Programmed Cell Death-1 Blockade Research Articles

TIM3 comes of age as an inhibitory receptor.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), a member of the TIM family, was originally identified as a receptor expressed on interferon-γ-producing CD4+ and CD8+ T cells. Initial data indicated that TIM3 functioned as a 'co-inhibitory' or 'checkpoint' receptor, but due to the lack of a definable inhibitory signalling motif, it was also suggested that TIM3 might act as a co-stimulatory receptor. Recent studies have shown that TIM3 is part of a module that contains multiple co-inhibitory receptors (checkpoint receptors), which are co-expressed and co-regulated on dysfunctional or 'exhausted' T cells in chronic viral infections and cancer. Furthermore, co-blockade of TIM3 and programmed cell death 1 (PD1) can result in tumour regression in preclinical models and can improve anticancer T cell responses in patients with advanced cancers. Here, we highlight the developments in understanding TIM3 biology, including novel ligand identification and the discovery of loss-of-function mutations associated with human disease. In addition, we summarize emerging data from human clinical trials showing that TIM3 indeed acts as a 'checkpoint' receptor and that inhibition of TIM3 enhances the antitumour effect of PD1 blockade.

Enhanced Therapeutic Efficacy of a Novel Oncolytic Herpes Simplex Virus Type 2 Encoding an Antibody Against Programmed Cell Death 1

The efficacy of immune checkpoint blockade therapy against immunologically “cold” tumors can be enhanced by applying the checkpoint inhibitors in combination with oncolytic viruses. Alternatively, the oncolytic virus construct has been modified to express factors that boost oncolytic virus function. We engineered a novel oncolytic herpes simplex virus 2 (HSV2) encoding an anti-human programmed cell death 1 (PD-1) monoclonal antibody (oHSV2-aPD1). This virus resulted in the detectable expression of a functional monoclonal antibody against human PD-1 by infecting eukaryotic cells. Therapeutic efficacy of oHSV2-aPD1 proved superior to unmodified oncolytic HSV2 treatment or PD-1 blockade alone and as effective as their combination in the poorly immunogenic melanoma models. Additionally, local oHSV2-aPD1 treatment induced a durable antitumor response and activated many immune effector cells and molecules both in the tumor microenvironment and in the systemic immune system. This provides support for combinatorial strategies involving local administration of an oncolytic HSV2 expressing a PD-1 inhibitor.

