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Supplemental figure legends 1-8, Supplementary tables 1-5, and Supplementary methods

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti–PD-1 alone was ineffective, the erdafitinib and anti–PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti–PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti–PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

Supplemental Figures 1-8

Several non-redundant features of the tumour microenvironment facilitate immunosuppression and limit anticancer immune responses. These include physical barriers to immune infiltration, the recruitment of suppressive immune cells and the upregulation of ligands on tumour cells that bind to inhibitory receptors on immune cells. Recent insights into the importance of the metabolic restrictions imposed by the tumour microenvironment on antitumour T cells have begun to inform immunotherapeutic anticancer strategies. Therapeutics that target metabolic restrictions, such as low glucose levels, a low pH, hypoxia and the generation of suppressive metabolites, have shown promise as combination therapies for different types of cancer. In this Review, we discuss the metabolic aspects of the antitumour T cell response in the context of immune checkpoint blockade, adoptive cell therapy and treatment with oncolytic viruses, and discuss emerging combination strategies. Immunotherapeutic approaches to cancer can be affected by metabolic restrictions that limit the potency of anticancer T cell responses. In this Review, DePeaux and Delgoffe discuss the metabolic features of the tumour microenvironment that limit anticancer immune responses, as well as emerging therapeutic approaches to target these.

Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8+ T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell-intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked: through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates.

While CD8+ cytotoxic T cells are clearly critical for identification and elimination of cancer cells, factors concentrated within the tumor microenvironment drive their altered differentiation to a hypofunctional, short-lived state termed exhaustion. Exhaustion is a progressive lineage, and it is now clear that the most terminally exhausted T (tTexh) cells are not the targets of checkpoint blockade immunotherapy but rather serve as factors that limit immunotherapeutic efficacy. Compared directly, tumor-infiltrating CD8+ tTexh cells bear notable phenotypic similarity to CD4+Foxp3+ regulatory T (Treg) cells, suggesting beyond loss of proinflammatory function, tTexh cells may be directly anti-functional and constrain tumor specific immunity. Here we show, when isolated from the same tumor microenvironment, terminally exhausted CD8+ T cells carried similar capacity to suppress T cell proliferation as CD4+Foxp3+ Treg cells. Unlike Treg cells, tTexh cells suppress solely via the ectonucleotidase CD39, which serves to deplete extracellular ATP (eATP) and produce an adenosinergic environment. CD39 expression in tTexh cells is driven by exposure to tumor hypoxia, such that therapeutic targeting of hypoxia limits tTexh suppression and support responses to immunotherapy. Specific deletion of CD39 in CD8+ T cells slows tumor progression and improves response to checkpoint blockade, highlighting a major role for immunosuppression by exhausted T cells in cancer. Thus, tTexh cells are an unappreciated immunoregulatory population and for full immunotherapeutic efficacy, strategies should be designed to either limit their generation, reprogram their immunosuppressive nature, or remove them from the tumor microenvironment.

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti–PD-1 alone was ineffective, the erdafitinib and anti–PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti–PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti–PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

The majority of patients with recurrent/metastatic squamous cell carcinoma of the head and neck (HNSCC) (R/M) do not benefit from anti-PD-1 therapy. Hypoxia induced immunosuppression may be a barrier to immunotherapy. Therefore, we examined the metabolic effect of anti-PD-1 therapy in a murine MEER HNSCC model as well as intratumoral hypoxia in R/M patients. In order to characterize the tumor microenvironment in PD-1 resistance, a MEER cell line was created from the parental line that are completely resistant to anti-PD-1. These cell lines were then metabolically profiled using seahorse technology and injected into C57/BL6 mice. After tumor growth, mice were pulsed with pimonidazole and immunofluorescent imaging was performed to analyze hypoxia and T cell infiltration. To validate the preclinical results, we analyzed tissues from R/M patients (n=36) treated with anti-PD-1 mAb, via immunofluorescent imaging for number of CD8+ T cells (CD8), Tregs and the percent area (CAIX) and mean intensity (I) of carbonic anhydrase IX in tumor. We analyzed disease control rate (DCR), progression free survival (PFS), and overall survival (OS) using proportional odds and proportional hazards (Cox) regression. We found that anti-PD-1 resistant MEER has significantly higher oxidative metabolism, while there was no difference in glycolytic metabolism. Intratumoral hypoxia was significantly increased and CD8+ T cells decreased in anti-PD-1 resistant tumors compared with parental tumors in the same mouse. In R/M patients, lower tumor hypoxia by CAIX/I was significantly associated with DCR (p=0.007), PFS, and OS, and independently associated with response (p=0.028) and PFS (p=0.04) in a multivariate model including other significant immune factors. During PD-1 resistance, tumor cells developed increased oxidative metabolism leading to increased intratumoral hypoxia and a decrease in CD8+ T cells. Lower tumor hypoxia was independently associated with increased efficacy of anti-PD-1 therapy in patients with R/M HNSCC. To our knowledge this is the first analysis of the effect of hypoxia in this patient population and highlights its importance not only as a predictive biomarker but also as a potential target for therapeutic intervention.

