Introduction: Multiple suppressive mechanisms within the tumor microenvironment (TME) contribute to blunt anti-tumor T cell responses. Among them, tumor-associated T cells have been phenotypically described to be functionally exhausted (or dysfunctional), reflecting to a hyporesponsive state of chronically stimulated T cells that express multiple inhibitory receptors (or immune checkpoint molecules), such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT), and exhibit a compromised cellular cytotoxicity. While immune checkpoint blockade therapies aimed at reversing the dysfunctional state T cell effector function have demonstrated clinical effectiveness, not all cancer patients achieve long-term disease control. This is due, at least in part, to the refractory nature of what are categorized as terminally exhausted CD8+ T cells to PD-1/PD-L blockade, for example. Considering that CD8+ T cell exhaustion and/or dysfunction is one of the major therapeutic challenges, we aim to uncover pathways that contribute to program T cells toward exhaustion/dysfunction during cancer progression. Methods and experimental procedures: CD47 (a.k.a. integrin-associated protein, IAP) is known for its role as a ‘‘don’t-eat-me’’ signal in malignant, transformed cells. Our recent study found that both human and murine tumor-associated CD8+ T cells derived from lesions of human and murine melanoma, and ovarian cancers exhibit a significant elevation of surface CD47. Surprisingly, the population of tumor-associated CD8+ T cells that express high level of CD47 also express high levels of TOX, the critical factor that drives differentiation of exhausted T (Tex) cells, as well as other known immune checkpoint molecules. Through naïve T cell adoptive transfer experiments paired with single cell-RNA sequencing analysis, we demonstrated that CD47 plays a key role in driving T cell exhaustion. We demonstrated that extracellular matrix protein thrombospondin-1 (TSP-1) is the ligand for CD47-mediated T cell exhaustion during tumor progression by selectively disrupting the interaction between TSP-1 and CD47. Mechanistically, we demonstrate through immunoblot experiments that engagement of TSP-1:CD47 results in activation of calcineurin-NFAT axis, a key modulatory pathway upstream of TOX that drives T cell exhaustion and dysfunction. Summary of new, unpublished data: Our study uncovered a novel role of TSP-1:CD47 interaction, besides chronic antigen stimulation, in driving CD8 T cells exhaustion via the calcineurin-NFAT pathway. Our findings have implications for the mechanism of action in CD47 targeting therapies. Concluding statement: Extracellular matrix protein thrombospondin-1 and CD47 expressed on T cells contributes to promote TOX-driven T cell exhaustion program in cancer. Citation Format: Chien-Huan Weng, Anais Assouvie, Lauren Dong, Jean-Christophe Beltra, Sadna Budhu, Levi Mangarin, Yacine Marouf, Lucia Morgado-Palacin, Cailian Liu, Sébastien Monette, Jonathan Khan, Isabell Schulze, Dmitriy Zamarin, Linda Hamadene, Fadi Samaan, Daniel Hirschhorn, Stephane Pourpe, David Schroder, Roberta Zappasodi, Pamela Holland, Niroshana Anandasabapathy, John Wherry, Jedd D Wolchok, Taha Merghoub. Thrombospondin-1:CD47 signaling contributes to the development of T cell exhaustion in cancer [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B056.

T cell exhaustion is a major barrier to effective cancer immunotherapy. Although immune checkpoint blockade can reinvigorate exhausted T cells, not all patients achieve long-term responses, partly due to the refractory nature of terminally exhausted T cells. Beyond persistent antigen stimulation, the environmental drivers of exhaustion remain to be thoroughly characterized. Here we identify CD47 upregulation in tumor-infiltrating exhausted CD8+ T cells in both human and murine tumors. We reveal a novel role for the extracellular matrix protein thrombospondin-1 (TSP-1) in engaging CD47 on T cells to promote exhaustion. This interaction activates calcineurin–NFAT signaling, inducing upregulation of TOX and expression of inhibitory receptors, and impairing effector function during tumor progression. Importantly, disrupting the TSP-1–CD47 axis prevents T cell exhaustion and enhances tumor control. Our findings identify a novel pathway promoting T cell dysfunction and suggest that targeting the TSP-1–CD47 axis is a promising strategy to enhance T cell immunity and immunotherapy efficacy. Merghoub, Wolchok and colleagues reveal a role of the extracellular matrix protein thrombospondin-1 (TSP-1) and CD47 in promoting T cell exhaustion during tumor progression in mice and humans.

