, rendering it immunocompetent and tumor-hostile. Of note, in contrast to TAAs and TSAs, TMAs also are expressed in non-transformed cells with consistent human leukocyte antigen (HLA) expression. Inflammation often induces HLA expression in malignant cells, so that targeting TMAs could additionally affect tumors with no or very low levels of surface HLA expression. This review defines the characteristics, differences, and advantages of TMAs compared with traditional tumor antigens and discusses the use of these antigens in immune modulatory vaccines as an attractive approach to immunotherapy. Different TMAs are expressed by different cells and could be combined in anti-cancer immunotherapies to attack tumor cells directly and modulate local immune cells to create a tumor-hostile microenvironment and inhibit tumor angiogenesis. Immune modulatory vaccines offer an approach for combinatorial therapy with additional immunotherapy including checkpoint blockade, cellular therapy, or traditional cancer vaccines. These combinations would increase the number of patients who can benefit from such therapeutic measures, which all have optimal efficiency in inflamed tumors., (© 2022. The Author(s).)

Introduction: Arginase-1 (ARG1) and Programed death ligand-1 (PD-L1) play a vital role in immunosuppression in myeloproliferative neoplasms (MPNs) and directly inhibit T-cell activation and proliferation. We previously identified spontaneous T-cell responses towards PD-L1 and ARG1 derived peptide epitopes in patients with MPNs. In the present First-in-Man study we tested dual vaccinations of ARG1- derived and PD-L1-derived peptides, combined with Montanide ISA-51 as adjuvant, in patients with Janus Kinase 2 (JAK2) V617F-mutated MPN., Methods: Safety and efficacy of vaccination with ARG1- derived and PD-L1-derived peptides with montanide as an adjuvant was tested in 9 patients with MPN The primary end point was safety and toxicity evaluation. The secondary end point was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT04051307)., Results: The study included 9 patients with JAK2 -mutant MPN of which 8 received all 24 planned vaccines within a 9-month treatment period. Patients reported only grade 1 and 2 vaccine related adverse events. No alterations in peripheral blood counts were identified, and serial measurements of the JAK2V617F allelic burden showed that none of the patients achieved a molecular response during the treatment period. The vaccines induced strong immune responses against both ARG1 and PD-L1- derived epitopes in the peripheral blood of all patients, and vaccine-specific skin-infiltrating lymphocytes from 5/6 patients could be expanded in vitro after a delayed-type hypersensitivity test. In two patients we also detected both ARG1- and PD-L1-specific T cells in bone marrow samples at the end of trial. Intracellular cytokine staining revealed IFNγ and TNFγ producing CD4 + - and CD8 + - T cells specific against both vaccine epitopes. Throughout the study, the peripheral CD8/CD4 ratio increased significantly, and the CD8 + TEMRA subpopulation was enlarged. We also identified a significant decrease in PD-L1 mRNA expression in CD14 + myeloid cells in the peripheral blood in all treated patients and a decrease in ARG1 mRNA expression in bone marrow of 6 out of 7 evaluated patients., Conclusion: Overall, the ARG1- and PD-L1-derived vaccines were safe and tolerable and induced strong T-cell responses in all patients. These results warrant further studies of the vaccine in other settings or in combination with additional immune-activating treatments., Competing Interests: MA is named as an inventor on various patent applications relating to therapeutic uses of PD-L1 and Arginase peptides. These patent applications are assigned to the company IO Biotech ApS, which is developing immune-modulating cancer treatments. MA is founder, shareholder and advisor of IO Biotech ApS. EM is an employee at IO Biotech ApS. IS is co-founder, shareholder and advisor of IO Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor NO declared a shared parent affiliation with the authors, MHA, JHG, MOH, EM, TL, HJG, DEF, UK, REM, NJ, IMS, at the time of review., (Copyright © 2023 Grauslund, Holmström, Martinenaite, Lisle, Glöckner, El Fassi, Klausen, Mortensen, Jørgensen, Kjær, Skov, Svane, Hasselbalch and Andersen.)

Competing Interests: MHA is founder, shareholder, and scientific advisor of IO Biotech.

