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Supplementary Figure from IDO Vaccine Ablates Immune-Suppressive Myeloid Populations and Enhances Antitumor Effects Independent of Tumor Cell IDO Status

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.

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.

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.

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.

BackgroundIO112 is an immune modulatory cancer therapy under preclinical development to target arginase-1-expressing tumor cells and immune inhibitory myeloid cells, such as myeloid derived suppressor cells (MDSCs), and tumor associated macrophages (TAMs). Arginase-1 acts as a metabolic immune regulator at the tumor site by reducing availability of L-arginine to the infiltrating immune cells thus reducing T cell functionality and proliferation. Previously, we demonstrated that IO112 triggers activation of spontaneous CD4+ and CD8+ T-cell responses against arginase-1, found in both cancer patients and healthy individuals.1 These T cells are present in the memory T cell compartment, and are activated in arginase-1 inducing conditions, such as presence of TH2 cytokines IL-4 or IL-13 in vitro.2 3 In this study we aimed to explore the role of arginase-1-specific T cells as immune modulators in immune homeostasis and tumor microenvironment for the development of IO112 immunomodulatory therapy.MethodsHuman arginase-1-specific T cells were isolated and expanded for functional characterization of reactivity against arginase-1 expressing target cells as well as subsequent phenotyping of the targeted arginase-1 positive cells. Syngeneic C57BL/6 mouse tumor models were used to assess the therapeutic efficacy of IO112.ResultsWe show that arginase-1-specific memory T cells specifically recognize arginase-1 expressing cells, such as mRNA transfected autologous dendritic cells (DCs) and B cells as well as M2 polarized macrophages in vitro. In addition, activated arginase-1-specific T cells produce pro-inflammatory cytokines IFNγ and TNFα. Secretion of TH1 cytokines by these T cells suggests that they may act as potent immune modulators in the tumor microenvironment, since many arginase-1 expressing myeloid cells are not terminally differentiated and they can be re-polarized to an immunostimulatory, M1-like phenotype. We also observed that targeting of M2-polarized arginase-1 expressing monocytic leukemia cell line THP-1 with arginase-1-specific CD4+ T cells induces upregulation of PD-L1 on the THP-1 cells. Furthermore, we demonstrate anti-tumor activity of IO112 in syngeneic mouse tumor models (B16 and MC38), both as monotherapy and in combination with anti-PD-1 treatment. The therapeutic effect was associated with increased immune infiltration in the IO112-treated mice compared to the control.ConclusionsWe demonstrate that arginase-1 specific T cells can influence the polarization of arginase-1-expressing immune cells. Our study provides evidence that IO112 immune therapy against arginase-1 is an attractive way of modulating the immune suppressive tumor microenvironment for therapeutic benefit. With this rationale, we are currently undertaking Investigational New Drug (IND) application enabling studies to explore this approach in a clinical setting.ReferencesMartinenaite E, Mortensen REJ, Hansen M, Holmström MO, Ahmad SM, Jørgensen NGD, Met Ö, Donia M, Svane IM, Andersen MH. Frequent adaptive immune responses against arginase-1. Oncoimmunology 2018;7(3):e1404215.Martinenaite E, Ahmad SM, Svane IM, Andersen MH. Peripheral memory T cells specific for Arginase-1. Cell Mol Immunol 2019;16(8):718–719.Martinenaite E, Ahmad SM, Bendtsen SK, Jørgensen MA, Weis-Banke SE, Svane IM, Andersen MH. Arginase-1-based vaccination against the tumor microenvironment: the identification of an optimal T-cell epitope. Cancer Immunol Immunother 2019;68(11):1901–1907.Ethics ApprovalThis study was approved by the Scientific Ethics Committee for The Capital Region of Denmark and Danish Ethics Committee on experimental animal welfare.

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.