Background Regulatory T (Treg) cells are vital for preventing autoimmunity but are a major barrier to robust cancer immunity as the tumor microenvironment (TME) recruits and promotes their function. The deregulated cellular metabolism of tumor cells leads to a metabolite-depleted, hypoxic, and acidic TME. While the TME impairs the effector function of highly glycolytic tumor infiltrating CD8 T cells, Treg cell suppressive function is maintained. Further, studies of in vitro induced and ex vivo Treg cells reveal a distinct metabolic profile compared to effector T cells. Thus, it may be that the altered metabolic landscape of the TME and the increased activity of intratumoral Treg cells are linked. Methods Flow cytometry, isotopic flux analysis, Foxp3 driven Cre-lox, glucose tracers, Seahorse extracellular flux analysis, RNA sequencing. Results Here we show Treg cells display heterogeneity in terms of their glucose metabolism and can engage an alternative metabolic pathway to maintain their high suppressive function and proliferation within the TME and other tissues. Tissue derived Treg cells (both at the steady state and under inflammatory conditions) show broad heterogeneity in their ability to take up glucose. However, glucose uptake correlates with poorer suppressive function and long-term functional stability, and culture of Treg cells in high glucose conditions decreased suppressive function. Treg cells under low glucose conditions upregulate genes associated with the uptake and metabolism of the glycolytic end-product lactic acid. Treg cells withstand high lactate conditions, and lactate treatment prevents the destabilizing effects of high glucose culture. Treg cells utilize lactate within the TCA cycle and generate phosphoenolpyruvate (PEP), a critical intermediate that can fuel intratumoral Treg cell proliferation in vivo. Using mice with a Treg cell-restricted deletion of lactate transporter Slc16a1 (MCT1) we show MCT1 is dispensable for peripheral Treg cell function but required intratumorally, resulting in slowed tumor growth and prolonged survival. Conclusions These data support a model in which Treg cells are metabolically flexible such that they can utilize ‘alternative’ metabolites present in the TME to maintain their suppressive identity. Further, our studies support the notion that tumors avoid immune destruction not only by depriving effector T cells of essential nutrients, but also by metabolically supporting regulatory T cells.

