Tumor Antigen-specific CD8 Research Articles

Abstract 2156: IL-18 pathway agonism expands intratumoral PD1+ Tcf1+ stem-like CD8+ cells and their polyfunctional effector progeny to promote anti-tumor immunity

Abstract Recombinant cytokines were the first modern immunotherapies to produce durable cures in metastatic cancer, but their application has been hampered by only modest efficacy and limited tolerability. A major limitation to cytokine therapies is their lack of selectivity for the small fraction of lymphocytes that specifically recognize tumors. To identify cytokine pathways with greater selectively for anti-tumor lymphocytes, we analyzed single-cell RNA-seq expression data and flow cytometric profiles of tumor-infiltrating lymphocytes (TILs). Among detectable cytokine pathways, Interleukin-18 (IL-18) and its receptors IL-18R1 and IL-18RAP were markedly upregulated in activated and dysfunctional TILs, suggesting the potential of IL-18R agonism to promote anti-tumor immunity. However, recombinant IL-18 therapy has consistently failed to demonstrate anti-tumor efficacy in clinical trials. We hypothesized this may be due to the upregulation of an ultra-high affinity inhibitory decoy receptor of IL-18, IL-18BP. Indeed, IL-18BP is highly expressed in the tumor microenvironment, which we found by immunohistochemical staining and qPCR analysis of IL-18BP levels in several human cancers and syngeneic murine tumor models. Using directed evolution with yeast-surface display, we engineered “decoy-resistant” IL-18 (DR-18), a variant of IL-18 that maintains signaling potential but is impervious to binding and inhibition by IL-18BP. In multiple syngeneic tumor models including YUMMER1.7, MC38, and CT26, we found that recombinant WT IL-18 was ineffective by itself or in combination with anti-PD-1 treatment, similar to the clinical experience of rIL-18 in patients. However, in the same models, DR-18 showed robust monoagent activity that was commensurate or superior to anti-PD-1 antibodies, and it produced added therapeutic synergism when combined with anti-PD-1. Single-cell RNA-seq profiling and flow cytometry analysis of DR-18 treated tumors indicated that DR-18 treatment drives the development of poly-functional effector CD8+ T cells and NK cells. Importantly, DR-18 expands the pool of intratumoral Tcf1+ memory precursor PD1+ CD8+ T cells and decreases the prevalence of exhausted CD8+ T cells that express TOX. Mechanistically, we demonstrate that tumor antigen specific CD8+ T cells were sufficient to mediate tumor regression using LCMV epitope GP33 specific P14 CD8+ T cells in a B16-GP33 model. Together, these results highlight DR-18 is able to remodel the tumor-immune landscape to bias immune cells towards potent anti-tumor effector cells and away from the T exhausted lineage. Therefore, we conclude that the IL-18 pathway is a powerful target for immunotherapeutic intervention and implicate the secreted checkpoint IL-18BP as an obstacle to effective IL-18 immunotherapy. Citation Format: Ting Zhou, Orr-El Weizman, William Damsky, Meaghan McGeary, Marcus W. Bosenberg, Aaron M. Ring. IL-18 pathway agonism expands intratumoral PD1+ Tcf1+ stem-like CD8+ cells and their polyfunctional effector progeny to promote anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2156.

Abstract 911: Anti-PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR mutant NSCLC