Anti-PD-1 (aPD1) therapy is an effective treatment for metastatic melanoma (MM); however, >50% of the patients progress due to resistance. Improved combination therapies enhancing aPD1 efficacy without leading to increased toxicity are needed. In this non-randomized phase I/II study (ClinicalTrials.gov: NCT03047928), we treated 30 aPD1 naive MM patients with a novel immune-modulatory vaccine (IO102/IO103) activating indoleamine 2,3-dioxygenase (IDO) and programmed death ligand-1 (PD-L1) specific T-cells combined with nivolumab. The vaccine has a dual mechanism of action, targeting both immunosuppressive cells and tumour cells expressing IDO/PD-L1. The vaccine was administered every two weeks for the first 6 injections and every fourth week thereafter, for up to 15 vaccines. Nivolumab was administered every two weeks (3mg/kg) for a maximum of 2 years. The primary endpoint was safety and feasibility; the systemic toxicity profile was comparable to nivolumab monotherapy, except for local and transient reactions at the vaccination site. Secondary endpoints were clinical efficacy and immunogenicity. An ORR of 80% (CI: 62.7-90.5%) was reached, with the majority of 43% (CI: 27.4-60.8%) achieving a complete response. After a median follow-up of 22.9 months, the median PFS was 26 months (CI: 15.4-69). Vaccine-specific responses were detected in vitro in blood from >93% of the patients on vaccination, suggesting the induction of memory response up to 6 months after the last vaccine. Vaccine reactive T-cells comprised both CD4+ and CD8+ T-cells. Paired biopsies from five patients with matched longitudinal PBMCs showed T-cell influx at the tumour site of peripherally expanded T-cells in responding patients and general enrichment of IDO and PD-L1 clones after treatment. These unprecedented clinical efficacy data support further validation in a larger randomized trial, to confirm the clinical potential of this first-in-class immunomodulating vaccine.

Simple Summary Basal cell carcinoma is the most common skin cancer and new treatments for patients with widespread and numerous tumors are lacking. In a previous study treating patients with multiple myeloma with a peptide vaccine called IO103 against an immune checkpoint molecule called programmed death ligand 1, two cases of basal cell carcinoma regressed. The aim of the present study was to assess the effect of vaccination with IO103 in ten patients with basal cell carcinoma. Patients were vaccinated with Montanide as adjuvant up to nine times during six months. Regression in tumor size of at least 30% was seen in five of 18 tumors, two of which showed complete regression. Vaccinations resulted in immune responses against the vaccine in blood samples from nine of ten patients and in skin samples from five of nine patients. The findings suggest that the vaccine may be effective against some basal cell carcinomas. Abstract 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.

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.

Background Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme that contributes to immunoregulation at many levels, including suppressing effector T cells and inducing/activating regulatory T cells. Thus far, several therapeutic approaches to target IDO1 enzymatic activity have shown promise in preclinical models, however, results from the first major clinical trial were disappointing. The present study seeks to provide preclinical PoC data for the conceptually unique idea of developing an IDO1-targeted vaccine based on our earlier findings that humans exhibit intrinsic T cell reactivity against IDO1 epitopes1 suggesting the existence of a T cell-mediated, counter-regulatory mechanism directed against cells that express IDO1. Materials and Methods IDO1-derived peptide vaccines were identified by measurement of vaccine-induced ex vivo response (IFNγ ELISpot) and demonstration of anti-tumor responses in CT26 tumor-bearing mice. To understand the vaccine’s mode of action, resected tumors were analyzed by immunofluorescence microscopy and flow cytometry. Results The CT26 colon carcinoma model was selected for these studies based on evidence of high levels of IDO1 expression and responsiveness to IDO1 inhibition reported for these tumors. In silico-predicted H2d MHC class I and II-restricted IDO1 peptide sequences were tested and vaccine candidates were chosen after confirming ex vivo response and anti-tumor response in CT26. Therapeutic treatment of established CT26 tumors with MHC class I- and II-directed, IDO1-derived peptide vaccines elicited anti-tumor responses when administered alone, and the effect was further pronounced when combined, suggesting distinct mechanisms of action. In addition, a combination of IDO1 vaccine with anti-PD-1 antibody produced a combinatorial anti-tumor response beyond what was achieved with either agent alone. Consistent with this observation, adoptive transfer of isolated CD8+ T cells from class I and CD4+ T cells from class II peptide-vaccinated responder mice delayed tumor growth in treatment naive mice. The class II-directed response was completely IDO1-dependent while the class I-directed response included an IDO1-independent component indicative of antigen spread. Examination into the tumors in vaccinated mice indicated that IDO1 vaccine treatment exerts its effect by selective reduction of IDO1 expression in the tumor microenvironment and concomitant expansion of activated CD4+ and CD8+ T cells. Conclusions As noted in humans, our data demonstrate that IDO1 is immunogenic in mice confirming that this endogenous protein is excluded from normal tolerance mechanisms. The observed immunotherapeutic efficacy of IDO1 peptide vaccines on their own and in combination with anti-PD-1 antibody support the rationale for ongoing clinical development of IDO1 peptide vaccine-based therapy. Future studies include further differentiation of the vaccine platform against other IDO1-targeting approaches, as well as decoding the underlying mechanism of cooperativity between anti-PD-1 antibody and IDO1 peptide vaccines. Reference Sorensen RB, Hadrup SR, Svane IM, Hjortso MC, Thor Straten P, Andersen MH. Indoleamine 2,3-dioxygenase specific, cytotoxic T cells as immune regulators. Blood 2011; 117(7): 2200–10 Disclosure Information S. Dey: None. E. Sutanto-Ward: None. K.L. Kopp: A. Employment (full or part-time); Significant; IO Biotech. J.B. DuHadaway: None. A. Mondal: None. I. Lecoq: A. Employment (full or part-time); Significant; IO Biotech. M. Zocca: A. Employment (full or part-time); Significant; IO Biotech. M.H. Andersen: A. Employment (full or part-time); Significant; IO Biotech. A.W. Pedersen: A. Employment (full or part-time); Significant; IO Biotech. A.J. Muller: F. Consultant/Advisory Board; Modest; IO Biotech.