MS01.01 Biological Mechanisms of Resistance

Immune checkpoint inhibitors targeting the programmed cell death 1 (PD-1) axis have transformed the management of non-small cell lung cancer (NSCLC). Despite the dramatic and sometimes durable activity of these agents, a majority of patients will progress on therapy.1 Patients who experience a de novo lack of response to initial therapy are deemed as having primary (intrinsic) resistance. By contrast, acquired resistance refers to patients who initially achieve an objective response to therapy but eventually progress over time. The mechanistic basis for primary versus acquired resistance to immune checkpoint inhibitors is still poorly defined. To date, studies into the biological mechanisms of resistance to immunotherapies have centered around preclinical studies and analyses of repeat biopsies obtained from patients at the time of disease progression.2 These efforts have been complemented by similar studies in other immuno-oncology settings (e.g., melanoma),3,4 since there is potential for shared biology underlying immunotherapy resistance across malignancies. Both tumor cell-intrinsic and tumor cell-extrinsic factors have been implicated in mediating resistance to immunotherapies.2 In the setting of primary resistance, disease progression is commonly driven by a lack of neoantigens sufficient to illicit an initial immune response, commonly due to a low tumor mutation burden.5 Alternatively, tumors may actually harbor antigens capable of generating an immune response, but defects in antigen processing may ultimately limit the presentation of these antigens on the cell surface via MHC. Defective antigen presentation may be due to loss of beta-2 microglobulin (B2M), elimination of transporters associated with antigen processing (TAP) proteins, or downregulation of HLA.2,3 In addition, intrinsic resistance to immune checkpoint inhibitors may be due to altered oncogenic cell signaling (e.g., WNT/B-catenin pathway) and/or tumor de-differentiation with impaired antigen expression. Molecular mechanisms of acquired resistance to checkpoint blockade likely include many of the same processes as those implicated in intrinsice resistance. Indeed, homozygous loss of B2M, which is required for stabilization of HLA on the cell surface, has been described in NSCLC at the time of acquired resistance to PD-1 pathway blockade.6 Additionally, neoantigen loss through elimination of specific tumor subclones (i.e., immunoediting), genetic alterations in interferon gamma (IFN) signaling, and upregulation of alternative checkpoints (e.g., TIM3) have also been purported to confer acquired resistance to checkpoint inhibitors.4,7,8 At present, the relative frequencies of these processes and the interplay among them within NSCLC and other malignancies remains to be defined. Moving forward, it will be critical to pursue more in-depth preclinical and clinical studies of resistance to PD-1 pathway blockade in NSCLC. Just as insights into the molecular mechanisms of resistance to targeted therapy have transformed the therapeutic landscape for oncogene-driven tumors, it will be imperative for immuno-oncology to develop a framework for understanding resistance to checkpoint inhibitors and apply this framework to clinical development of next-generation immuno-onclogy agents. This is especially critical due to the sheer number of immuno-oncology combinations currently in clinical testing. Ultimately, knowledge of the mechanisms of resistance to immune checkpoint inhibitors may help better inform the rationale design of trials evaluating PD-1 inhibitor combinations. 1. Garon EB, et al. J Clin Oncol 2019 Jun 2 [Epub ahead of print]. 2. Sharma P, et al. Cell 2017;168(4):707-23. 3. Sade-Feldman M, et al. Nat Commun 2017;8(1):1136. 4. Zaretsky JM, et al. NEJM 2016;375:819-29. 5. Rizvi NA, et al. Science 2015;348(6230):124-128. 6. Gettinger S, et al. Cancer Discov 2017:7:1420-35. 7. Anagnostou V, et al. Cancer Discov 2017;7(3):264-76. 8. Koyama S, et al. Nat Commun 2016;7:10501. Immunotherapy, resistance, PD-1 blockade

Efficacy of chemotherapy following nivolumab in metastatic gastric cancer

Abstract Background Immuno-checkpoint inhibitors, nivolumab targeting programmed cell death 1 (PD-1) can result in significant benefit to gastric cancer. Some recent data about non-small cell lung cancer suggest anti-PD-1 therapy may enhance sensitivity to subsequent chemotherapy. To date, there are no reports on treatment efficacy after failure of PD-1 blockade in advanced gastric cancer. Therefore, we aimed to report the efficacy of chemotherapy after progressive disease on nivolumab. Methods We have retrospectively reviewed data of all patients in metastatic gastric cancer who received chemotherapy in our hospital and investigated the efficacy and adverse events of chemotherapy after progression on nivolumab. Results Between September 2017 and January 2019, 29 patients received nivolumab for metastatic gastric cancer. 11 patients received subsequent treatment following nivolumab. The median age was 70. Performance status (0/1/2); 2/8/1. Previous treatment regimens (2/3/4/5); 4/3/2/1. Chemotherapy regimen (irinotecan / nab-paclitaxel±ramcirumab / CAPOX±trastuzumab / SOX+trastuzumab); 3/5/2/1. Overall Response rate was 27%. 2 patients for nab-PTX+ramcirumab and 1patient of CAPOX+trastuzumab had a PR, 1patient for nab-PTX+ramcirumab had a SD, 5 patients had a PD and 1 patient had a NE. Median PFS was 3.3 (95% CI. 0.8-5.5) months. Median OS was 8.0 (95%CI. 2.3-11.6) months. One patient developed Gr3 dysfunction of liver maybe due to nivolumab. Other adverse events were mild. Conclusions We evaluated the efficacy of chemotherapy after progression on nivolumab. Further investigation is warranted to reveal an immunotherapy-induced chemosensitization effect.

CAR-T 19 combined with reduced-dose PD-1 blockade therapy for treatment of refractory follicular lymphoma: A case report.