Abstract It has been reported that NSCLC patients with EGFR mutation do not respond to anti-PD-1(L1) therapies as well as patients without the mutation. We have previously shown that EGFR mutant tumors express more CD73 compared to EGFR wildtype tumors and we hypothesized that this may contribute to the resistance to anti-tumor immunity and anti-PD-1(L1) therapies. To understand how CD73 expression in EGFR mutant tumors may confer immunosuppression, we compared EGFR mutant cell lines (PC9, HCC2935) and wildtype cell lines (H1666, H1355) in in vitro T cell killing assays. Furthermore, we investigated the effect of anti-CD73 antibody, either as a monotherapy or in combination with anti-PD-L1 antibody, in promoting tumor growth inhibition (TGI) using a xenograft mouse model. In this model, NSG mice were implanted with MART1-expressing PC9 cells and treated with MART1-specific CD8+ T cells together with anti-PD-L1, anti-CD73 or combination. We showed that CD73 expression is regulated by EGFR signaling in NSCLC, possibly through activation of Protein Kinase C (PKC). EGFR mutant cell lines were significantly more resistant to T cells killing compared to wildtype cell lines in vitro, by suppressing T cell proliferation and function as indicated by Ki-67 and granzyme B expression. In the xenograft mouse model using MART1-expressing PC9 cells, anti-PD-L1 did not promote TGI compared to isotype control, which closely mimicked the limited therapeutic effect of anti-PD-1(L1) antibodies on EGFR mutant NSCLC. Although anti-CD73 alone did not reduce tumor size, mice treated with anti-CD73 and anti-PD-L1 combination therapy had significantly smaller tumors compared to mice treated with isotype control (~38%, p<0.0001), anti-PD-L1 alone (~32%, p<0.0001) or anti-CD73 alone (~32%, p<0.01). The combination therapy increased the number of MART1-specific CD8+ T cells in the tumor compared to isotype control (~280%, p<0.01), anti-PD-L1 alone (~556%, p<0.001) and anti-CD73 alone (~270%, p<0.01). The combination therapy also increased the frequency of CD62L+CD45RO+CCR7+ phenotype in CD8+ T cells in the spleen compared to isotype control (~120%, p<0.001), but not in the tumor, as well as enhancing IFNγ production by tumor antigen-specific CD8+ T cells compared to isotype control (~224%, p<0.001). Together, these results further support the ongoing clinical trials evaluating combination of anti-CD73 and anti-PD-L1 in treating EGFR mutant NSCLC while suggesting the increased central memory T cells may play a role in response to therapy. Citation Format: Eric Tu, Kelly McGlinchey, Yelena Lazdun, James Kurasawa, Susan Wilson, Leslie Wetzel, Karen Coffman, Zachary Cooper, Katie Streicher. Anti-PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR mutant NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 911.

Abstract 3381: Identifying tumor antigen-specific CD4+ T cell dysfunctional states by single cell transcriptomics in immunotherapy-treated cancer patients

Abstract Dysfunctional T cell states resulting from continuous exposure to self shared tumor antigens or neoantigens are key to understanding immune mechanisms that prevent proper antitumor immunity. We previously showed that repeated administration of recombinant tumor antigen vaccines without immunological adjuvant emulates a tolerized-like immune response that cannot be rescued even after in vivo re-challenge with adjuvant. We used bulk and single-cell RNA-seq of purified antigen-specific CD4+ T-cells (i.e., the orchestrator of immunity) from 12 ovarian and non-small cell lung cancer patients receiving NY-ESO-1 or MAGE-A3 vaccines with or without adjuvant, over a period of 13 weeks. First, using bulk RNA-seq data, we derived novel temporal, tolerization CD4+ T cell expression and clonotype evolution signatures using differential expression and VDJ-deconvolution analyses, highlighting significant association with cell cycle and immune activation. Then, we projected these signatures onto several recently published single-cell RNA-seq data profiling exhausted tumor-infiltrating and peripheral blood lymphocytes, finding reasonable predictive power (AUC~0.8) to classify dysfunctional T-cell states that could distinguish exhaustion from tolerance. Next, we characterized in depth the transcriptional profiles and heterogeneity of our antigen-specific CD4+ T-cells pre- and post-treatment at the single-cell level using combined scRNAseq and scTCRseq. Finally, we computationally inferred the regulatory axis of tolerized T-cells by deriving transcription factor signaling networks upstream of gene expression. In conclusion, we characterized transcriptional signatures of tumor antigen-specific CD4+ T-cells and derived a specific tolerization profile, distinguishable from exhaustion, and conserved across datasets. Through comprehensive identification of genes and pathways in a human experimental setting for induction of tolerization and exhaustion, these findings provide relevant and promising avenues to reverse dysfunctional CD4+ T cell states. Citation Format: Edgar Kozlova, Shingo Eikawa, Sacha Gnjatic, Bojan Losic. Identifying tumor antigen-specific CD4+ T cell dysfunctional states by single cell transcriptomics in immunotherapy-treated cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3381.

Abstract 959: Cellular dynamics of productive anti-tumor response mediating long-term rejection of disseminated lung tumors after systemic therapy with oncolytic adenovirus