Background Blockade of co-inhibitory ‘checkpoint’ molecules, PD-1 and CTLA-4, has induced impressive clinical responses in advanced tumors; yet only in a subset of patients.1–3 Limited success with checkpoint blockade therapy suggests other cell extrinsic or intrinsic mechanisms may be dampening an effective immune response. Cytotoxic CD8+ T cells (CTL) encountering chronic antigen and metabolic restriction can differentiate to a terminally exhausted state (Texh), marked by hyporesponsiveness and metabolic, epigenetic, and transcriptional dysfunction.4–8 While enrichment of this population in tumor is a negative prognostic factor,9–10 it remains unclear whether Texh are simply non-functional or instead possess tolerogenic or suppressive properties. Transcriptional profiling of tumor-infiltrating PD-1int (progenitor exhausted) CTL versus PD-1hiTIM-3+ (terminally exhausted; Texh), reveals that exhausted cells express a pattern of genes associated with immune suppression. We hypothesize that Texh potentiate the suppressive microenvironment of solid tumor by autoregulation and inhibition of local immune responses. Methods T cell populations were isolated from murine melanoma–B16-F10 or a lab-generated melanoma clone of the spontaneous BREF/PTEN model–by expression of inhibitory receptors and assayed in tandem in microsuppression assays. Murine melanoma clones with inhibited oxidative metabolism were generated by CRISPR-Cas9 deletion and validated for ablated mitochondrial respiration by extracellular flux analysis. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery. Results When sorted directly from tumor, PD-1hiTim3+ Texh, but not progenitor exhausted PD-1int CTL, induce marked suppression of T cell effector responses, comparable to Foxp3+ Treg from the same environment. Expression of the ectonucleotidase, CD39, is uniquely expressed in Texh and increases as T cells differentiate towards exhaustion. Genetic deletion of CD39 in Texh eliminates the regulatory phenotype of tumor-infiltrating Texh and enforced CD39 expression on effector T cells can inhibit T cell receptor signaling and downstream function. CD39 expression correlates with exposure to hypoxia and Texh sorted from tumors engineered to be less hypoxic displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, contributes to generation of immunosuppressive adenosine, and has been previously associated with terminal exhaustion.11–13 Conclusions Our data support a model that as CTL progress to terminal exhaustion, hypoxic exposure enforces the upregulation of CD39, providing Texh a mechanism to suppress proinflammatory processes. These findings suggest Texh are not solely dysfunctional but rather are deleterious to anti-tumor immunity and may need to be drastically reprogrammed or deleted in order to alleviate immunosuppressive functions. References Wolchok JD. et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl. J. Med 2017; 377, 1345–1356. Hellmann MD, et al. Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012): results of an open-label, phase 1, multicohort study. Lancet Oncol 2017; 18, 31–41. Robert C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 2015; 372, 2521–2532. Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336. Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421. Blackburn SD, Shin H, Freeman GJ & Wherry EJ. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc. Natl. Acad. Sci 2008;105:15016–15021. Pauken KE, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354:1160–1165. Najjar YG, et al. Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma. JCI Insight. 2019; 4. Loo K, et al. Partially exhausted tumor-infiltrating lymphocytes predict response to combination immunotherapy. JCI Insight 2017; 2. Daud AI, et al. Tumor immune profiling predicts response to anti-PD-1 therapy in human melanoma. J. Clin. Invest 2016;126:3447–3452. . Duhen T, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat. Commun 2018;9:2724. Canale FP, et al. CD39 Expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128. Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11, e1005177.

BackgroundOncolytic viruses are an underappreciated immunotherapy capable of inflaming the tumor microenvironment (TME), vaccinating a patient against their own tumor, and delivering gene therapy to the TME. However, apart from the oncolytic HSV T-vec, these therapies have not seen widespread use, due in part to incomplete understanding of their immunologic mechanisms of action. We sought to determine features of oncolytic vaccinia virus (VV) response and resistance using subclones of the HPV+ head and neck cancer model MEER rendered sensitive or resistant to VV.MethodsA VV sensitive MEER tumor resisting treatment was serially passaged in mice and treated with VV until a stably resistant line was generated (Fig1). Sensitive or resistant MEER tumors were implanted, treated with a single intratumoral dose of VV, and harvested 4–7 days later for cytometric analysis. A genetically encoded TGFβ inhibitor was recombined into oncolytic VV (VV-TGFβi).ResultsWe used serial in vivo passaging to generate a VV-resistant MEER line (MEERvvR) from one sensitive to VV (MEERvvS, figure 1) and compared their immune infiltrate. While VV promoted acute cytokine production and cytotoxicity in conventional T cells, the major determining factor between sensitivity and resistance was the phenotype of Treg cells. At baseline, Treg cells in MEERvvS had lower Nrp1 expression and higher IFNγ-STAT1 signaling compared to MEERvvR, indicative of Treg 'fragility'. VV treatment induced MEERvvS Treg cells to become immunostimulatory and produce IFNγ (figure 2). RNAseq revealed MEERvvR produced more TGFβ than MEERvvS cells, suggesting these tumors directly stabilize Treg cells. To determine if MEERvvR could be sensitized to VV, we engineered oncolytic vaccinia to produce a genetically-encoded TGFβ inhibitor which binds TGFβRII, preventing TGFβ1-3 binding (VV-TGFβi). When MEERvvR were treated with VV-TGFβi, elite responses were restored, with commensurate increase in survival (figure 3) associated with increased STAT1 signaling in Treg cells.ConclusionsResistance to oncolytic vaccinia is controlled by Treg cell phenotype; tumors harboring more fragile Treg cells respond exquisitely to VV. An oncolytic vaccinia engineered to produce a novel TGFβi could remodel the TME to be less supportive of Tregs, rendering resistant tumors sensitive to VV. Our data highlight the importance of Treg cell status in resistance to oncolytic virus therapy and suggest TGFβ can be effectively targeted through an inhibitor encoded within the virus. Importantly, this TME directed production of the TGFβi carries no toxicity previously associated with systemic TGFβ inhibition, suggesting a viral approach to TGFβ inhibition can be an effective strategy support broader immunotherapy response.Abstract 743 Figure 1Strategy used to generate a vaccinia resistant MEER (MEERvvR) from vaccinia sensitive MEER (MEERvvS)Abstract 743 Figure 2IFNγ production in Treg cells in MEERvvS and MEERvvR after treatment with PBS or control vaccinia (VV-Ctrl)Abstract 743 Figure 3Survival of VV-resistant MEER treated with PBS, control vaccinia (VV-Ctrl), or vaccinia engineered to deliver a potent inhibitor of TGFβ (VV-TGFβi)