Background: Pancreatic ductal adenocarcinoma (PDAC) is associated with very poor survival, making it the third and fourth leading cause of all cancer-related deaths in the USA and European Union, respectively. The tumor microenvironment (TME) in PDAC is highly immunosuppressive and desmoplastic, which could explain the limited therapeutic effect of immunotherapy in PDAC. One of the key molecules that contributes to immunosuppression and fibrosis is transforming growth factor-β (TGFβ). The aim of this study was to target the immunosuppressive and fibrotic TME in PDAC using a novel immune modulatory vaccine with TGFβ-derived peptides in a murine model of pancreatic cancer., Methods: C57BL/6 mice were subcutaneously inoculated with Pan02 PDAC cells. Mice were treated with TGFβ1-derived peptides (major histocompatibility complex (MHC)-I and MHC-II-restricted) adjuvanted with Montanide ISA 51VG. The presence of treatment-induced TGFβ-specific T cells was assessed by ELISpot (enzyme-linked immunospot). Changes in the immune infiltration and gene expression profile in tumor samples were characterized by flow cytometry, reverse transcription-quantitative PCR (RT-qPCR), and bulk RNA sequencing., Results: Treatment with immunogenic TGFβ-derived peptides was safe and controlled tumor growth in Pan02 tumor-bearing mice. Enlargement of tumor-draining lymph nodes in vaccinated mice positively correlated to the control of tumor growth. Analysis of immune infiltration and gene expression in Pan02 tumors revealed that TGFβ-derived peptide vaccine increased the infiltration of CD8 + T cells and the intratumoral M1/M2 macrophage ratio, it increased the expression of genes involved in immune activation and immune response to tumors, and it reduced the expression of myofibroblast-like cancer-associated fibroblast (CAF)-related genes and genes encoding fibroblast-derived collagens. Finally, we confirmed that TGFβ-derived peptide vaccine actively modulated the TME, as the ability of T cells to proliferate was restored when exposed to tumor-conditioned media from vaccinated mice compared with media from untreated mice., Conclusion: This study demonstrates the antitumor activity of TGFβ-derived multipeptide vaccination in a murine tumor model of PDAC. The data suggest that the vaccine targets immunosuppression and fibrosis in the TME by polarizing the cellular composition towards a more pro-inflammatory phenotype. Our findings support the feasibility and potential of TGFβ-derived peptide vaccination as a novel immunotherapeutic approach to target immunosuppression in the TME., Competing Interests: Competing interests: MHA has made an invention based on the use of transforming growth factor-β-derived peptides for vaccinations. A patent application directed to the invention is owned by the company IO Biotech ApS and lists MHA as the sole inventor. MHA is advisor and shareholder at IO Biotech. IL and EM are employees at IO Biotech. The additional authors declare no competing financial interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Chronic lymphocytic leukemia (CLL) patients with unmutated immunoglobulin heavy chain (IgHV) are at risk of early disease progression compared to patients with mutated IgHV. As a preventive strategy, we treated 19 previously untreated CLL patients with unmutated IgHV in a phase 1/2 trial (clinicaltrials.gov, NCT03939234) exploring the efficacy and toxicity of a therapeutic cancer vaccine containing peptides derived from programmed death ligand 1 (PD-L1) and ligand 2 (PD-L2), hoping to restore immunological control of the disease. According to the International Workshop on Chronic lymphocytic Leukemia (iwCLL) response criteria, no patients obtained a response; however, during follow-up, one patient had complete normalization of the peripheral lymphocyte count and remained in biochemical remission after a follow-up time of 15 months. At the end of treatment, one patient had progressed, and 17 patients had stable disease. During follow-up with a median time of 23.5 months since inclusion, seven patients had progressed, and eight patients had stable disease. The median time to first treatment (TTFT) from diagnosis was 90.3 months with a median follow-up time of 50.1 months. This apparent favorable outcome in TTFT needs to be investigated in a randomized setting, as our population may have been biased. More than 80% of patients obtained vaccine-specific immune responses, confirming the immunogenicity of the vaccine. The vaccine was generally well tolerated with only grade I-II adverse events. Although there were some signs of clinical effects, the vaccine seems to be insufficient as monotherapy in CLL, possibly due to a high tumor burden. The efficacy of the vaccine should preferably be tested in combination with novel targeted therapies or as a consolidating treatment., Competing Interests: The first author UK was employed at National Center for Cancer Immune Therapy CCIT-DK, supported by a research agreement with IO biotech APS during the trial. Further, the study is part of a PhD thesis by UK, who answers to MA, his primary supervisor and employer. MA is named as an inventor on various patent applications relating to therapeutic uses of PD-L1 and PD-L2 peptides. These patent applications are assigned to the company IO Biotech, which is developing immune-modulating cancer treatments. MA and IS have cofounded IO Biotech and are shareholders and active advisors in the company. CN received research funding and/or consultancy fees outside this study from AbbVie, AstraZeneca, Janssen, CSL Behring, BeiGene, Genmab, Takeda, and Octapharma. Large parts of the study have taken place at CCIT-DK, which is an organization that has an economical interest in the tested vaccines according to the patent laws in Denmark. JG, SW-B, SA, MH, MOH, and ÖM were employed at CCIT-DK during the trial, answering to either IMS or MHA. EM is an employee at IO Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Klausen, Grauslund, Jørgensen, Ahmad, Jonassen, Weis-Banke, Martinenaite, Pedersen, Lisle, Gang, Enggaard, Hansen, Holmström, Met, Svane, Niemann, Pedersen and Andersen.)

Background: Arginase-1-producing cells inhibit T cell-mediated anti-tumor responses by reducing L-arginine levels in the tumor microenvironment. T cell-facilitated elimination of arginase-1-expressing cells could potentially restore L-arginine levels and improve anti-tumor responses. The activation of arginase-1-specific T cells may convert the immunosuppressive tumor microenvironment and induce or strengthen local Th1 inflammation. In the current clinical study, we examined the safety and immunogenicity of arginase-1-based peptide vaccination., Methods: In this clinical phase I trial, ten patients with treatment-refractory progressive solid tumors were treated. The patients received an arginase-1 peptide vaccine comprising three 20-mer peptides from the ARG1 immunological "hot spot" region in combination with the adjuvant Montanide ISA-51. The vaccines were administered subcutaneously every third week (maximum 16 vaccines). The primary endpoint was to evaluate safety assessed by Common Terminology Criteria for Adverse Events 4.0 and laboratory monitoring. Vaccine-specific immune responses were evaluated using enzyme-linked immune absorbent spot assays and intracellular cytokine staining on peripheral blood mononuclear cells. Clinical responses were evaluated using Response Evaluation Criteria in Solid Tumors 1.1., Results: The vaccination was feasible, and no vaccine-related grade 3-4 adverse events were registered. Nine (90%) of ten patients exhibited peptide-specific immune responses in peripheral blood mononuclear cells. Six (86%) of the seven evaluable patients developed a reactive T cell response against at least one of the ARG1 peptides during treatment. A phenotypic classification revealed that arginase-1 vaccine-specific T cells were both CD4+ T cells and CD8+ T cells. Two (20%) of ten patients obtained stable disease for respectively four- and seven months on vaccination treatment., Conclusion: The peptide vaccine against arginase-1 was safe. Nine (90%) of ten patients had measurable peptide-specific responses in the periphery blood, and two (20%) of ten patients attained stable disease on protocol treatment., Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT03689192, identifier NCT03689192., Competing Interests: MA has various patent applications in relation to the therapeutic uses of ARG1 peptides. The patents are allocated to the company IO Biotech. MA is a founder, advisor, and shareholder for IO Biotech. EM, AP, and EE are employees at IO Biotech. IS has lectured for or had advisory board relationships with MSD, Sanofi Aventis, BMS, Pierre Fabre, Novartis, TILT Biotherapeutics, IO Biotech, and Novo Nordisk. IS has received research grants from Lytix biopharma, IO Biotech, BMS, Adaptimmune, and TILT Biotherapeutics. IS is a co-founder and shareholder for the company IO Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lorentzen, Martinenaite, Kjeldsen, Holmstroem, Mørk, Pedersen, Ehrnrooth, Andersen and Svane.)