Abstract Currently, some of the most advanced and clinically effective solid cancer treatment approaches are based on counteracting immune checkpoint inhibitory pathways (ICI) to activate cytotoxic activity of tumor antigen-specific CD8+ T cells. Unfortunately, the use of ICI targeting drugs provides clinical benefits only to a minority of cancer patients. Although major efforts are put forth to improve understanding of CD8+ T cell-centric adaptive immune mechanisms to increase the the proportion of cancer patients responding to this therapy, the effector functions of innate immunity in mediating tumor rejection remain poorly understood. Using a CD8+ T cell-deficient mouse model and a disseminated human lung cancer model, allowing for a productive replication of human oncolytic adenovirus, AVID-317, in this study we analyzed the recruitment and activation states of hematopoietic and stromal cells that enter the lungs of tumor-bearing mice after systemic oncolytic virus therapy and their correlation with the efficacy of anti-tumor response. Intravenous administration of AVID-317 resulted in a significant suppression of tumor growth and extension of survival of mice with disseminated tumors in the lungs. However, the longitudinal analyses showed that two distinct cohorts of mice could be identified within 21 days after the beginning of the therapy - one where mice responded by a complete rejection of disseminated lung tumors (complete responders) and the other, where mice exhibited unimpeded tumor progression (non-responders). Multi-dimensional flow cytometry analysis revealed that after oncolytic virus treatment, there were quantitative and qualitative differences in tumor-infiltrating cells recovered from the lungs of responder and non-responder mice. After oncolytic virotherapy, the tumor infiltrating cells in non-responder mice were significantly enriched for inflammatory myeloid Ly-6G-, Ly6C-, and CD11b-expressing cells, which also expressed high levels of CD80/CD86 and MHC class II proteins, indicating their highly activated state. In contrast, in responder mice, the tumor infiltrating cells were significantly enriched for B cells, NKT cells, and Ly-6C- and Sca-1-positive stromal cells. The influx of B cells, NK, and NKT cells in the lungs after systemic AVID-317 therapy significantly correlated with tumor burden reduction. Although the relative abundance of NK cells was not different between responder and non-responder mice, the single cell transcriptomics analysis showed that NK cells in responder mice expressed higher levels of KLRG1 and KLRC1 terminal effector markers and perforin. Taken together, our data provides evidence that a systemic therapy with oncolytic adenovirus promotes significant alteration of the tumor microenvironment, leading to the recruitment of lymphocytes, namely B cells, NK cells, and NKT cells, with effector functions, which mediate long-lasting rejection of disseminated lung tumors via innate immune mechanisms. Citation Format: Jia Yao, Svetlana Atasheva, Cedrick B. Young, Dmitry M. Shayakhmetov. Cellular dynamics of productive anti-tumor response mediating long-term rejection of disseminated lung tumors after systemic therapy with oncolytic adenovirus [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 959.

Abstract NG12: Mechanisms governing efficacy of combination CD40 agonist and anti-PD-L1 in pancreatic ductal adenocarcinoma

Abstract NG12: Mechanisms governing efficacy of combination CD40 agonist and anti-PD-L1 in pancreatic ductal adenocarcinoma

The Expression of Adenosine A2B Receptor on Antigen-Presenting Cells Suppresses CD8+ T-cell Responses and Promotes Tumor Growth.

Accumulating evidence suggests that inhibiting adenosine-generating ecto-enzymes (CD39 and CD73) and/or adenosine A2A or A2B receptors (R) stimulates antitumor immunity and limits tumor progression. Although activating A2ARs or A2BRs causes similar immunosuppressive and protumoral functions, few studies have investigated the distinct role of A2BR in cancer. Here, we showed that A2BR expression by hematopoietic cells was primarily responsible for promoting tumor growth. Deletion of A2BR profoundly enhanced anticancer T-cell immunity. Although T-cell A2BR plays an insignificant role for A2BR-mediated immunosuppression and tumor promotion, A2BR deficiency in tumor-bearing mice caused increased infiltration of myeloid and CD103+ dendritic cells, which was associated with more effective cross-priming of adoptively transferred tumor antigen-specific CD8+ T cells. A2BR deletion also intrinsically favored accumulation of myeloid and CD11bdim antigen-presenting cells (APC) in the tumor microenvironment. Both myeloid-specific or CD11c-specific conditional deletion of A2BR delayed primary tumor growth. Myeloid, but not CD11c-specific conditional, depletion delayed lung metastasis. Pharmacologic blockade of A2BR improved the antitumor effect of adoptive T-cell therapy. Overall, these results suggested that A2BR expression on myeloid cells and APCs indirectly suppressed CD8+ T-cell responses and promoted metastasis. These data provide a strong rationale to combine A2BR inhibition with T-cell-based immunotherapy for the treatment of tumor growth and metastasis.

Involvement of the M-CSF/IL-34/CSF-1R pathway in malignant pleural mesothelioma.