Identifying epitopes that T cells respond to is critical for understanding T cell-mediated immunity. Traditional multimer and other single cell assays often require large blood volumes and/or expensive HLA-specific reagents and provide limited phenotypic and functional information. Here, we present the Rap id T CR: E pitope R anker (RAPTER) assay, a single cell RNA sequencing (scRNA-SEQ) method that uses primary human T cells and antigen presenting cells (APCs) to assess functional T cell reactivity. Using hash-tag oligonucleotide (HTO) coding and T cell activation-induced markers (AIM), RAPTER defines paired epitope specificity and TCR sequence and can include RNA- and protein-level T cell phenotype information. We demonstrate that RAPTER identified specific reactivities to viral and tumor antigens at sensitivities as low as 0.15% of total CD8 + T cells, and deconvoluted low-frequency circulating HPV16-specific T cell clones from a cervical cancer patient. The specificities of TCRs identified by RAPTER for MART1, EBV, and influenza epitopes were functionally confirmed in vitro. In summary, RAPTER identifies low-frequency T cell reactivities using primary cells from low blood volumes, and the resulting paired TCR:ligand information can directly enable immunogenic antigen selection from limited patient samples for vaccine epitope inclusion, antigen-specific TCR tracking, and TCR cloning for further therapeutic development.

Multiple suppressive mechanisms within the tumor microenvironment (TME) are capable of blunting anti-tumor T cell responses. These include engagement of inhibitory receptors expressed in tumor-associated, exhausted CD8 T cells, such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). While immune checkpoint blockade therapies aimed at reversing the dysfunctional state of tumor-associated T cells have demonstrated clinical effectiveness, not all cancer patients achieve long-term disease control. This is due, at least in part, to the refractory nature of what are categorized as terminally exhausted CD8 T cells to be reinvigorated by, for example, PD-1/PD-L1 blockade. As CD8 T cell exhaustion (or dysfunction) is a major therapeutic challenge, gaps in our understanding of cellular and molecular mechanisms underlying the T cell exhaustion (or dysfunction) program in cancer warrant further study of pathways that program T cells toward exhaustion (or dysfunction). Through comprehensive immune profiling of tumor-infiltrating T lymphocytes (TILs), we found that CD47 expression in CD8 TILs isolated from melanoma patients significantly correlates with expression of several checkpoint inhibitory molecules (e.g., TIM-3, PD-1 and LAG-3). Additionally, our re-analysis of single cell data from melanoma patients revealed that terminally exhausted T cells (Tex) and TCF7hi Tex precursor cells exhibit high levels of CD47 transcripts, suggesting phenotypic association of CD47 with T cell exhaustion. We confirmed our observations in murine B16-F10 melanoma where CD47 expression is significantly upregulated in exhausted CD8 TILs. We also show that CD47 functions as a negative regulator for T cell proliferation and function during T cell priming. To address the role of CD47 during the development of CD8 T cell exhaustion/dysfunction in cancer, we performed adoptive T cell transfer of the naïve-sorted Cd47+/+ (WT) and Cd47+/- (Het) antigen specific Pmel-1 CD8 T cells (but not Cd47-deficient Pmel-1 CD8 T cells as they would be subject to innate immune clearance) into B16 tumor-bearing mice and found that Cd47-Het Pmel-1 CD8 TILs, as compared to the Cd47-WT Pmel-1 CD8 TILs, exhibit less expression of exhaustion-related genes (e.g. Pdcd1, Lag3 and Tox), and increased expression of genes associated with T cell activation and proliferation (e.g. Mki67, Lck, Cd69, Gzma, Gzmk). We further confirmed that thrombospondin-1 (TSP-1), as an extracellular matrix protein and a ligand of CD47, contributes to driving the differentiation of CD8 T cells toward exhaustion. Our data highlight for the first time the potential of extracellular matrix protein TSP-1 in programming CD8 T cell exhaustion in cancer through its interaction with CD47 expressed on CD8 T cells. Citation Format: Chien-Huan Weng, Fadi Samaan, Sadna Budhu, Levi Mangarin, Sébastien Monette, Cailian Liu, Stephane Pourpe, Linda Hamadene, Hong Zhong, Xia Yang, David Schroder, Roberta Zappasodi, Pamela Holland, Jedd D. Wolchok, Taha Merghoub. Potential role of CD47 in T cell exhaustion program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6150.