Background: High expression of the metabolic enzyme arginase-2 (ARG2) by cancer cells, regulatory immune cells, or cells of the tumor stroma can reduce the availability of arginine (L-Arg) in the tumor microenvironment (TME). Depletion of L-Arg has detrimental consequences for T cells and leads to T-cell dysfunction and suppression of anticancer immune responses. Previous work from our group has demonstrated the presence of proinflammatory ARG2-specific CD4 T cells that inhibited tumor growth in murine models on activation with ARG2-derived peptides. In this study, we investigated the natural occurrence of ARG2-specific CD8 T cells in both healthy donors (HDs) and patients with cancer, along with their immunomodulatory capabilities in the context of the TME., Materials and Methods: A library of 15 major histocompatibility complex (MHC) class I-restricted ARG2-derived peptides were screened in HD peripheral blood mononuclear cells using interferon gamma (IFN-γ) ELISPOT. ARG2-specific CD8 T-cell responses were identified using intracellular cytokine staining and ARG2-specific CD8 T-cell cultures were established by enrichment and rapid expansion following in vitro peptide stimulation. The reactivity of the cultures toward ARG2-expressing cells, including cancer cell lines and activated regulatory T cells (Tregs), was assessed using IFN-γ ELISPOT and a chromium release assay. The Treg signature was validated based on proliferation suppression assays, flow cytometry and quantitative reverse transcription PCR (RT-qPCR). In addition, vaccinations with ARG2-derived epitopes were performed in the murine Pan02 tumor model, and induction of ARG2-specific T-cell responses was evaluated with IFN-γ ELISPOT. RNAseq and subsequent GO-term and ImmuCC analysis was performed on the tumor tissue., Results: We describe the existence of ARG2-specific CD8 + T cells and demonstrate these CD8 + T-cell responses in both HDs and patients with cancer. ARG2-specific T cells recognize and react to an ARG2-derived peptide presented in the context of HLA-B8 and exert their cytotoxic function against cancer cells with endogenous ARG2 expression. We demonstrate that ARG2-specific T cells can specifically recognize and react to activated Tregs with high ARG2 expression. Finally, we observe tumor growth suppression and antitumorigenic immunomodulation following ARG2 vaccination in an in vivo setting., Conclusion: These findings highlight the ability of ARG2-specific T cells to modulate the immunosuppressive TME and suggest that ARG2-based immunomodulatory vaccines may be an interesting option for cancer immunotherapy., Competing Interests: Competing interests: MHA is named as an inventor on various patent applications relating to the therapeutic use of arginase peptides, including ARG2 peptides, for vaccination. These patent applications have been transferred to the company IO Biotech ApS whose purpose is to develop immunomodulatory vaccines for cancer treatment. MHA is cofounder, shareholder and scientific advisor of IO Biotech ApS. IMS is cofounder, shareholder and clinical advisor of IO Biotech ApS. The remaining authors declare no conflicts of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

CCL22 is a macrophage-derived immunosuppressive chemokine that recruits regulatory T cells through the CCL22:CCR4 axis. CCL22 was shown to play a key role in suppressing anti-cancer immune responses in different cancer types. Recently, we showed that CCL22-specific T cells generated from cancer patients could kill CCL22-expressing tumor cells and directly influence the levels of CCL22 in vitro . The present study aimed to provide a rationale for developing a CCL22-targeting immunotherapy. Vaccination with CCL22-derived peptides induced CCL22-specific T-cell responses in both BALB/c and C57BL/6 mice, assessed by interferon-γ secretion ex vivo . Anti-tumor efficacy of the peptides was evaluated in mouse models engrafted with syngeneic tumor models showing a reduced tumor growth and prolonged survival of the treated mice. Vaccination induced changes in the cellular composition of immune cells that infiltrated the tumor microenvironment assessed with multicolor flow cytometry. In particular, the infiltration of CD8 + cells and M1 macrophages increased, which increased the CD8/Treg and the M1/M2 macrophage ratio. This study provided preclinical evidence that targeting CCL22 with CCL22 peptide vaccines modulated the immune milieu in the tumor microenvironment. This modulation led to an augmentation of anti-tumor responses. This study provided a rationale for developing a novel immunotherapeutic modality in cancer., Competing Interests: ILM, KLK, MC, EM and AWP are employed by IO Biotech. M-BZ is an employee and a shareholder in IO Biotech. MHA is Founder, a shareholder and a Scientific Advisory Board member in IO Biotech. The other authors declare no direct conflicts of interest., (© 2022 IO Biotech ApS. Published with license by Taylor & Francis Group, LLC.)

Myeloproliferative neoplasms (MPN) are chronic cancers of the hematopoietic stem cells in the bone marrow, and patients often harbor elevated numbers of circulating platelets (PLT). We investigated the frequencies of circulating PLT-lymphocyte aggregates in MPN patients and the effect of PLT-binding on CD8 T cell function. The phenotype of these aggregates was evaluated in 50 MPN patients and 24 controls, using flow cytometry. In vitro studies compared the proliferation, cytokine release, and cytoxicity of PLT-bound and PLT-free CD8 T cells. Frequencies of PLT-CD8 T cell aggregates, were significantly elevated in MPN patients. Advanced disease stage and CALR mutation associated with the highest aggregate frequencies with a predominance of PLT-binding to antigen-experienced CD8 T cells. PLT-bound CD8 T cells showed reduction in proliferation and cytotoxic capacity. Our data suggest that CD8 T cell responses are jeopardized in MPN patients. JAK2 and CALR exon 9 mutations - the two predominant driver mutations in MPN - are targets for natural T cell responses in MPN patients. Moreover, MPN patients have more infections compared to background. Thus, PLT binding to antigen experienced CD8 T cells could play a role in the inadequacy of the immune system to control MPN disease progression and prevent recurrent infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Carnaz Simões, Holmström, Aehnlich, Rahbech, Radziwon-Balicka, Zamora, Wirenfeldt Klausen, Skov, Kjær, Ellervik, Fassi, Vidal, Hasselbalch, Andersen and thor Straten.)

The immunosuppressive tumor microenvironment (TME) does not allow generation and expansion of antitumor effector cells. One of the potent immunosuppressive factors present in the TME is the indoleamine-pyrrole 2,3-dioxygenase (IDO) enzyme, produced mainly by cancer cells and suppressive immune cells of myeloid origin. In fact, IDO+ myeloid-derived suppressor cells (MDSC) and dendritic cells (DC) tend to be more suppressive than their IDO- counterparts. Hence, therapeutic approaches that would target the IDO+ cells in the TME, while sparing the antigen-presenting functions of IDO- myeloid populations, are needed. Using an IDO-specific peptide vaccine (IDO vaccine), we explored the possibility of generating effector cells against IDO and non-IDO tumor-derived antigens. For this, IDO-secreting (B16F10 melanoma) and non-IDO-secreting (TC-1) mouse tumor models were employed. We showed that the IDO vaccine significantly reduced tumor growth and enhanced survival of mice in both the tumor models, which associated with a robust induction of IDO-specific effector cells in the TME. The IDO vaccine significantly enhanced the antitumor efficacy of non-IDO tumor antigen-specific vaccines, leading to an increase in the number of total and antigen-specific activated CD8+ T cells (IFNγ+ and granzyme B+). Treatment with the IDO vaccine significantly reduced the numbers of IDO+ MDSCs and DCs, and immunosuppressive regulatory T cells in both tumor models, resulting in enhanced therapeutic ratios. Together, we showed that vaccination against IDO is a promising therapeutic option for both IDO-producing and non-IDO-producing tumors. The IDO vaccine selectively ablates the IDO+ compartment in the TME, leading to a significant enhancement of the immune responses against other tumor antigen-specific vaccines., (©2022 The Authors; Published by the American Association for Cancer Research.)