BackgroundMalignant pleural mesothelioma (MPM) is a rare and aggressive cancer related to asbestos exposure. The tumor microenvironment content, particularly the presence of macrophages, was described as crucial for the development of the disease. This work aimed at studying the involvement of the M-CSF (CSF-1)/IL-34/CSF-1R pathway in the formation of macrophages in MPM, using samples from patients.MethodsPleural effusions (PEs), frozen tumors, primary MPM cells and MPM cell lines used in this study belong to biocollections associated with clinical databases. Cytokine expressions were studied using real-time PCR and ELISA. The Cancer Genome Atlas database was used to confirm our results on an independent cohort. An original three-dimensional (3D) coculture model including MPM cells, monocytes from healthy donors and a tumor antigen-specific cytotoxic CD8 T cell clone was used.ResultsWe observed that high interleukin (IL)-34 levels in PE were significantly associated with a shorter survival of patients. In tumors, expression of CSF1 was correlated with ‘M2-like macrophages’ markers, whereas this was not the case with IL34 expression, suggesting two distinct modes of action of these cytokines. Expression of IL34 was higher in MPM cells compared with primary mesothelial cells. Particularly, high expression of IL34 was observed in MPM cells with an alteration of CDKN2A. Finally, using 3D coculture model, we demonstrated the direct involvement of MPM cells in the formation of immunosuppressive macrophages, through activation of the colony stimulating factor-1 receptor (CSF1-R) pathway, causing the inhibition of cytotoxicity of tumor antigen-specific CD8+ T cells.ConclusionsThe M-CSF/IL-34/CSF-1R pathway seems strongly implicated in MPM and could constitute a therapeutic target to act on immunosuppression and to support immunotherapeutic strategies.

Lipo-Based Vaccines as an Approach to Target Dendritic Cells for Induction of T- and iNKT Cell Responses.

In this study we developed a liposome-based vaccine containing palmitoylated synthetic long peptides (SLP) and alpha galactosylceramide (αGC) to specifically target dendritic cells (DC) for activation of both innate (invariant natural killer T-cells [iNKT]) and adaptive (CD8+ T-cells) players of the immune system. Combination of model tumor specific antigens (gp100/MART-1) formulated as a SLP and αGC in one liposome results in strong activation of CD8+ and iNKT, as measured by IFNγ secretion. Moreover, addition of lipo-Lewis Y (LeY) to the liposomes for C-type lectin targeting increased not only uptake by monocyte-derived dendritic cells (moDC), dermal dendritic cells and Langerhans cells but also enhanced gp100-specific CD8+ T- and iNKT cell activation by human skin-emigrated antigen presenting cells in an ex vivo explant model. Loading of moDC with liposomes containing LeY also showed priming of MART-126−35L specific CD8+ T-cells. In conclusion, chemically linking a lipid tail to a glycan-based targeting moiety and SLP combined with αGC in one liposome allows for easy generation of vaccine formulations that target multiple skin DC subsets and induce tumor antigen specific CD8+ T- and iNKT cells. These liposomes present a new vaccination strategy against tumors.

OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity.

Plasmacytoid DCs (pDCs), the major producers of type I interferon, are principally recognized as key mediators of antiviral immunity. However, their role in tumor immunity is less clear. Depending on the context, pDCs can promote or suppress antitumor immune responses. In this study, we identified a naturally occurring pDC subset expressing high levels of OX40 (OX40+ pDC) enriched in the tumor microenvironment (TME) of head and neck squamous cell carcinoma. OX40+ pDCs were distinguished by a distinct immunostimulatory phenotype, cytolytic function, and ability to synergize with conventional DCs (cDCs) in generating potent tumor antigen-specific CD8+ T cell responses. Transcriptomically, we found that they selectively utilized EIF2 signaling and oxidative phosphorylation pathways. Moreover, depletion of pDCs in the murine OX40+ pDC-rich tumor model accelerated tumor growth. Collectively, we present evidence of a pDC subset in the TME that favors antitumor immunity.

A phase Ia/Ib dose-escalation study of intravenously administered SB 11285 alone and in combination with nivolumab in patients with advanced solid tumors.