Background CCL22 is a macrophage-derived chemokine that exerts immunosuppressive functions by the recruitment of regulatory T cells (Treg) through the CCL22/CCR4 axis. It has been described to play a key role in the suppression of anti-cancer immunity in different cancer types including ovarian, breast, or pancreatic cancer and is thought to promote the suppression of anti-cancer immunity by Treg recruitment. Recently, we described that CCL22-specific T cells generated from cancer patients can kill CCL22-expressing tumor cells and directly influence the level of CCL22 in vitro.1 In this study, we provide PoC data for a CCL22-targeting vaccine by assessing the immunotherapeutic efficacy of this approach in syngeneic mouse tumor models. Materials and Methods Peptide vaccines that induce expansion of CCL22-specific T cells were identified by measurement of vaccine-induced ex vivo response (IFNγ ELISpot) in BALB/c and C57BL/6 mice. The antitumor efficacy was evaluated in CT26, Pan02 and B16 syngeneic models. To investigate the vaccine’s mode of action, the tumor immune infiltration was analyzed through flow cytometry and qPCR. Results Vaccination with CCL22-specific peptide vaccines induced expansion of primarily CD8+, CCL22-specific T cell responses (assessed by ex vivo IFNγ ELISpot). Treatment with CCL22 vaccines reduced tumor growth and increased survival in CT26, Pan02 and B16 tumor models. Assessment of gene expression in the tumors indicated that vaccination leads to a reduction of CCL22 expression in the tumor microenvironment (TME), as well as the expression of other immune-suppressive molecules such as IDO. Furthermore, vaccinated mice harbored an increased CD8+ T cell infiltration with a concomitant increase in M1/M2 ratio within the TME. Conclusions This study provides evidence that targeting CCL22 expressing cells by vaccination induces immune modulation in the TME, leading to augmentation of anti-tumor responses - thus provides a rationale for a novel immunotherapeutic approach in cancer. Disclosure Information I. Lecoq: A. Employment (full or part-time); Modest; IO Biotech. K.L. Kopp: A. Employment (full or part-time); Modest; IO Biotech. R. Christensen: A. Employment (full or part-time); Modest; IO Biotech. E. Martinenaite: A. Employment (full or part-time); Modest; IO Biotech. A.W. Pedersen: A. Employment (full or part-time); Modest; IO Biotech. M.H. Andersen: A. Employment (full or part-time); Modest; IO Biotech.

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 H2d 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.