Galectin-3 (Gal3) can be expressed by many cells in the tumor microenvironment (TME), including cancer cells, cancer-associated fibroblasts, tumor-associated macrophages, and regulatory T cells (Tregs). In addition to immunosuppression, Gal3 expression has been connected to malignant cell transformation, tumor progression, and metastasis. In the present study, we found spontaneous T-cell responses against Gal3-derived peptides in PBMCs from both healthy donors and cancer patients. We isolated and expanded these Gal3-specific T cells in vitro and showed that they could directly recognize target cells that expressed Gal3. Finally, therapeutic vaccination with a long Gal3-derived peptide epitope, which induced the expansion of Gal3-specific CD8 + T cells in vivo , showed a significant tumor-growth delay in mice inoculated with EO771.LMB metastatic mammary tumor cells. This was associated with a significantly lower percentage of both Tregs and tumor-infiltrating Gal3 + cells in the non-myeloid CD45 + CD11b - compartment and with an alteration of the T-cell memory populations in the spleens of Gal3-vaccinated mice. These results suggest that by activating Gal3-specific T cells by an immune-modulatory vaccination, we can target Gal3-producing cells in the TME, and thereby induce a more immune permissive TME. This indicates that Gal3 could be a novel target for therapeutic cancer vaccines., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Anti-programmed death (PD)-1 (aPD1) therapy is an effective treatment for metastatic melanoma (MM); however, over 50% of patients progress due to resistance. We tested a first-in-class immune-modulatory vaccine (IO102/IO103) against indoleamine 2,3-dioxygenase (IDO) and PD ligand 1 (PD-L1), targeting immunosuppressive cells and tumor cells expressing IDO and/or PD-L1 (IDO/PD-L1), combined with nivolumab. Thirty aPD1 therapy-naive patients with MM were treated in a phase 1/2 study ( https://clinicaltrials.gov/ , NCT03047928). The primary endpoint was feasibility and safety; the systemic toxicity profile was comparable to that of nivolumab monotherapy. Secondary endpoints were efficacy and immunogenicity; an objective response rate (ORR) of 80% (confidence interval (CI), 62.7-90.5%) was reached, with 43% (CI, 27.4-60.8%) complete responses. After a median follow-up of 22.9 months, the median progression-free survival (PFS) was 26 months (CI, 15.4-69 months). Median overall survival (OS) was not reached. Vaccine-specific responses assessed in vitro were detected in the blood of >93% of patients during vaccination. Vaccine-reactive T cells comprised CD4 + and CD8 + T cells with activity against IDO- and PD-L1-expressing cancer and immune cells. T cell influx of peripherally expanded T cells into tumor sites was observed in responding patients, and general enrichment of IDO- and PD-L1-specific clones after treatment was documented. These clinical efficacy and favorable safety data support further validation in a larger randomized trial to confirm the clinical potential of this immunomodulating approach., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Expression of the L-arginine catabolizing enzyme arginase 1 (ARG1) is a central immunosuppressive mechanism mediated by tumor-educated myeloid cells. Increased activity of ARG1 promotes the formation of an immunosuppressive microenvironment and leads to a more aggressive phenotype in many cancers. Intrinsic T-cell immunity against ARG1-derived epitopes in the peripheral blood of cancer patients and healthy subjects has previously been demonstrated. To evaluate the antitumor efficacy of ARG1-derived peptide vaccines as a monotherapy and as a combinational therapy with checkpoint blockade, different in vivo syngeneic mouse tumor models were utilized. To evaluate the antitumor effects, flow cytometry analysis and IHC were performed on tumors, and ELISPOT assays were performed to characterize immune responses. We show that ARG1-targeting therapeutic vaccines were able to activate endogenous antitumor immunity in several in vivo syngeneic mouse tumor models and to modulate the cell composition of the tumor microenvironment without causing any associated side effects or systemic toxicity. ARG1-targeting vaccines in combination with anti-PD-1 also resulted in increased T-cell infiltration, decreased ARG1 expression, reduced suppressive function of tumor-educated myeloid cells, and a shift in the M1/M2 ratio of tumor-infiltrating macrophages. These results indicated that the induced shift toward a more proinflammatory microenvironment by ARG1-targeting immunotherapy favors effective tumor control when combined with anti-PD-1 checkpoint blockade. Our data illustrate the ability of ARG1-based immune modulatory vaccination to elicit antigen-specific immunosurveillance and imply the feasibility of this novel immunotherapeutic approach for clinical translation., (©2021 American Association for Cancer Research.)

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8 + and CD4 + tumor-specific reactive TILs in vitro , using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8 + TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4 + and CD8 + tumor-specific reactive TILs. In situ , the combined detection of TNFRSF9 , TNF , and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro , which can be rapidly adopted in most cancer immunology laboratories., Competing Interests: MD has received honoraria for lectures from Roche and Novartis (past two years). IMS has received honoraria for consultancies and lectures from Novartis, Roche, Merck, and Bristol-Myers Squibb; a restricted research grant from Novartis; and financial support for attending symposia from Bristol-Myers Squibb, Merck, Novartis, Pfizer, and Roche. HDR is currently an employee at Novo Nordisk. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Draghi, Chamberlain, Khan, Papp, Lauss, Soraggi, Radic, Presti, Harbst, Gokuldass, Kverneland, Nielsen, Westergaard, Andersen, Csabai, Jönsson, Szallasi, Svane and Donia.)