BackgroundWhile CD8+ cytotoxic T cells are clearly critical for identification and elimination of cancer cells, factors concentrated within the tumor microenvironment drive altered differentiation of these cells to a hypofunctional, short-lived state termed T cell exhaustion1 (figure 1a). Exhaustion is a progressive lineage, and it is now clear that terminally exhausted T (tTexh) cells are not the targets of checkpoint blockade immunotherapy but may serve as factors that limit immunotherapeutic efficacy.2–6 Compared directly, tumor-infiltrating CD8+ tTexh cells bear notable phenotypic similarity to CD4+Foxp3+ regulatory T (Treg) cells in expression of immunosuppressive molecules suggesting beyond loss of effector function, tTexh cells may be directly anti-functional and constrain tumor-specific immunity. Thus, we hypothesize that tTexh cells potentiate the suppressive microenvironment of solid tumor and that strategies to limit their generation or reprogram their immunosuppressive nature will improve control of tumor progression.MethodsT cell populations were isolated from murine tumor lines, B16-F10 melanoma, Ptenflox/floxBrafLSL.V600ETyr2Cre.ERT2–derived Clone 24 melanoma, MEER head and neck carcinoma, and MC38 adenocarcinoma. T cell-specific CD39 (Entpd1) deletion was accomplished by crossing Entpd1flox/flox mice to Cd4Cre or E8iGFP-Cre-ERT2. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery. Tumor hypoxia was alleviated by CRISPR-Cas9-directed deletion of mitochondrial genes in B16-F10 or by treatment with axitinib or metformin.ResultsWhen sorted directly from tumor, CD8+PD-1hiTim-3+ tTexh cells, but not progenitor PD-1intTim-3– pTexh cells, induce marked suppression of T cell effector responses, comparable to CD4+Foxp3+ Treg cells from the same environment (figure 1b-c). The ectonucleotidase, CD39, increases as cells progressively differentiate and is associated with terminal exhaustion.7 8 CD8+ T cell-restricted deletion of CD39 restricts regulatory functions of tTexh cells (figure 1b), improving tumor control and augmenting response to checkpoint blockade (figure 1d). CD39 expression correlates with hypoxia exposure and tTexh cells sorted from tumors engineered to be less hypoxic or treated with hypoxia-mitigating agents displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, supports adenosine generation, and limits therapeutic efficacy.ConclusionsOur data support a model that as CD8+ T cells progress to terminal exhaustion, hypoxia exposure enforces the upregulation of CD39, providing tTexh cells a mechanism to suppress proinflammatory processes and promote tumor progression. These findings suggest tTexh cells are not solely dysfunctional but rather are deleterious to antitumor immunity and may need to be drastically reprogrammed or depleted to improve patient outcomes.ReferencesBlank CU, et al. Defining “T cell exhaustion”. Nat Rev Immunol 2019;19:665–674.Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol 2019;20:326–336.Blackburn SD, et al. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc Natl Acad Sci USA 2008;105:15016–15021.Sade-Feldman M, et al. Defining T Cell states associated with response to checkpoint immunotherapy in Melanoma. Cell 2018;175:998–1013.e20.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Siddiqui I, et al. Intratumoral Tcf1+PD-1+CD8+ T Cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 2019;50:195–211.e10.Canale FP, et al. CD39 expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128.Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11:e1005177.Abstract 679 Figure 1(a) Schematic depicting differentiation of CD8+ T cells to terminal exhaustion in cancer and subsequent suppression of local immune responses by expression of ectonucleosidase, CD39; (b) When assayed directly ex vivo, CD8+ terminally exhausted T (tTexh) cells, but not progenitor exhausted T (pTexh) cells, suppress effector functions as effectively as CD4+Foxp3+ Treg isolated from the same environment. Deletion of CD39 alleviates tTexh-mediated suppression; (c) CD8+ T cell suppression correlates with expression of CD39 on tTexh from various tumor models. (d) CD8+ T cell-specific deletion of CD39 slows tumor growth and improves immune response to checkpoint blockade-resistant tumors. Data are pooled from ≥3 experiments. Statistics are two-way ANOVA with multiple comparisons or Pearson correlation. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Summary Immunotherapy can reinvigorate dormant responses to cancer, but response rates remain low. Oncolytic viruses, which replicate in cancer cells, induce tumor lysis and immune priming, but their immune consequences are unclear. We profiled the infiltrate of aggressive melanomas induced by oncolytic Vaccinia virus using RNA sequencing and found substantial remodeling of the tumor microenvironment, dominated by effector T cell influx. However, responses to oncolytic viruses were incomplete due to metabolic insufficiencies induced by the tumor microenvironment. We identified the adipokine leptin as a potent metabolic reprogramming agent that supported antitumor responses. Leptin metabolically reprogrammed T cells in vitro, and melanoma cells expressing leptin were immunologically controlled in mice. Engineering oncolytic viruses to express leptin in tumor cells induced complete responses in tumor-bearing mice and supported memory development in the tumor infiltrate. Thus, leptin can provide metabolic support to tumor immunity, and oncolytic viruses represent a platform to deliver metabolic therapy.