TPS3162 Background: Activation of the Stimulator of Interferon Genes (STING) pathway in immune cells in the tumor microenvironment (TME) and tumor cells results in the induction of innate and adaptive immunity and subsequent activation of cytotoxic T cells and NK cells for durable anti-tumor responses. SB 11285 is a novel agonist of the STING pathway leading to the activation of tumor-resident APCs and priming of tumor antigen specific CD8+ T cells. In our preclinical studies using multiple tumor-derived cell lines, SB 11285 has been observed to cause the induction of cytokines, such as INF-b, INF- a, TNFa and others consistent with engagement of the STING target, as well as tumor cell death by STING-mediated apoptosis. SB 11285 reduced tumor volumes in multiple rodent tumor models when administered intravenously, intraperitoneally or intratumorally as monotherapy or in combination with checkpoint inhibitors such as anti-CTLA-4 or anti-PD-1 antibody. Systemic administration could additionally facilitate trafficking of newly activated CD8+T cells from periphery into the tumor site. Methods: This open-label, multicenter phase 1a/1b clinical trial (NCT04096638) aims to enroll approximately 110 patients in the dose escalation (Part 1) and expansion cohorts (Part 2). Part 1 of the trial is a dose escalation study with IV SB 11285 monotherapy followed by combination with the checkpoint inhibitor nivolumab. Part 1 Dose Escalation of the study will evaluate ascending doses of intravenously administered SB 11285 with respect to dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), recommended phase 2 dose (RP2D) and the pharmacokinetic (PK)/pharmacodynamic profile as monotherapy and in combination with nivolumab. SB 11285, with a starting dose of 0.3μg/kg, will be administered as monotherapy weekly on Days 1, 8, 15, and 22 of repeated 28-day cycles in escalating doses and in combination with nivolumab administered on Q4W schedule. Part 2 Expansion Cohorts of the study will explore initial signs of efficacy in pre-specified tumor types (such as Melanoma, Head and Neck squamous cell carcinoma) using the recommended phase 2 dose (RP2D) of SB 11285 in combination with nivolumab. In addition, the biological effects of SB 11285 will be evaluated by changes in immune cell types and activation state, serum cytokines, and gene expression patterns indicative of activation of the immune compartment. The trial is being conducted at multiple sites in the U.S . Clinical trial information: NCT04096638 .

Glioblastoma infiltration of both tumor- and virus-antigen specific cytotoxic T cells correlates with experimental virotherapy responses

Abstract The mode of action for oncolytic viruses (OVs) in cancer treatment is thought to depend on a direct initial cytotoxic effect against infected tumor cells and subsequent activation of immune cell responses directed against the neoplasm. To study both of these effects in a mouse model of glioblastoma (GBM), we employed murine GBM cells engineered to constitutively express the type I Herpes Simplex Virus (HSV1) HSV-1 receptor, nectin-1, to allow for more efficient infection and replication by oncolytic HSV (oHSV). These cells were further engineered with a surrogate tumor antigen to facilitate assays of T cell activity. We utilized MRI-based volumetric to measure GBM responses after injection with the oHSV and bioluminescent imaging (BLI) to determine oHSV replicative kinetics in the injected tumor mass. We found increased infiltration of both surrogate tumor antigen- and oHSV antigen-specific CD8+ T cells within 7 days after oHSV injection. There was no increase in tumor infiltrating CD8+ T cells expressing “exhaustion” markers, yet oHSV infection led to a reduction in PD-1+ CD8+ T cells in injected GBMs and an increase in IFNγ+ CD8+ T cells. There was a significant direct correlation between oHSV-mediated reduction in GBM volume, oHSV intratumoral gene activity and increased infiltration of both viral and tumor antigen-specific CD8+ T cells. These findings imply that both intratumoral viral replication and CD8+ T cell cytotoxicity against both tumor and viral antigens are important in oHSV-mediated GBM therapy.

CD103+ cDC1 vaccine efficacy in murine breast cancer is inhibited by STAT3

Abstract Conventional dendritic cells (cDCs) are a crucial immune population, which includes multiple subtypes that coordinate adaptive immunity and tolerance. Migratory type 1 cDC1s (CD103+ cDC1s in mice) are required to induce CD8+ T cell-mediated anti-tumor immunity, yet whether and how tumors regulate CD103+ cDC1 function is largely unknown. Many tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10, require Signal Transducer and Activator of Transcription 3 (STAT3) for intracellular signaling; therefore, we sought to determine whether STAT3 inhibits CD103+ cDC1-mediated anti-tumor immunity. Indeed, we found that in vitro-derived STAT3-deficient (Stat3Δ/Δ) CD103+ cDC1sare resistant to IL-10 – mediated inhibition of Toll-like receptor 3 (TLR3)-agonist induced maturation. When used as an anti-tumor vaccine, Stat3Δ/Δ CD103+ cDC1s inhibited breast tumor growth and increased mouse survival to a greater degree than STAT3-sufficient CD103+ cDC1s. Vaccination with Stat3Δ/Δ CD103+ cDC1s increased tumor antigen-specific CD8+ T cells and IFN-g+ CD4+ T cells in tumors and tumor-draining lymph nodes compared to controls. Moreover, IL-10 receptor-deficient CD103+ cDC1s restrained tumor growth as effectively as Stat3Δ/Δ CD103+ cDC1s. Taken together, our results demonstrate STAT3 suppresses CD103+ cDC1 maturation and vaccine efficacy in breast cancer. The data also suggest IL-10 is an important factor in the murine mammary tumor environment mediating immunosuppressive STAT3 signaling in CD103+ cDC1s. Thus, STAT3 inhibition may provide an effective strategy for improving the anti-tumor efficacy of CD103+ cDC1-based vaccines.