Cells in the tumor microenvironment of Follicular lymphoma (FL) express checkpoint molecules such as programmed death ligands 1 and 2 (PD-L1 and PD-L2) and are suppressing anti-tumor immune activity. Stimulation of peripheral blood mononuclear cells (PBMC) with PD-L1 (IO103) or PD-L2 (IO120) peptides can activate specific T cells inducing anti-regulatory functions including cytotoxicity against PD-L1/PD-L2-expressing cells. In this study, we vaccinated eight FL patients with PD-L1 and PD-L2 peptides following treatment with standard chemotherapy. Patients experienced grade 1-2 injection site reaction (5/8) and mild flu-like symptoms (6/8). One patient experienced neutropenia and thrombocytopenia during pseudo-progression. Enzyme-linked immunospot detected vaccine-specific immune responses in PBMC from all patients, predominately toward PD-L1. The circulating immune composition was stable during treatment; however, we observed a reduction regulatory T cells, however, not significant. One patient achieved a complete remission during vaccination and two patients had pseudo-progression followed by long-term disease regression. Further examination of these early signs of clinical efficacy of the dual-epitope vaccine in a larger study is warranted., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Introduction: Lynch syndrome is a multi-tumor syndrome characterized by mismatch repair deficiency (MMR-d), microsatellite instability (MSI), and increased tumor-infiltrating lymphocytes (TILs) making these tumors candidates for treatment with immune checkpoint inhibitors. However, response may depend on tumor-induced immune evasion mechanisms, e.g. loss of Beta-2-Microglobulin (B2M) or upregulation of programmed death protein ligand 1 (PD-L1). We investigated the immune response and B2M and PD-L1 expression in Lynch syndrome-associated ovarian cancers., Methods: We successfully analyzed 30 Lynch syndrome-associated epithelial ovarian cancers collected through the Danish Hereditary Non-Polyposis Colorectal Cancer (HNPCC) register. MMR-d, MSI, immune response (CD3, CD8, and CD68), and immune evasion mechanisms (B2M and PD-L1) were investigated. Statistical associations between these markers were evaluated in addition to survival in relation to B2M/PD-L1., Results: Of the 29 evaluable tumors, 27 were MMR-d (93.1%). Likewise of 26 evaluable tumors, 14 were MSI (53.8%). MMR-d/MMR-proficiency associated with MSI/MSS in 60.0%. Half of the ovarian tumors presented with high levels of TILs. Loss of B2M expression was observed in 46.7% of the tumors, while expression of PD-L1 was seen in 28.0% of the cases. There was no association between B2M/PD-L1 and MSI/TILs/survival. Loss of B2M was often seen in tumors with low TILs (p = 0.056 or p = 0.059 for CD3 and CD8 positive cells, respectively)., Conclusion: MMR-d, MSI, and TILs are also seen in Lynch syndrome-associated ovarian cancers making these potential candidates for checkpoint-based immunotherapy. The clinical impact from immune evasion through loss of B2M needs to be investigated further in larger cohorts., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interests to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The calreticulin ( CALR ) exon 9 mutations that are identified in 20% of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN) generate immunogenic antigens. Thus, therapeutic cancer vaccination against mutant CALR could be a new treatment modality in CALR -mutant MPN., Methods: The safety and efficacy of vaccination with the peptide CALRLong36 derived from the CALR exon 9 mutations was tested in a phase I clinical vaccination trial with montanide as adjuvant. Ten patients with CALR mut MPN were included in the trial and received 15 vaccines over the course of one year. The primary end point was evaluation of safety and toxicity of the vaccine. Secondary endpoint was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT03566446)., Results: Patients had a median age of 59.5 years and a median disease duration of 6.5 years. All patients received the intended 15 vaccines, and the vaccines were deemed safe and tolerable as only two grade three AE were detected, and none of these were considered to be related to the vaccine. A decline in platelet counts relative to the platelets counts at baseline was detected during the first 100 days, however this did not translate into neither a clinical nor a molecular response in any of the patients. Immunomonitoring revealed that four of 10 patients had an in vitro interferon (IFN)-γ ELISPOT response to the CALRLong36 peptide at baseline, and four additional patients displayed a response in ELISPOT upon receiving three or more vaccines. The amplitude of the immune response increased during the entire vaccination schedule for patients with essential thrombocythemia. In contrast, the immune response in patients with primary myelofibrosis did not increase after three vaccines., Conclusion: Therapeutic cancer vaccination with peptide vaccines derived from mutant CALR with montanide as an adjuvant, is safe and tolerable. The vaccines did not induce any clinical responses. However, the majority of patients displayed a marked T-cell response to the vaccine upon completion of the trial. This suggests that vaccines directed against mutant CALR may be used with other cancer therapeutic modalities to enhance the anti-tumor immune response., Competing Interests: MOH, HH, and MA have filed a patent regarding the CALR exon 9 mutations as a target for cancer immune therapy. The patent has been transferred to University Hospital Zealand, Zealand Region and Copenhagen University Hospital at Herlev, Capital Region according to Danish Law concerning inventions made at public research institutions. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Handlos Grauslund, Holmström, Jørgensen, Klausen, Weis-Banke, El Fassi, Schöllkopf, Clausen, Gjerdrum, Breinholt, Kjeldsen, Hansen, Koschmieder, Chatain, Novotny, Petersen, Kjær, Skov, Met, Svane, Hasselbalch and Andersen.)

Antitumor activity of immune checkpoint blocking antibodies against programmed death 1 (PD-1) in basal cell carcinoma (BCC) has been described. IO103 is a peptide vaccine against the major PD-1 ligand PD-L1. A phase IIa study of vaccination with IO103 and Montanide adjuvant was conducted in patients with resectable BCC (NCT03714529). Vaccinations were given six times every 2 weeks (q2w), followed by three vaccines q4w in responders. Primary endpoints were clinical responses of target tumors, change in target tumor size and immune responses to the vaccine. Secondary endpoint was safety. One tumor per patient was designated target tumor and biopsied twice during the course of vaccination. Synchronous non-target BCCs were not biopsied during vaccinations. Ten patients were vaccinated (six patients received six vaccinations and four patients received nine vaccinations). A partial response (PR) was seen in two target tumors. Two complete responses (CR) and one PR were observed in eight non-target tumors in four patients. No tumors progressed. Related adverse events were grade 1 and reversible. Immune responses against IO103 were induced in blood samples from nine of ten and skin-infiltrating lymphocytes from five of the nine patients. The regressions seen in non-target tumors suggest that IO103 may be effective against a subtype of BCC.

Transforming growth factor-beta (TGFβ) is a highly potent immunosuppressive cytokine. Although TGFβ is a tumor suppressor in early/premalignant cancer lesions, the cytokine has several tumor-promoting effects in advanced cancer; abrogation of the antitumor immune response is one of the most important tumor-promoting effects. As several immunoregulatory mechanisms have recently been shown to be targets of specific T cells, we hypothesized that TGFβ is targeted by naturally occurring specific T cells and thus could be a potential target for immunomodulatory cancer vaccination. Hence, we tested healthy donor and cancer patient T cells for spontaneous T-cell responses specifically targeting 38 20-mer epitopes derived from TGFβ1. We identified numerous CD4 + and CD8 + T-cell responses against several epitopes in TGFβ. Additionally, several ex vivo responses were identified. By enriching specific T cells from different donors, we produced highly specific cultures specific to several TGFβ-derived epitopes. Cytotoxic CD8 + T-cell clones specific for both a 20-mer epitope and a 9-mer HLA-A2 restricted killed epitope peptide were pulsed in HLA-A2 + target cells and killed the HLA-A2 + cancer cell lines THP-1 and UKE-1. Additionally, stimulation of THP-1 cancer cells with cytokines that increased TGFβ expression increased the fraction of killed cells. In conclusion, we have shown that healthy donors and cancer patients harbor CD4 + and CD8 + T cells specific for TGFβ-derived epitopes and that cytotoxic T cells with specificity toward TGFβ-derived epitopes are able to recognize and kill cancer cell lines in a TGFβ-dependent manner.