Outcomes remain poor for patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL). While chimeric antigen receptor (CAR) T-cell therapy has revolutionised treatment, a significant proportion of patients relapse or fail to respond. Odronextamab is a CD20 × CD3 bispecific antibody that has demonstrated durable responses and a manageable safety profile in patients with R/R B-NHL in a first-in-human trial (NCT02290951). Here, we document two patients with diffuse large B-cell lymphoma refractory to CART-cell therapy. Both achieved complete responses that remain ongoing for ≥2 years following odronextamab. Neither patient experienced Grade ≥3 cytokine release syndrome or Grade ≥3 neurological adverse events during treatment.

The majority of JAK2 V617F -negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin ( CALR ), resulting in a common carboxyl-terminal mutant fragment (CALR MUT ), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALR MUT -specific T cells are rare in patients with CALR MUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALR MUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALR MUT neoepitopes with high affinity are underrepresented in patients with CALR MUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALR MUT MPN would not efficiently respond to a CALR MUT fragment cancer vaccine but would when immunized with a modified CALR MUT heteroclitic peptide vaccine approach. We found that heteroclitic CALR MUT peptides specifically designed for the MHC-I alleles of patients with CALR MUT MPN efficiently elicited a CALR MUT cross-reactive CD8 + T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALR MUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8 + T cell response to the CALR MUT fragment upon immunization with a CALR MUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide–based cancer vaccines in patients with CALR MUT MPN.

Tryptophan catabolism by the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase 2 (IDO/TDO) promotes immunosuppression across different cancer types. The tryptophan metabolite L-Kynurenine (Kyn) interacts with the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) to drive the generation of Tregs and tolerogenic myeloid cells and PD-1 up-regulation in CD8 + T cells. Here, we show that the AHR pathway is selectively active in IDO/TDO-overexpressing tumors and is associated with resistance to immune checkpoint inhibitors. We demonstrate that IDO-Kyn-AHR-mediated immunosuppression depends on an interplay between Tregs and tumor-associated macrophages, which can be reversed by AHR inhibition. Selective AHR blockade delays progression in IDO/TDO-overexpressing tumors, and its efficacy is improved in combination with PD-1 blockade. Our findings suggest that blocking the AHR pathway in IDO/TDO expressing tumors would overcome the limitation of single IDO or TDO targeting agents and constitutes a personalized approach to immunotherapy, particularly in combination with immune checkpoint inhibitors.