Anti-CD19 chimeric antigen receptor T cell (CAR-T) therapy has changed the typical outcomes of relapsed/refractory B-cell leukemia and lymphoma. However, treatment effectiveness for patients with relapsed/refractory B-cell non-Hodgkin lymphoma has been less satisfactory compared with patients with B-cell acute lymphoblastic leukemia. The present study described a case of refractory follicular lymphoma. A high expression of programmed cell death 1 (PD-1) was measured on CD3+ T cells (80.90%) in peripheral blood samples obtained from the patient enrolled in this study, indicating that treatment with autologous CAR-T 19 cell therapy may not be successful. Therefore, a therapy regimen consisting of CAR-T 19 cells in combination with a reduced dose of nivolumab (1.5 mg/kg) for PD-1 blockade was used. A low dose of PD-1 blockade therapy was used to reduce the adverse effects associated with the combination of a PD-1 inhibitor and CAR-T 19 cells. This salvage therapy resulted in remission that lasted for >10 months.

Immune microenvironment modulation unmasks therapeutic benefit of radiotherapy and checkpoint inhibition

BackgroundImmune checkpoint inhibitors (ICIs) for solid tumors, including those targeting programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), have shown impressive clinical efficacy, however, most patients do not achieve durable responses. One major therapeutic obstacle is the immunosuppressive tumor immune microenvironment (TIME). Thus, we hypothesized that a strategy combining tumor-directed radiation with TIME immunomodulation could improve ICI response rates in established solid tumors.MethodsUsing a syngeneic mouse model of human papillomavirus (HPV)-associated head and neck cancer, mEER, we developed a maximally effective regimen combining PD-1 and CTLA-4 inhibition, tumor-directed radiation, and two existing immunomodulatory drugs: cyclophosphamide (CTX) and a small-molecule inducible nitric oxide synthase (iNOS) inhibitor, L-n6-(1-iminoethyl)-lysine (L-NIL). We compared the effects of the various combinations of this regimen on tumor growth, overall survival, establishment of immunologic memory, and immunologic changes with flow cytometry and quantitative multiplex immunofluorescence.ResultsWe found PD-1 and CTLA-4 blockade, and radiotherapy alone or in combination, incapable of clearing established tumors or reversing the unfavorable balance of effector to suppressor cells in the TIME. However, modulation of the TIME with cyclophosphamide (CTX) and L-NIL in combination with dual checkpoint inhibition and radiation led to rejection of over 70% of established mEER tumors and doubled median survival in the B16 melanoma model. Anti-tumor activity was CD8+ T cell-dependent and led to development of immunologic memory against tumor-associated HPV antigens. Immune profiling revealed that CTX/L-NIL induced remodeling of myeloid cell populations in the TIME and tumor-draining lymph node and drove subsequent activation and intratumoral infiltration of CD8+ effector T cells.ConclusionsOverall, this study demonstrates that modulation of the immunosuppressive TIME is required to unlock the benefits of ICIs and radiotherapy to induce immunologic rejection of treatment-refractory established solid tumors.

PD-L1 expression is associated with ALK positivity and STAT3 activation, but not outcome in patients with systemic anaplastic large cell lymphoma.

The programmed cell death 1 (PD-1) pathway is a recently recognized mechanism of tumor immune evasion. In this study, programmed cell death ligand 1 (PD-L1) expression was evaluated in 95 patients with systemic anaplastic large cell lymphoma: 45 ALK+ and 50 ALK-. ALK+ anaplastic large cell lymphoma was more often positive for PD-L1 than ALK- anaplastic large cell lymphoma (76% vs 42%, p = 0.002). ALK- anaplastic large cell lymphoma showed a strong correlation between PD-L1 expression and STAT3 activation (measured by pSTAT3Tyr705) (r = 0.8, p < 0.0001). In contrast, the PD-L1/pSTAT3 correlation was weaker in ALK+ anaplastic large cell lymphoma (r = 0.4, p = 0.08). In ALK- anaplastic large cell lymphoma, the PD-L1+ subgroup was more often EMA positive (69% vs 20%, p = 0.02) and tended to be less often CD2+ (50% vs 83%, p = 0.059). In ALK+ anaplastic large cell lymphoma, PD-L1 was not associated with pathologic features (all p > 0.05). Negative ALK status and high IPI score (≥3) were associated with shorter overall survival (p = 0.009 and p = 0.0005, respectively). Overall survival was not different between patients with PD-L1+ vs PD-L1- anaplastic large cell lymphoma (p = 0.44), regardless of ALK status and International Prognostic Index (IPI) score. We conclude that PD-L1 expression is more common in ALK+ anaplastic large cell lymphoma than ALK- anaplastic large cell lymphoma. In ALK- anaplastic large cell lymphoma, PD-L1 is strongly correlated with STAT3 activation and is associated with more frequent EMA and less frequent CD2 expression. PD-L1 has no prognostic significance in predicting the outcome of patients with systemic anaplastic large cell lymphoma, regardless of ALK status. PD-L1 expression on the anaplastic large cell lymphoma cells suggests these patients as potential candidates for PD-1 blockade immunotherapy.