Background: To characterise disease course and remission in a longitudinal observational study of newly diagnosed, initially treatment-naïve patients with seropositive rheumatoid arthritis (RA). Methods: Patients with early untreated seropositive RA were recruited from 28 UK centres. Multiple clinical and laboratory measures were collected every 3 months for up to 18 months. Disease activity was measured using the 28-joint Disease Activity Score with C-reactive protein (DAS28-CRP) and Simplified Disease Activity Index (SDAI). Logistic regression models examined clinical predictors of 6-month remission and latent class mixed models characterised disease course. Results: We enrolled 275 patients of whom 267 met full eligibility and provided baseline data. According to SDAI definition, 24.3% attained 6-month remission. Lower baseline Health Assessment Questionnaire (HAQ) and SDAI predicted 6-month remission ( p = 0.013 and 0.011). Alcohol intake and baseline prescribing of methotrexate with a second disease-modifying antirheumatic drug (DMARD; vs monotherapy without glucocorticoids) were also predictive. Three distinct SDAI trajectory subpopulations emerged; corresponding to an inadequate responder group (6.5%), and higher and lower baseline activity responder groups (22.4% and 71.1%). Baseline HAQ and Short Form-36 Health Survey – Mental Component Score (SF-36 MCS) distinguished these groups. In addition, a number of baseline clinical predictors correlated with disease activity severity within subpopulations. Beneficial effects of alcohol intake were found across subpopulations. Conclusion: Three distinct disease trajectory subpopulations were identified. Differential effects of functional and mental well-being, alcohol consumption, and baseline RA medication prescribing on disease activity severity were found across subpopulations. Heterogeneity across trajectories cannot be fully explained by baseline clinical predictors. We hypothesise that biological markers collected early in disease course (within 6 months) may help patient management and better targeting of existing and novel therapies.

ObjectivesTo identify predictors of remission and disease activity patterns in patients with rheumatoid arthritis (RA) using individual participant data (IPD) from clinical trials.MethodsPhase II and III clinical trials completed between 2002 and 2012 were identified by systematic literature review and contact with UK market authorisation holders. Anonymised baseline and follow-up IPD from non-biological arms were amalgamated. Multiple imputation was used to handle missing outcome and covariate information. Random effects logistic regression was used to identify predictors of remission, measured by the Disease Activity Score 28 (DAS28) at 6 months. Novel latent class mixed models characterised DAS28 over time.ResultsIPD of 3290 participants from 18 trials were included. Of these participants, 92% received methotrexate (MTX). Remission rates were estimated at 8.4%(95%CI 7.4%to9.5%) overall, 17%(95%CI 14.8%to19.4%) for MTX-naïve patients with early RA and 3.2% (95% CI 2.4% to 4.3%) for those with prior MTX exposure at entry. In prior MTX-exposed patients, lower baseline DAS28 and MTX reinitiation were associated with remission. In MTX-naïve patients, being young, white, male, with better functional and mental health, lower baseline DAS28 and receiving concomitant glucocorticoids were associated with remission. Three DAS28 trajectory subpopulations were identified in MTX-naïve and MTX-exposed patients. A number of variables were associated with subpopulation membership and DAS28 levels within subpopulations.Conclusions Predictors of remission differed between MTX-naïve and prior MTX-exposed patients at entry. Latent class mixed models supported differential non-biological therapy response, with three distinct trajectories observed in both MTX-naïve and MTX-exposed patients. Findings should be useful when designing future RA trials and interpreting results of biomarker studies.

CCL22 is a macrophage-derived chemokine that exerts immunosuppressive functions by the recruitment of regulatory T cells (Tregs) through the CCL22/CCR4 axis. It is expressed by cells in the tumor microenvironment (TME) in many different cancer tissues including breast, lung, and pancreatic cancer and is thought to promote the suppression of anti-cancer immunity. Recently, we described that CCL22-specific T-cells from cancer patients can kill CCL22-expressing tumor cells, and activation of CCL22-specific T cells may directly influence the CCL22 concentration in the TME, suggesting that CCL22 may be an interesting target for an immune modulatory vaccine. Thus, in the present study we have targeted CCL22-expressing cells by activation of CCL22-specific effector T cells by peptide vaccination in tumor models. We present here that CCL22-targeting peptide vaccination leads to expansion of predominantly CD8+, IFNγ-secreting CCL22-specific T cells in both BALB/c and C57BL/6 mice. Furthermore, CCL22 vaccination results in delayed tumor growth and enhanced survival in tumor harboring hosts in CT26 and Pan02 models. Preliminary assessment of gene expression in the tumors reveals that CCL22 vaccination leads to a reduction of CCL22 expression, alongside other immune suppressive molecules, such as IDO1 in the TME. Characterization of vaccine-mediated changes of cellular composition of the TME is currently ongoing. In conclusion, our findings provide a rationale for a novel cancer immunotherapy to target the immune suppressive TME. Citation Format: Inés Lecoq, Katharina L. Kopp, Rikke Christensen, Ayako W. Pedersen, Evelina Martinenaite, Mai-Britt Zocca, Mads H. Andersen. Therapeutic potential of an immune modulatory CCL22-based vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5020.