Targeting MerTK Enhances Adaptive Immune Responses After Radiation Therapy

The role of MerTK, a member of the Tyro3-Axl-MerTK family of receptor tyrosine kinase, in the immune response to radiation therapy (RT) is unclear. We investigated immune-mediated tumor control after RT in murine models of colorectal and pancreatic adenocarcinoma using MerTK wild-type and knock-out hosts and whether inhibition of MerTK signaling with warfarin could replicate MerTK knock-out phenotypes. Wild-type and MerTK-/- BALB/c mice were grafted in the flanks with CT26 tumors and treated with computed tomography guided RT. The role of macrophages and CD8 T cells in the response to radiation were demonstrated with cell depletion studies. The role of MerTK in priming immune responses after RT alone and with agonist antibodies to the T cell costimulatory molecule OX40 was evaluated in a Panc02-SIY model antigen system. The effect of warfarin therapy on the in-field and abscopal response to RT was demonstrated in murine models of colorectal adenocarcinoma. The association between warfarin and progression-free survival for patients treated with SABR for early-stage non-small cell lung cancer was evaluated in a multi-institutional retrospective study. MerTK-/- hosts had better tumor control after RT compared with wild-type mice in a macrophage and CD8 T cell-dependent manner. MerTK-/- mice showed increased counts of tumor antigen-specific CD8 T cells in the peripheral blood after tumor-directed RT alone and in combination with agonist anti-OX40. Warfarin therapy phenocopied MerTK-/- for single-flank tumors treated with RT and improved abscopal responses for RT combined with anti-CTLA4. Patients on warfarin therapy when treated with SABR for non-small cell lung cancer had higher progression-free survival rates compared with non-warfarin users. MerTK inhibits adaptive immune responses after SABR. Because warfarin inhibits MerTK signaling and phenocopies genetic deletion of MerTK in mice, warfarin therapy may have beneficial effects in combination with SABR and immune therapy in patients with cancer.

Targeting tumors with IL-21 reshapes the tumor microenvironment by proliferating PD-1intTim-3-CD8+ T cells.

The lack of sufficient functional tumor-infiltrating lymphocytes in the tumor microenvironment (TME) is one of the primary indications for the poor prognosis of patients with cancer. In this study, we developed an Erbitux-based IL-21 tumor-targeting fusion protein (Erb-IL21) to prolong the half-life and improve the antitumor efficacy of IL-21. Compared with Erb-IL2, Erb-IL21 demonstrated much lower toxicity in vivo. Mechanistically, Erb-IL21 selectively expanded functional cytotoxic T lymphocytes but not dysfunctional CD8+ T cells in the TME. We observed that the IL-21-mediated antitumor effect largely depended on the existing intratumoral CD8+ T cells, instead of newly migrated CD8+ T cells. Furthermore, Erb-IL21 overcame checkpoint blockade resistance in mice with advanced tumors. Our study reveals that Erb-IL21 can target IL-21 to tumors and maximize the antitumor potential of checkpoint blockade by expending a subset of tumor antigen-specific CD8+ T cells to achieve effective tumor control.

Abstract PR05: Blockade of AHR activation by IDO/TDO-derived kynurenine restricts cancer immune suppression