Targeted therapies against activated oncogenes, such as receptor tyrosine kinases, have significantly prolonged non-small cell lung cancer (NSCLC) patient survival, but the development of resistance limits the durability of clinical response. Genetic alterations which constitutively activate Fibroblast Growth Factor Receptors (FGFR) have been observed in patients with NSCLC. Erdafitinib (JNJ-42756493), an orally bioavailable pan-FGFR inhibitor discovered as part of a collaboration between Janssen and Astex Pharmaceuticals, has been shown to inhibit FGFR signaling pathways resulting in cell death and tumor growth inhibition in both in vitro and in vivo models of FGFR pathway aberration. Further, erdafitinib has been shown to have favorable pharmaceutical properties with manageable side effects in humans and several clinical trials are currently underway. One potential strategy to enhance the durability of response to targeted therapies, such as FGFR inhibitors, is to couple them with immunotherapy. In this setting, T cell responses primed and activated by increased antigen release resulting from the tumor cell targeted therapy could be enhanced and maintained by T-cell directed checkpoint blockade. To test this hypothesis, we evaluated erdafitinib in combination with an anti-programmed death-1 (PD-1) blocking antibody in an autochthonous FGFR2K660N/p53 genetically engineered mouse model (GEMM) of lung cancer, in which tumors develop within the context of an intact immune microenvironment. Cohorts of tumor bearing FGFR2K660N/p53 mutant mice treated with erdafitinib with or without anti-PD-1 showed significant tumor regressions compared to control and anti-PD-1 alone groups. Despite lack of differences in acute tumor responses between erdafitinib monotherapy and combination therapy, we observed significant survival benefit in the combination group erdafitinib alone (median survival 19.7 weeks vs 13.4 weeks, p Citation Format: Sangeetha Palakurthi, Mari Kuraguchi, Sima Zacharek, Jeff Liu, Dennis Bonal, Wei Huang, Kristin Depeaux, Abha Dhaneshwar, Sam Regan, Dyane Bailey, Martha Gowaski, Mei Zheng, Roderick Bronson, Catherine Ferrante, Enrique Zudaire, Sylvie Laquerre, Mark Bittinger, Kirschmeier Paul, Kathryn Packman, Raluca I. Verona, Kwok-Kin Wong, Matthew V. Lorenzi. Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B27.