Background: We evaluated the clinical and immunological effects of dendritic cell (DC) vaccination of patients with NSCLC. Autologous DCs were pulsed with a MAGE containing allogeneic melanoma cell lysate (MelCancerVac?, Dandrit Biotech,Copenhagen,Denmark). Imiquimod cream, proleukin and celecoxib were used as adjuvants to the vaccines. The objective of the study was to evaluate specific T cell response in vitro by IFNg EliSpot. Secondary objectives were overall survival, response and quality of life (QoL). Results: Twenty-two patients initiated the vaccination program consisting of ten vaccinations. Seven patients remained in stable disease (SD) three months after the first vaccination. After ten vaccinations (six months), four patients still showed SD and continued vaccinations on a monthly basis. These four patients received a total of 12, 16, 26 and 35 vaccinations, respectively. Five patients showed an unexpectedly prolonged survival. The treatment was well tolerated and only minor adverse events were reported. Quality of life did not change during the study period. In four of the seven patients with SD, vaccine-specific T cells were detected by IFNγ EliSpot assays, whereas only one patient with progressive disease (PD) showed vaccine-specific responses. Conclusion: This DC-based vaccine trial has indicated a correlation between vaccine-specific immunity and sustained SD. Furthermore, we observed an unexpectedly prolonged survival in some patients, which may indicate delayed effect of DC vaccination after completion of the treatment. A prospective randomized phase-IIb or -III is needed to further evaluate the use of MelCancerVac? vaccine treatment in patients with progressive NSCLC.

Tumors evade immune surveillance using several mechanisms such as induction of regulatory T-cells (Tregs) and upregulation of indolemine-2,3-dioxygenase (IDO) enzyme. IDO is an intracellular enzyme that mediates its immunosuppressive effects by depleting tryptophan leading to accumulation of kynurenine and in turn reducing the proliferation of effector cells while activating Tregs. IDO is expressed by several suppressive immune cells including Tregs, suppressive antigen presenting cells (APCs), as well as by tumor cells. These immunosuppressive effects of IDO make it an attractive target for cancer immunotherapy, including a target for vaccine therapy. To test the anti-tumor immune effect of IDO vaccine, we evaluated the anti-tumor effect of an IDO-derived MHC I restricted peptide (IDO-pep) vaccine in the low IDO-expressing (TC-1) and high IDO-expressing B16 (melanoma) mouse tumor models. For this, mice were treated with three doses of IDO-peptides in combination with the respective tumor-specific vaccines (E7 for TC-1 and gp100 for B16) at an interval of seven days, followed by assessment of tumor growth and mice survival. We observed a significant delay in the tumor growth and prolonged survival in both tumor models when IDO vaccine was combined with tumor-specific antigen vaccine. We found that tumor-infiltration of antigen-specific CD8+ T-cells was significantly enhanced following the addition of IDO vaccine. Furthermore, while CD4+ T-cells increased in the tumor microenvironment (TME), there was a significant reduction in the numbers of Treg cells. We also found a significant reduction in the numbers of IDO-expressing myeloid-derived suppressor cells (MDSCs), dendritic cells as well as macrophages out of the total CD45+CD11b+ myeloid cells in the TME after the administration of IDO-pep in combination with tumor antigen-specific vaccine. In conclusion, our findings suggest that IDO-pep vaccine enhances the specific anti-tumor immune response by increasing the tumor antigen-specific effector T-cell infiltration in the TME through inhibition of immune suppressive cells. These results also suggest that MHC I restricted IDO peptide treatment is a promising strategy to improve cancer immunotherapy. Citation Format: Rahul Nandre, Vivek Verma, Pankaj Gaur, Pooja Vir, Hua Wang, Baolin Kang, Ayako Wakatsuki Pedersen, Mai-Britt Zocca, Mads Hald Andersen, Seema Gupta, Samir Khleif. IDO vaccine enhances antigen-specific anti-tumor effects by reducing IDO-expressing antigen presenting cells and MDSCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3763.