Abstract Immune checkpoint blockade (ICB) has been shown to convey significant clinical activity across a spectrum of malignancies, yet there is now recognition that multiple mechanisms of resistance can impair response. The catabolism of tryptophan into metabolites known as kynurenines (Kyn) by enzymes such as indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO) plays a major suppressive role. Recently it was shown that Kyn acts as an endogenous agonist of the Aryl hydrocarbon receptor (AhR). In order to gain a better understanding of this pathway, we sought to characterize the mechanisms of immunosuppression associated with AhR and evaluate its potential as therapeutic target. Gene-expression analysis of IDO-overexpressing melanomas (B16-IDO vs. B16-WT) demonstrated reduced expression levels of Type 1 inflammatory genes, including IFNy, TNF, GzmB, and CD40. In addition, B16-IDO presents higher infiltration of tumor-associated macrophages TAMs, which upregulate the AHR as well as classic AhR-regulated genes (Cyp1a1 and Cyp1b1) and are differentially skewed towards an immunosuppressive M2 phenotype. Tumor-antigen specific CD8+T cells show reduced expression of activation markers (GzmB and CD44) and proliferation rate when primed by Kyn-treated antigen-presenting cells. In addition, TAMs from B16-IDO tumors suppressed activation of CD8+T cells in vitro and their depletion delayed tumor growth. When B16-IDO cells are implanted in mice depleted of Foxp3 expressing cells, TAMs do not accumulate. Treatment of B16-IDO tumors with an AhR-specific antagonist (CH-223191) upregulates MHC II in APCs, activation markers in CD8s, and reduced frequency of T-regs in B16-IDO tumors. AhR inhibition slows tumor growth and prolongs survival of tumors with active IDO/TDO/Kyn pathway (B16-IDO and B16-TDO), and this is enhanced when PD-1 blockade is used in combination. In summary, our findings demonstrate that targeting the Kyn pathway through AhR inhibition could overcome key suppressive mechanisms and sensitize tumors to ICB. This abstract is also being presented as Poster A57. Citation Format: Luis F. Campesato, Sadna Budhu, Mathieu Gigoux, Jeremy Tchaicha, Stephane Pourpe, Cailian Liu, Dmitriy Zamarin, Mark G. Manfredi, Karen McGovern, Jedd D. Wolchok, Taha Merghoub. Blockade of AHR activation by IDO/TDO-derived kynurenine restricts cancer immune suppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR05.

Immune involvement of the contralateral hemisphere in a glioblastoma mouse model

BackgroundGlioblastoma (GBM) is the most common and deadliest form of brain cancer in adults. Standard treatment, consisting of surgery and radiochemotherapy, only provides a modest survival benefit and is incapable...

Glioblastoma infiltration of both tumor- and virus-antigen specific cytotoxic T cells correlates with experimental virotherapy responses

The mode of action for oncolytic viruses (OVs) in cancer treatment is thought to depend on a direct initial cytotoxic effect against infected tumor cells and subsequent activation of immune cell responses directed against the neoplasm. To study both of these effects in a mouse model of glioblastoma (GBM), we employed murine GBM cells engineered to constitutively express the type I Herpes Simplex Virus (HSV1) HSV-1 receptor, nectin-1, to allow for more efficient infection and replication by oncolytic HSV (oHSV). These cells were further engineered with a surrogate tumor antigen to facilitate assays of T cell activity. We utilized MRI-based volumetrics to measure GBM responses after injection with the oHSV and bioluminescent imaging (BLI) to determine oHSV replicative kinetics in the injected tumor mass. We found increased infiltration of both surrogate tumor antigen- and oHSV antigen-specific CD8+ T cells within 7 days after oHSV injection. There was no increase in tumor infiltrating CD8+ T cells expressing “exhaustion” markers, yet oHSV infection led to a reduction in PD-1+ CD8+ T cells in injected GBMs and an increase in IFNγ+ CD8+ T cells. There was a significant direct correlation between oHSV-mediated reduction in GBM volume and increased infiltration of both viral and tumor antigen-specific CD8+ T cells, as well as oHSV intratumoral gene activity. These findings imply that CD8+ T cell cytotoxicity against both tumor and viral antigens as well as intratumoral oHSV gene expression are important in oHSV-mediated GBM therapy.

Abstract A51: Impact of CaMKK2 inhibition in tumor-associated myeloid cells on CD8+ cytotoxic T-cell recruitment into mammary tumors