Background: Immune checkpoint blockade with monoclonal antibodies targeting programmed death 1 (PD-1) and its ligand PD-L1 has played a major role in the rise of cancer immune therapy. We have identified naturally occurring self-reactive T cells specific to PD-L1 in both healthy donors and cancer patients. Stimulation with a PD-L1 peptide (IO103), activates these cells to exhibit inflammatory and anti-regulatory functions that include cytotoxicity against PD-L1-expressing target cells. This prompted the initiation of the present first-in-human study of vaccination with IO103, registered at clinicaltrials.org (NCT03042793)., Methods: Ten patients with multiple myeloma who were up to 6 months after high dose chemotherapy with autologous stem cell support, were enrolled. Subcutaneous vaccinations with IO103 with the adjuvant Montanide ISA 51 was given up to fifteen times during 1 year. Safety was assessed by the common toxicity criteria for adverse events (CTCAE). Immunogenicity of the vaccine was evaluated using IFNγ enzyme linked immunospot and intracellular cytokine staining on blood and skin infiltrating lymphocytes from sites of delayed-type hypersensitivity. The clinical course was described., Results: All adverse reactions to the PD-L1 vaccine were below CTCAE grade 3, and most were grade 1-2 injection site reactions. The total rate of adverse events was as expected for the population. All patients exhibited peptide specific immune responses in peripheral blood mononuclear cells and in skin-infiltrating lymphocytes after a delayed-type hypersensitivity test. The clinical course was as expected for the population. Three of 10 patients had improvements of responses which coincided with the vaccinations., Conclusion: Vaccination against PD-L1 was associated with low toxicity and high immunogenicity. This study has prompted the initiation of later phase trials to assess the vaccines efficacy., Clinical Trial Registration: clinicaltrials.org, identifier NCT03042793., (Copyright © 2020 Jørgensen, Klausen, Grauslund, Helleberg, Aagaard, Do, Ahmad, Olsen, Klausen, Breinholt, Hansen, Martinenaite, Met, Svane, Knudsen and Andersen.)

The RAS mutations are the most frequently occurring somatic mutations in humans, and several studies have established that T cells from patients with RAS -mutant cancer recognize and kill RAS -mutant cells. Enhancing the T cell response via therapeutic cancer vaccination against mutant RAS results in a clinical benefit to patients; thus, T cells specific to RAS mutations are effective at battling cancer. As the theory of cancer immuno-editing indicates that healthy donors may clear malignantly transformed cells via immune-mediated killing, and since T cells have been shown to recognize RAS -mutant cancer cells, we investigated whether healthy donors harbor T-cell responses specific to mutant RAS. We identified strong and frequent responses against several epitopes derived from the RAS codon 12 and codon 13 mutations. Some healthy donors demonstrated a response to several mutant epitopes, and some, but not all, exhibited cross-reactivity to the wild-type RAS epitope. In addition, several T cell responses were identified against mutant RAS epitopes in healthy donors directly ex vivo. Clones against mutant RAS epitopes were established from healthy donors, and several of these clones did not cross-react with the wild-type epitope. Finally, CD45RO + memory T cells from healthy donors demonstrated a strong response to several mutant RAS epitopes. Taken together, these data suggest that the immune system in healthy donors spontaneously clears malignantly transformed RAS -mutant cells, and the immune system consequently generates T-cell memory against the mutations.

Adoptive cell therapy (ACT), based on treatment with autologous tumor infiltrating lymphocyte (TIL)-derived or genetically modified chimeric antigen receptor (CAR) T cells, has become a potentially curative therapy for subgroups of patients with melanoma and hematological malignancies. To further improve response rates, and to broaden the applicability of ACT to more types of solid malignancies, it is necessary to explore and define strategies that can be used as adjuvant treatments to ACT. Stimulation of endogenous dendritic cells (DCs) alongside ACT can be used to promote epitope spreading and thereby decrease the risk of tumor escape due to target antigen downregulation, which is a common cause of disease relapse in initially responsive ACT treated patients. Addition of checkpoint blockade to ACT and DC stimulation might further enhance response rates by counteracting an eventual inactivation of infused and endogenously primed tumor-reactive T cells. This review will outline and discuss therapeutic strategies that can be utilized to engage endogenous DCs alongside ACT and checkpoint blockade, to strengthen the anti-tumor immune response., (Copyright © 2020 Hübbe, Jæhger, Andresen and Andersen.)

Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPN) are neoplastic diseases of the hematopoietic stem cells in the bone marrow. MPN are characterized by chronic inflammation and immune dysregulation. Of interest, the potent immunostimulatory cytokine interferon-α has been used to treat MPN for decades. A deeper understanding of the anti-cancer immune response and of the different immune regulatory mechanisms in patients with MPN has paved the way for an increased perception of the potential of cancer immunotherapy in MPN. Therapeutic vaccination targeting the driver mutations in MPN is one recently described potential new treatment modality. Furthermore, T cells can directly react against regulatory immune cells because they recognize proteins like arginase and programmed death ligand 1 (PD-L1). Therapeutic vaccination with arginase or PD-L1 therefore offers a novel way to directly affect immune inhibitory pathways, potentially altering tolerance to tumor antigens like mutant CALR and mutant JAK2. Other therapeutic options that could be used in concert with therapeutic cancer vaccines are immune checkpoint-blocking antibodies and interferon-α. For more advanced MPN, adoptive cellular therapy is a potential option that needs more preclinical investigation. In this review, we summarize current knowledge about the immune system in MPN and discuss the many opportunities for anti-cancer immunotherapy in patients with MPN.