Historically, the development of cancer vaccines has focused on the central role of tumor antigens in eliciting tumor-specific immune responses, with limited success. Recent advances with checkpoint blockade approaches have brought about a renewed appreciation of the importance of targeting immune suppression in cancer patients. Here we discuss a novel approach to cancer immunotherapy, namely to target recently described T cells that uniquely control cells with immune suppressive functions. Accumulating evidence support the existence of self-reactive T cells that are specific to antigens derived from immunoregulatory proteins ("immunoregulatory antigens"), such as indoleamine 2,3-dioxygenase (IDO) and PD-L1. Vaccination approaches to potentiate these T cells have proven safe with minimal toxicity in the clinical phase I trials conducted thus far. Given that immunoregulatory antigens can be new targets for cancer immunotherapy, we propose here that they could be considered as a new class of tumor antigens. Targeting such antigens has advantages over targeting classical tumor antigens, as there is no requirement for identification of relevant antigens that are specific for the cancer type, and the targets are genetically stable. Furthermore, targeting immunoregulatory antigen-specific T cells potentially has dual mode of actions (I) targeting immune suppression and thereby potentiating anti-tumor effector T cell responses and (II) direct killing of immunoregulatory antigen-expressing tumor cells.

Dendritic cells (DCs) exposed to tumor antigens followed by treatment with T(h)1-polarizing differentiation signals have paved the way for the development of DC-based cancer vaccines. Critical parameters for assessment of the optimal functional state of DCs and prediction of the vaccine potency of activated DCs have in the past been based on measurements of differentiation surface markers like HLA-DR, CD80, CD83, CD86, and CCR7 and the level of secreted cytokines like interleukin-12p70. However, the level of these markers does not provide a complete picture of the DC phenotype and may be insufficient for prediction of clinical outcome for DC-based therapy. We therefore looked for additional biomarkers by investigating the differential expression of microRNAs (miRNAs) in mature DCs relative to immature DCs. A microarray-based screening revealed that 12 miRNAs were differentially expressed in the two DC phenotypes. Of these, four miRNAs, hsa-miR-155, hsa-miR-146a, hsa-miR-125a-5p, and hsa-miR-29a, were validated by real-time polymerase chain reaction and northern blotting. The matured DCs from 12 individual donors were divided into two groups of highly and less differentiated DCs, respectively. A pronounced difference at the level of miRNA induction between these two groups was observed, suggesting that quantitative evaluation of selected miRNAs potentially can predict the immunogenicity of DC vaccines.

Dendritic cells (DC) are potent antigen presenting cells capable of inducing immune responses. DC are widely used as vaccine adjuvant in experimental clinical settings. DC-based vaccines are normally generated using a standard 8day DC protocol (SDDC). In attempts to shorten the vaccine production we have developed fast DC protocol by comparing two different fast DC protocols with SDDC. DC were evaluated by FACS analysis, and the optimal profile was considered: CD14(low), CD80(high), CD83(high), CD86(high), CCR7(high), HLA class I and II(high). FACS profiles were used as the selection criteria together with yield and morphology. Two fast DC protocols fulfilled these criteria and were selected for functional analysis. Our results demonstrate that DC generated within 5days or 48h are comparable with SDDC both phenotypically and functionally. However, we found that 48h DC were more susceptible than SDDC to the IL-10 inducing stimulus of TLR ligands (R848 and LPS). Thus to determine the clinical relevance of fast DC protocols in cancer settings, small phase I trials should be conducted monitoring regulatory T cells carefully.

Dendritic cells (DCs), the most potent antigen-presenting cells of the immune system, express nuclear receptors for 1,25-dihydroxyvitamin D(3) (VD3) and they are one of its main targets. In the presence of VD3, DCs differentiate into a phenotype that resembles semimature DCs, with reduced T cell costimulatory molecules and hampered IL-12 production. These VD3-modulated DCs induce T cell tolerance in vitro using multiple mechanisms such as rendering T cells anergic, dampening of Th1 responses, and recruiting and differentiating regulatory T cells. Due to their ability to specifically target pathological T cells, VD3-modulated DCs are safe and potentially more effective alternatives to currently available immunoregulatory therapies. While a number of considerations remain, including the establishment of a reliable quality control measure to ensure the safety and efficacy of VD3-DCs in vivo and the optimal frequency, dose, and route of DC administration to achieve therapeutic effects in humans, adoptive VD3-DC transfer represents one of the most promising approaches to future treatment of autoimmune diseases.