BACKGROUND: Odronextamab (REGN1979) is a first-in-class, hinge-stabilized, fully human CD20 x CD3 IgG4-based bispecific antibody that binds to CD20-expressing cells and CD3 on T cells, targeting CD20+ cells via T-cell-mediated cytotoxicity independent of T-cell receptor recognition. Patients with relapsed/refractory B-cell non-Hodgkin lymphoma were treated with odronextamab in a first-in-human, Phase 1 study (NCT02290951). Patient biopsies were analyzed to investigate the association of clinical response and relapse with B- and T-cell markers. METHODS: Tumor biopsies collected at baseline and at disease progression were analyzed by semi-quantitative CD20 chromogenic immunohistochemistry (IHC). B-cell antigen and immune cell multiplex immunofluorescence was also performed. Bulk tumor tissue nucleic acid isolates were analyzed by whole exome DNA sequencing. Peripheral blood mononuclear cells isolated from baseline samples were analyzed by highly multiplexed flow cytometry T-cell immunophenotyping assays. A single biopsy sample of fresh tumor tissue obtained at disease progression was analyzed by flow cytometry and single cell RNA sequencing. RESULTS: At baseline, higher levels of tumor-infiltrating CD4 and CD8 T cells were present in patients with a complete or partial response to odronextamab (N=13) compared with patients with no response (N=14); median (interquartile range) in CD4 responders vs non-responders was 2496 cells/mm2 (869-3940) vs 475 cells/mm2 (208-2714), and in CD8 responders vs non-responders was 3289 cells/mm2 (1981-7060) vs 489 cells/mm2 (110-3811), respectively. However, clinical responses were also observed in patients with low levels of baseline T-cell infiltration. Clinical efficacy was also associated with systemic T-cell immunophenotypic subsets in peripheral blood at baseline, including T-cell subset distribution, co-stimulatory molecule expression, and checkpoint molecule expression. Patient response to odronextamab was independent of the baseline overall frequency of intratumoral CD20+ cells (N=51) or intensity of CD20 expression (N=28) in tumor cells. The presence of CD20(-)/Pax5(+) lymphoma cell subsets at baseline, a potential source of CD20(-) disease escape, did not preclude durable clinical responses (N=27). However, loss of CD20 expression was observed in 6/9 biopsy samples taken at relapse. Genomic analysis of relapse samples identified CD20 gene mutations in 3/8 cases (two truncating and one c-terminal); these three patients were treated at an active odronextamab dose level, and had experienced response before progressing. In one case with available repeat samples, CD20 mutation observed at disease progression had not been detected at Week 5. Loss of CD20 expression by IHC was observed in two cases in the absence of a CD20 gene mutation, suggesting an alternative epigenetic molecular mechanism of CD20 loss. Single-cell analysis of a fresh tissue sample from a case of progressive disease, occurring after a prior complete response, showed a complete loss of CD20 expression on the cell surface while maintaining abundant tumor-infiltrating effector T cells. CONCLUSIONS: Preliminary analyses suggest that high levels of baseline tumor-infiltrating T cells may be associated with clinical response to odronextamab. Systemic T-cell homeostasis at baseline may be a potential predictor of clinical benefit with odronextamab, and further investigation is warranted. Although baseline CD20 expression level did not correlate with efficacy, loss of CD20 expression was observed frequently in progressive disease. Several CD20 gene mutations were detected in patient samples at clinical progression, suggesting potential target antigen-dependent disease escape. A newly detected CD20 mutation at disease progression suggests resistance may not always be mediated by outgrowth of pre-existing CD20(-) disease subclones. The use of baseline CD20 expression as a predictive biomarker is not supported at this time. Durable responses observed in patients with CD20(-) subclones suggest a bystander immune effect may be induced by odronextamab. CD20 loss appears to be an important mechanism of treatment resistance, which may help inform future clinical development strategies. Additional mechanisms of resistance are under investigation, including an evaluation of the immune inhibitory microenvironment at disease progression. Disclosures Brouwer-Visser: Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Fiaschi:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Deering:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Dhanik:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Cygan:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Zhang:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Jeong:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Pourpe:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Boucher:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Hamon:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Bannerji:Sanofi-Pasteur: Other: Spouse is employee; AbbVie: Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc and Pharmacyclics LLC, an AbbVie Company: Research Funding; Regeneron Pharmaceuticals: Research Funding. Duell:Morphosys: Research Funding. Advani:Celgene, Forty Seven, Inc., Genentech/Roche, Janssen Pharmaceutical, Kura, Merck, Millenium, Pharmacyclics, Regeneron, Seattle Genetics: Research Funding; Astra Zeneca, Bayer Healthcare Pharmaceuticals, Cell Medica, Celgene, Genentech/Roche, Gilead, KitePharma, Kyowa, Portola Pharmaceuticals, Sanofi, Seattle Genetics, Takeda: Consultancy. Flink:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Chaudhry:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Sirulnik:Regeneron Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Murphy:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Weinreich:Regeneron Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Yancopoulos:Regeneron Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Thurston:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Ambati:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Jankovic:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure: The biomarker data described in the abstract will report on use of odronextamab in a Phase 1 clinical trial of patients with B-NHL