Abstract The development of cancer immunotherapy has reached an important inflection point, whereby such therapies have been administered with considerable clinical success in many cancers. However, despite some success in triple-negative breast cancer, the overall efficacy of existing immunotherapies in breast cancer has remained disappointing. This is largely due to low levels of tumor antigen-specific CD8+ tumor-infiltrating lymphocytes (TILs) in breast tumors, as a result of which mammary tumors are unable to establish a potent antitumor immune response. The efficacy of immune checkpoint inhibitors positively correlates with the intratumoral content of cytotoxic T cells and on the activity of processes that increase trafficking of these cells into the tumor microenvironment (TME). This study has been undertaken to identify and exploit mechanisms that facilitate recruitment of TILs to tumors to develop strategies rendering weakly immunogenic mammary tumors sensitive to contemporary immunotherapies. Our studies have shown that the serine/threonine kinase Calcium/Calmodulin Kinase Kinase 2 (CaMKK2) is expressed in all breast tumor subtypes, and its overexpression is directly correlated to poor clinical outcome. In previous studies we reported that primary tumor growth was dramatically reduced when tumors were propagated syngenically in Camkk2−/− mice or mice harboring a myeloid-specific CaMKK2 knockout as compared to wild-type mice. Taking this observation forward, we conducted immune cell profiling, which revealed that relative to WT mice, the number of cytotoxic CD8+T cells was significantly increased in tumors derived from Camkk2−/− mice. These results pointed to a cell-extrinsic function of CaMKK2 in breast cancer whereby depletion of CaMKK2 in stromal cells facilitated changes in the immune cell repertoire, specifically the increase of intratumoral effector T cells, thereby resulting in a more immunogenic tumor microenvironment. Furthermore, we found evidence of increased CXCL10 expression in bone marrow-derived macrophages (BMDMs) generated from mice harboring a myeloid-specific CaMKK2 knockout as compared to wild-type mice. Given the role of the CXCL10-CXCR3 signaling arc in intratumoral lymphocyte recruitment, our studies directly point to the role of myeloid-specific CaMKK2 in attenuating lymphocyte trafficking into mammary tumors by inhibiting the expression of promigratory chemokines. Indeed, transwell assays showed significantly higher number of T cells migrating towards supernates collected from Camkk2−/− BMDM than those from WT mice. These results were supported by additional data from a second study using antigen-specific CD8+T cells (OTI-T). We anticipate that our study will open up a new avenue of research into facilitating the entry of activated effector T cells into the tumor microenvironment, thereby enhancing the efficacy of established immune checkpoint inhibitors in cancer immunotherapy. Citation Format: Debarati Mukherjee, Robert Baldi, Ching-Yi Chang, Luigi Racioppi, Donald P. McDonnell. Impact of CaMKK2 inhibition in tumor-associated myeloid cells on CD8+ cytotoxic T-cell recruitment into mammary tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A51.

Development and characterization of a novel anti-OX40 antibody for potent immune activation.

With the great success of anti-CTLA-4 and anti-PD-1 therapeutics in cancer immunotherapy, tumor necrosis factor receptor superfamily members have been recognized as ideal targets to provide co-stimulatory signals in combination with immune checkpoint blocking antibodies. Among these is OX40 (CD134), a co-stimulatory molecule expressed by activated immune cells. Recently, several anti-OX40 agonistic monoclonal antibodies, pogalizumab as the most advanced, have entered early phase clinical trials. Using ayeast platform and multiple screening methods, we identified a fully human anti-OX40 antibody (IBI101) with distinct modes of action. Unlike pogalizumab, IBI101 partially blocks the binding of OX40 to its ligand OX40L and exhibits both FcγR-dependent and independent agonistic activities in NF-κB luciferase reporter assays. IBI101 also promotes T cell activation and proliferation in vitro. These unique properties partially explain the more potent anti-tumor activity of IBI101 than that of pogalizumab in humanized NOG mice bearing LoVo tumors. In addition, IBI101 shows efficacious anti-tumor activity in mice when administrated alone or in combination with anti-PD-1 antibodies. In human OX40 knock-in mice bearing MC38 colon carcinoma, IBI101 treatment induces tumor antigen-specific CD8+ T-cell responses, decreases immunosuppressive regulatory T cells in tumor, and enhances the immune response to PD-1 inhibition. Preclinical studies of IBI101 in non-human primates demonstrate typical pharmacokinetic characteristics of an IgG antibody and no drug-related toxicity. Collectively, IBI101 has desirable preclinical attributes which support its clinical development for cancer treatment.

Genetically stable poliovirus vectors activate dendritic cells and prime antitumor CD8 T cell immunity

Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses can provide a context optimal for generating antigen-specific CD8 T cells, as they have natural tropism for dendritic cells, preeminent inducers of CD8 T cell immunity; elicit Th1-promoting inflammation; and lack interference with innate or adaptive immunity. However, notorious genetic instability and underlying neuropathogenicity has hampered poliovirus-based vector applications. Here we devised a strategy based on the polio:rhinovirus chimera PVSRIPO, devoid of viral neuropathogenicity after intracerebral inoculation in human subjects, for stable expression of exogenous antigens. PVSRIPO vectors infect, activate, and induce epitope presentation in DCs in vitro; they recruit and activate DCs with Th1-dominant cytokine profiles at the injection site in vivo. They efficiently prime tumor antigen-specific CD8 T cells in vivo, induce CD8 T cell migration to the tumor site, delay tumor growth and enhance survival in murine tumor models.