Background: The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which subverts T-cell immunity at multiple levels, is itself subject to inherent T-cell reactivity. This intriguing deviation from central tolerance has been interpreted as counterbalancing IDO1-mediated immunosuppression. Based on this hypothesis, clinical studies employing an IDO1 peptide-based vaccine approach for cancer treatment have been initiated, but there remains a pressing need to further investigate the immunological ramifications of stimulating the anti-IDO1 T-cell response in this manner., Methods: CT26 colon carcinoma tumors were evaluated for expression of IDO1 protein by western blot analysis, immunofluorescence microscopy and flow cytometry. Mouse IDO1-derived peptides, predicted to bind either major histocompatibility complex (MHC) class I or II of the H2 d BALB/c strain, were emulsified in 50% Montanide for prophylactic or therapeutic vaccine treatment of CT26 tumor-bearing mice initiated either 7 days prior to or following tumor cell injection, respectively. In some therapeutic treatment experiments, administration of programmed cell death protein 1-binding antibody (anti-PD1 antibody) or epacadostat was concurrently initiated. Tumor size was determined by caliper measurements and comparative tumor growth suppression was assessed by longitudinal analyses of tumor growth data. For adoptive transfer, T cells from complete responder animals were isolated using paramagnetic beads and fluorescence-activated cell sorting., Results: This study identifies mouse MHC class I-directed and II-directed, IDO1-derived peptides capable of eliciting antitumor responses, despite finding IDO1 expressed exclusively in tumor-infiltrating immune cells. Treatment of established tumors with anti-PD1 antibody and class I-directed but not class II-directed IDO1 peptide vaccines produced an enhanced antitumor response. Likewise, class I-directed and II-directed IDO1 peptides elicited an enhanced combinatorial response, suggesting distinct mechanisms of action. Consistent with this interpretation, adoptive transfer of isolated CD8 + T cells from class I and CD4 + T cells from class II peptide-vaccinated responder mice delayed tumor growth. The class II-directed response was completely IDO1-dependent while the class I-directed response included an IDO1-independent component consistent with antigen spread., Conclusions: The in vivo antitumor effects demonstrated with IDO1-based vaccines via targeting of the tumor microenvironment highlight the utility of mouse models for further exploration and refinement of this novel vaccine-based approach to IDO1-directed cancer therapy and its potential to improve patient response rates to anti-PD1 therapy., Competing Interests: Competing interests: IO Biotech, which provided financial support for this project, is conducting IDO1 peptide vaccine-based clinical trials. KLK, IL and AWP are employed by IO Biotech, M-BZ is a Founder, Chief Executive Officer and a shareholder in IO Biotech, MHA is a Founder, Chief Scientific Officer and shareholder in IO Biotech and AJM is a Scientific Advisory Board member and shareholder in IO Biotech., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

One way that tumors evade immune destruction is through tumor and stromal cell expression of arginine-degrading enzyme arginase-2 (ARG2). Here we describe the existence of pro-inflammatory effector T-cells that recognize ARG2 and can directly target tumor and tumor-infiltrating cells. Using a library of 34 peptides covering the entire ARG2 sequence, we examined reactivity toward these peptides in peripheral blood mononuclear cells from cancer patients and healthy individuals. Interferon-γ ELISPOT revealed frequent immune responses against several of the peptides, indicating that ARG2-specific self-reactive T-cells are natural components of the human T-cell repertoire. Based on this, the most immunogenic ARG2 protein region was further characterized. By identifying conditions in the microenvironment that induce ARG2 expression in myeloid cells, we showed that ARG2-specific CD4T-cells isolated and expanded from a peripheral pool from a prostate cancer patient could recognize target cells in an ARG2-dependent manner. In the 'cold' in vivo tumor model Lewis lung carcinoma, we found that activation of ARG2-specific T-cells by vaccination significantly inhibited tumor growth. Immune-modulatory vaccines targeting ARG2 thus are a candidate strategy for cancer immunotherapy., Competing Interests: MHA has made an invention based on the use of ARG2 for vaccinations. The rights of the invention have been transferred to Copenhagen University Hospital Herlev, according to the Danish Law of Public Inventions at Public Research Institutions. The capital region has licensed the rights to the company IO Biotech ApS, whose purpose is to develop immune-modulating vaccines for cancer treatments. The patent application was filed by IO Biotech ApS. MHA is a shareholder and board member of IO Biotech ApS. IMS is a shareholder of IO Biotech ApS. EM and AWP are employed at IO Biotech ApS., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Sézary syndrome (SS) is a heterogeneous leukemic subtype of cutaneous T-cell lymphoma (CTCL) with generalized erythroderma, lymphadenopathy, and a poor prognosis. Advanced disease is invariably associated with severe immune dysregulation and the majority of patients die from infectious complications caused by microorganisms such as, Staphylococcus aureus, rather than from the lymphoma per se. Here, we examined if staphylococcal enterotoxins (SE) may shape the phenotype of malignant SS cells, including expression of the regulatory T-cell-associated marker FOXP3. Our studies with primary and cultured malignant cells show that SE induce expression of FOXP3 in malignant cells when exposed to nonmalignant cells. Mutations in the MHC class II binding domain of SE-A (SEA) largely block the effect indicating that the response relies at least in part on the MHC class II-mediated antigen presentation. Transwell experiments show that the effect is induced by soluble factors, partly blocked by anti-IL-2 antibody, and depends on STAT5 activation in malignant cells. Collectively, these findings show that SE stimulate nonmalignant cells to induce FOXP3 expression in malignant cells. Thus, differences in exposure to environmental factors, such as bacterial toxins may explain the heterogeneous FOXP3 expression in malignant cells in SS.

Cutaneous T-cell lymphoma (CTCL) represents a heterogeneous group of potentially devastating primary skin malignancies. Despite decades of intense research efforts, the pathogenesis is still not fully understood. In the early stages, both clinical and histopathological diagnosis is often difficult due to the ability of CTCL to masquerade as benign skin inflammatory dermatoses. Due to a lack of reliable biomarkers, it is also difficult to predict which patients will respond to therapy or progress towards severe recalcitrant disease. In this review, we discuss recent discoveries concerning dysregulated microRNA (miR) expression and putative pathological roles of oncogenic and tumor suppressive miRs in CTCL. We also focus on the interplay between miRs, histone deacetylase inhibitors, and oncogenic signaling pathways in malignant T cells as well as the impact of miRs in shaping the inflammatory tumor microenvironment. We highlight the potential use of miRs as diagnostic and prognostic markers, as well as their potential as therapeutic targets. Finally, we propose that the combined use of miR-modulating compounds with epigenetic drugs may provide a novel avenue for boosting the clinical efficacy of existing anti-cancer therapies in CTCL., Competing Interests: The authors declare no conflict of interest