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 checkpoint blockade (ICB) results in clinical benefit for a subset of cancer patients, yet multiple mechanisms of resistance can impair optimal response. The catabolism of tryptophan into metabolites known as kynurenines (Kyn) by the expression of enzymes such as IDO or TDO is a frequent phenomenon that plays a suppressive role in tumor immunity. Recently it was shown that Kyn acts as agonist of the aryl hydrocarbon receptor (AHR). Here we sought to characterize the mechanisms of immune suppression associated with the AHR pathway and to evaluate its potential as therapeutic target. RNAseq analysis of human cancers revealed a correlation between the expressions of AHR-related genes with markers associated with immunotherapy resistance (PD-1, FOXP3, CD206). By using IDO or TDO-overexpressing variants of a melanoma cell model (B16-F10), we found that myeloid cells, such as tumor-associated macrophages (TAMs) and dendritic cells (DCs), present up-regulation of the AHR. IDO-expressing tumors (B16-IDO) show higher myeloid cell infiltration, which present a tolerogenic phenotype. Tumor-antigen specific CD8T cells present reduced expression of activation markers and proliferation rate when primed by Kyn-treated BMDCs. Treatment of B16-IDO-bearing mice with an AHR-specific antagonist (CH-223191) leads to an increase of MHC II in TAMs, of activation markers in CD8 T cells and reduced frequency of T-regs. AHR inhibition delays progression of tumors with an active IDO/TDO/Kyn pathway (B16-IDO and B16-TDO), and efficacy is further improved when ICB 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.

12060 Background: NSCLCs often harbor mutations in the KRAS oncogene and require improved treatment to provide durable disease control. Targeted therapy via MEK inhibition shows promising but temporary control of tumor growth but ultimately fails with a quick rebound of tumor growth. As the RAS-RAF-MEK-ERK pathway is evolutionarily conserved in most cells, MEK inhibition may cause broader effects in the tumor microenvironment. Methods: We examined the effect of novel pulsatile versus standard continuous MEK inhibition on lymphocytes and myeloid cells. KRAS tumor bearing mice were treated with selumetinib and T cells were analyzed for phenotypic changes. Tumor progression and survival with and without the addition of checkpoint blockade therapy was also monitored. In vitro experiments were also conducted with murine bone marrow-derived macrophages subsequently exposed to pulsatile or continuous selumetinib treatment. Results: MEK inhibition in T cells shows significant effects on T cell activation and proliferation. CD8+ T cell phenotypes increased CTLA-4, Ki67 and 4-1BB expression when treated with pulsatile compared to continuous MEK inhibitor, suggesting greater activation and proliferation status. Pulsatile MEK inhibitor therapy with CTLA-4 checkpoint blockade showed improved overall survival compared to continuous treatment in a mouse model of Kras mutant lung cancer. We extended our investigation to other tumor microenvironment components particularly myeloid cells. In vitro experiments revealed that pulsatile versus continuous treatment with the clinical MEK1/2 inhibitor selumetinib differentially effects macrophage viability and pro-inflammatory cytokine production. Conclusions: Pulsatile MEK inhibition improves T cell activation and prolongs survival in combination with anti-CTLA-4, compared with continuous treatment. It also has a modulating effect on myeloid cells compared to the standard continuous dosing. Optimizing MEK inhibition scheduling to target cancer cells and activate immune infiltrates will provide the best therapy for KRAS mutant NSCLC. These data will contribute to informing design immune modulation with targeted therapies in patients.