Dendritic cells (DCs), the most potent antigen-presenting cells of the immune system, express nuclear receptors for 1,25-dihydroxyvitamin D(3) (VD3) and they are one of its main targets. In the presence of VD3, DCs differentiate into a phenotype that resembles semimature DCs, with reduced T cell costimulatory molecules and hampered IL-12 production. These VD3-modulated DCs induce T cell tolerance in vitro using multiple mechanisms such as rendering T cells anergic, dampening of Th1 responses, and recruiting and differentiating regulatory T cells. Due to their ability to specifically target pathological T cells, VD3-modulated DCs are safe and potentially more effective alternatives to currently available immunoregulatory therapies. While a number of considerations remain, including the establishment of a reliable quality control measure to ensure the safety and efficacy of VD3-DCs in vivo and the optimal frequency, dose, and route of DC administration to achieve therapeutic effects in humans, adoptive VD3-DC transfer represents one of the most promising approaches to future treatment of autoimmune diseases.

SummaryThe clinical use of dendritic cells (DCs) to induce antigen-specific immune tolerance has been hampered by the lack of a widely acknowledged method for generating human regulatory DCs but even more so by the non-existence of reliable markers. Thus, we set out to find reliable markers that can be measured with simple methods to identify regulatory DCs that are applicable for future clinical studies. Human DCs were generated from peripheral blood monocytes in the presence of 1α,25-dihydroxyvitamin D3 (VD3), which gave rise to a phenotype that resembles immature DCs, with the exception of high CD14 and reduced CD1a on the cell surface. These VD3-treated DCs exert a long-lasting inefficient T cell stimulation and induce T cell hyporesponsiveness with regulatory potential. Importantly, such VD3-treated DCs were readily distinguishable from untreated DCs by low levels of interleukin-23 secretion and low expression of miR-155 upon exposure to maturation stimuli. Furthermore, VD3-treated DCs showed over-expression of miR-378. All these features can be used as robust markers for quality control of VD3-treated regulatory DCs in future clinical studies.

Dendritic cells (DCs) are bone marrow-derived professional antigen presenting cells. Due to their role as potent inducers of immune responses, these cells are widely used as adjuvant in experimental clinical settings for cancer immune therapy. We have developed a DC-based vaccine using autologous blood monocytes loaded with allogeneic tumor cell lysate rich in cancer/testis antigens. This vaccine has at present been tested for activity in three phase II clinical trials including two cohorts of patients with advanced colorectal cancer (CRC) and one cohort of patients with advanced non-small-cell-lung-cancer (NSCLC). In the present paper we retrospectively compare the maturation profile based on surface marker expression on DCs generated from the three patient cohorts and between cancer patient cohorts and a cohort of healthy donors. Vaccines were generated under cGMP conditions and phenotypic profiles of DC were analyzed by flow cytometry and the obtained data were used as a basis to set guideline values for our quality control of GMP produced DC vaccines. Each vaccine batch was analyzed for the expression of the surface maturation and differentiation molecules CD14, CD1a, CD83, CD86, MHC class II and CCR7, and the optimal expression pattern is considered as CD14(low), CD1a, CD83(high), CD86(high), MHC class II(high) and CCR7(high). In accordance with data from other studies including other types of cancer patients, especially breast cancer patients, we found that the maturation status of the DC batches depends on cancer type and correlates with clinical status of cancer patients included.

ObjectivesAn interferon (IFN) gene signature (IGS) is present in approximately 50% of early, treatment naive rheumatoid arthritis (eRA) patients where it has been shown to negatively impact initial response to treatment. We wished to validate this effect and explore potential mechanisms of action.MethodsIn a multicentre inception cohort of eRA patients (n=191), we examined the whole blood IGS (MxA, IFI44L, OAS1, IFI6, ISG15) with reference to circulating IFN proteins, clinical outcomes and epigenetic influences on circulating CD19+ B and CD4+ T lymphocytes.ResultsWe reproduced our previous findings demonstrating a raised baseline IGS. We additionally showed, for the first time, that the IGS in eRA reflects circulating IFN-α protein. Paired longitudinal analysis demonstrated a significant reduction between baseline and 6-month IGS and IFN-α levels (pPARP9, STAT1,andEPSTI1, associated with baseline IGS/IFNα levels. Differentially methylated CPG sites implicated altered transcription factor binding in B cells (GATA3, ETSI, NFATC2, EZH2) and T cells (p300, HIF1α).ConclusionsOur data suggest that, in eRA, IFN-α can cause a sustained, epigenetically mediated, pathogenic increase in lymphocyte activation and proliferation, and that the IGS is, therefore, a robust prognostic biomarker. Its persistent harmful effects provide a rationale for the initial therapeutic targeting of IFN-α in selected patients with eRA.