Staphylococcus aureus and its toxins have been linked to disease progression and mortality in advanced stages of cutaneous T-cell lymphoma (CTCL). CD8 + T cells play a crucial role in anti-cancer responses and high CD8 + T cell numbers in tumor lesions are associated with a favorable prognosis in CTCL. Here, we show that CD8 + T cells from both healthy donors and Sézary syndrome patients are highly susceptible to cell death induced by Staphylococcal alpha-toxin, whereas malignant T cells are not. Importantly, alpha-toxin almost completely blocks cytotoxic killing of CTCL tumor cells by peptide-specific CD8 + T cells, leading to their escape from induced cell death and continued proliferation. These findings suggest that alpha-toxin may favor the persistence of malignant CTCL cells in vivo by inhibiting CD8 + T cell cytotoxicity. Thus, we propose a novel mechanism by which colonization with Staphylococcus aureus may contribute to cancer immune evasion and disease progression in CTCL., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

There is mounting evidence that the immune system can spontaneously clear malignant lesions before they manifest as overt cancer, albeit this activity has been difficult to demonstrate in humans. The calreticulin (CALR) exon 9 mutations are driver mutations in patients with chronic myeloproliferative neoplasms (MPN), which are chronic blood cancers. The CALR mutations generate a neo-antigen that is recognized by patient T cells, and T cells isolated from a patient with a CALR-mutation can recognize and kill autologous CALR-mutant cells. Surprisingly, healthy individuals display frequent and strong T cell responses to the CALR neo-antigens too. Furthermore, healthy individuals display immune responses to all parts of the mutant CALR epitope, and the CALR neo-epitope specific responses are memory T cell responses. These data suggest that although healthy individuals might acquire a CALR mutation, the mutant cells can be eliminated by the immune system. Additionally, a small fraction of healthy individuals harbor a CALR exon 9 mutation. Four healthy individuals carrying CALR mutations underwent a full medical examination including a bone marrow biopsy after a median follow up of 6.2 years. None of these patients displayed any signs of CALR-mutant MPN. Additionally, all healthy individuals displayed strong CALR neo-epitope specific T cell responses suggesting that these healthy individuals retained their CALR-mutant cells in the editing stage for several years. Thus, we suggest that CALR-mutant MPN could be a disease model of cancer immuno-editing, as we have demonstrated that CALR-mutant MPN displays all three stages described in the theory of cancer immuno-editing.

L-arginine depletion by regulatory cells and cancer cells expressing arginase-1 (Arg-1) is a vital contributor to the immunosuppressive tumor microenvironment in patients with cancer. We have recently described the existence of pro-inflammatory effector T cells that recognize Arg-1. Hence, Arg-1-specific self-reactive T cells are a naturally occurring part of the memory T-cell repertoire of the human immune system. Here, we further characterize a highly immunogenic epitope from Arg-1. We describe frequent T-cell-based immune responses against this epitope in patients with cancer, as well as in healthy donors. Furthermore, we show that Arg-1-specific T cells expand in response to the T H 2 cytokine interleukin (IL)-4 without any specific stimulation. Arg-1-specific memory T H 1 cells that respond to increased IL-4 concentration may, therefore, drive the immune response back into the T H 1 pathway. Arg-1-specific T cells thus appear to have an important function in immune regulation. Because Arg-1 plays an important role in the immunosuppressive microenvironment in most cancers, an immune modulatory vaccination approach can readily be employed to tilt the balance away from immune suppression in these settings.

Few trials testing the clinical efficacy of therapeutic cancer vaccination have been successful, and therapeutic cancer vaccines are yet to enter the clinic for treatment of haematological cancers. The review summarises the present knowledge of the interplay between cancer and the immune system. These novel insights have uncovered knowledge, which can be used to enhance the effect of therapeutic cancer vaccines in haematology. Immune checkpoint inhibitors, immunomodulating agents, radiotherapy and vaccination against regulatory mechanisms can potentially increase the clinical effect of cancer vaccines for haematological cancer.

Objective: Given a proposed role for PD-L1 + and IL-10-producing B-cell subsets in promoting certain cancers, we sought to characterize the frequency and phenotype of B cells in patients with chronic myeloproliferative neoplasms (MPNs) and the influence of ruxolitinib and interferon-α2 therapy., Methods: We analyzed B-cell frequencies and phenotype in patients with MPNs (n = 107), before and during treatment with ruxolitinib (n = 29), interferon-α2 (n = 21), or the two drugs in combination (COMBI; n = 42) and healthy donors (HDs; n = 52) using flow cytometry., Results: Myelofibrosis patients had lower lymphocyte counts and proportions of B cells than patients with essential thrombocythemia or polycythemia vera and HDs. The B-cell count correlated inversely with JAK2-V617F allele burden and spleen size and increased after ruxolitinib or COMBI treatment. The proportions of PD-L1 + B cells and PD-1 + B cells were significantly higher in patients with myelofibrosis or polycythemia vera than in HDs and decreased during ruxolitinib and COMBI treatment. The proportions of TNF-α + and IL-6 + B cells were elevated in myelofibrosis patients. The proportion of IL-6 + B cells decreased, and the proportion of IL-10 + B cells increased during ruxolitinib treatment., Conclusion: B-cell frequency and phenotype were altered in MPN patients. Ruxolitinib therapy had marked effects on both frequency and phenotype., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

PD-L1-specific T cells are a natural part of the T-cell repertoire in humans. Hence, we have previously described spontaneous CD8 + and CD4 + T-cell reactivity against PD-L1 in the peripheral blood of patients with various cancers as well as in healthy donors. It is well described that the expression of the PD-L1 protein is introduced in cells by pro-inflammatory cytokines, e.g. IFN-γ. In the current study, we were able to directly link inflammation with PD-L1-specific T cells by showing that inflammatory mediators such as IFN-γ generate measurable numbers of PD-L1-specific T cells in human PBMCs as well as in in vivo models. These PD-L1-specific T cells can vigorously modulate the cell compartments of the local environment. PD-L1-specific T cells may be important for immune homeostasis by sustaining the ongoing inflammatory response by the suppression of regulatory cell function both directly and indirectly., Competing Interests: Conflict of interest: Mads Hald Andersen has filed several patent applications based on the use of PD-L1 for vaccination. The rights of the patent applications have been transferred to Copenhagen University Hospital, Herlev, according to the Danish Law of Public Inventions at Public Research Institutions. The capital region has licensed these patents to the company IO Biotech ApS. MHA is a shareholder and board member of the IO Biotech ApS. The other authors declare “no conflict of interest”., (Copyright: © 2019 Munir et al.)