Irreversible exhaustion of tumor-associated T cells is an important factor limiting the efficacy of PD-1 blockade in a number of cancers. Recent data suggest that PD-1 blockade functions largely by augmenting T cell costimulation. Given that activated APCs provide necessary T-cell costimulation, we hypothesized that enforced activation of intratumoral APCs would prime tumor-specific T cells and synergize with PD-1 blockade to overcome T-cell exhaustion. After screening various agents in syngeneic murine models, we found that dual CD40/TLR4 activation within a single tumor triggers a systemic tumor-specific CD8 T-cell response that is dependent on BATF3+ dendritic cells. Remarkably, this approach abolishes exhausted PD-1+ intratumoral T cells in treated as well as distant tumors while sparing more proximal T cells outside the tumors. In addition to treating large established tumors, this approach also confers persistent immunity allowing animals to reject reimplanted tumors 90 days after treatment. Dual CD40/TLR4 activation within a single tumor is thus a promising method for overcoming tumor-associated T-cell exhaustion in a manner that may provide durable systemic control of metastatic human cancers while sparing healthy tissues. Citation Format: Danny Khalil, Luis Felipe Campesato, Sadna Budhu, Yanyun Li, Caitlin Jones, Nathan Suek, Cailian Liu, Billel Gasmi, Rachel Giese, Stephane Pourpe, Taha Merghoub, Jedd D. Wolchok. Defined factors overcome T-cell exhaustion via abscopal effect [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 5009.

Although with notable impact on cancer treatment, immune-checkpoint blockade (ICB) have multiple mechanisms of resistance. The catabolism of Tryptophan into metabolites known as Kynurenines (Kyn) by enzymes such as IDO or TDO plays a major suppressive role in different tumor types. 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) present reduced expression levels of Type-1 inflammatory genes, including IFNy, TNFa, Granzyme B and CD40. In addition, B16-IDO presents higher infiltration of TAMs, which up-regulate classic AhR-regulated genes (Cyp1a1 and Cyp1b1) and are differentially skewed towards an immunosuppressive M2-phenotype. Tumor-antigen specific CD8+T cells present reduced expression of activation markers (GzmB and CD44) and proliferation rate when primed by Kyn-treated BMDMs. Also, B16-IDO TAMs suppressed activation of CD8+T cells in vitro and their depletion abrogated tumor growth. Implantation of B16-IDOs in FoxP3-depleted mice prevents TAMs accumulation. Treatment of B16-IDO tumors with an AhR-specific antagonist (CH-223191) up-regulates MHC II in APCs, activation markers in CD8s and reduced frequency of T-regs. AhR inhibition slows down tumor growth and prolongs survival, which is improved in combination with PD-1 blockade. In summary, our findings demonstrate that targeting the Kyn pathway through AhR-inhibition represents a promising approach in cancer patients who are resistant to ICB.

We present an exceptional case of a patient with high-grade serous ovarian cancer, treated with multiple chemotherapy regimens, who exhibited regression of some metastatic lesions with concomitant progression of other lesions during a treatment-free period. Using immunogenomic approaches, we found that progressing metastases were characterized by immune cell exclusion, whereas regressing and stable metastases were infiltrated by CD8+ and CD4+ T cells and exhibited oligoclonal expansion of specific T cell subsets. We also detected CD8+ T cell reactivity against predicted neoepitopes after isolation of cells from a blood sample taken almost 3 years after the tumors were resected. These findings suggest that multiple distinct tumor immune microenvironments co-exist within a single individual and may explain in part the heterogeneous fates of metastatic lesions often observed in the clinic post-therapy. VIDEO ABSTRACT.