In situ vaccination with defined factors overcomes T cell exhaustion in distant tumors.

Irreversible T cell exhaustion limits the efficacy of programmed cell death 1 (PD-1) blockade. We observed that dual CD40-TLR4 stimulation within a single tumor restored PD-1 sensitivity and that this regimen triggered a systemic tumor-specific CD8+ T cell response. This approach effectively treated established tumors in diverse syngeneic cancer models, and the systemic effect was dependent on the injected tumor, indicating that treated tumors were converted into necessary components of this therapy. Strikingly, this approach was associated with the absence of exhausted PD-1hi T cells in treated and distant tumors, while sparing the intervening draining lymph node and spleen. Furthermore, patients with transcription changes like those induced by this therapy experienced improved progression-free survival with anti-PD-1 treatment. Dual CD40-TLR4 activation within a single tumor is thus an approach for overcoming resistance to PD-1 blockade that is unique in its ability to cause the loss of exhausted T cells within tumors while sparing nonmalignant tissues.

IL-17C-mediated innate inflammation decreases the response to PD-1 blockade in a model of Kras-driven lung cancer

Chronic obstructive pulmonary disease (COPD) is associated with neutrophilic lung inflammation and CD8 T cell exhaustion and is an important risk factor for the development of non-small cell lung cancer (NSCLC). The clinical response to programmed cell death-1 (PD-1) blockade in NSCLC patients is variable and likely affected by a coexisting COPD. The pro-inflammatory cytokine interleukin-17C (IL-17C) promotes lung inflammation and is present in human lung tumors. Here, we used a Kras-driven lung cancer model to examine the function of IL-17C in inflammation-promoted tumor growth. Genetic ablation of Il-17c resulted in a decreased recruitment of inflammatory cells into the tumor microenvironment, a decreased expression of tumor-promoting cytokines (e.g. interleukin-6 (IL-6)), and a reduced tumor proliferation in the presence of Haemophilus influenzae- (NTHi) induced COPD-like lung inflammation. Chronic COPD-like inflammation was associated with the expression of PD-1 in CD8 lymphocytes and the membrane expression of the programmed death ligand (PD-L1) independent of IL-17C. Tumor growth was decreased in Il-17c deficient mice but not in wildtype mice after anti-PD-1 treatment. Our results suggest that strategies targeting innate immune mechanisms, such as blocking of IL-17C, may improve the response to anti-PD-1 treatment in lung cancer patients.

PD-1 blockade with pembrolizumab for classical Hodgkin lymphoma after autologous stem cell transplantation

AbstractAutologous stem cell transplantation (ASCT) remains the standard of care for patients with relapsed/refractory (RR) classical Hodgkin lymphoma (cHL) who respond to salvage chemotherapy. However, relapse after ASCT remains a frequent cause of treatment failure, with poor subsequent prognosis. Because cHL is uniquely vulnerable to programmed cell death-1 (PD-1) blockade, PD-1 blockade given as consolidation after ASCT could improve ASCT outcomes. We therefore conducted a multicohort phase 2 study of pembrolizumab in patients with RR cHL after ASCT, hypothesizing that it would improve the progression-free survival (PFS) at 18 months after ASCT (primary end point) from 60% to 80%. Pembrolizumab was administered at 200 mg IV every 3 weeks for up to 8 cycles, starting within 21 days of post-ASCT discharge. Thirty patients were treated on this study. The median age was 33 years, and 90% were high-risk by clinical criteria. Seventy-seven percent completed all 8 cycles. Toxicity was manageable, with 30% of patients experiencing at least 1 grade 3 or higher adverse event (AE), and 40% at least 1 grade 2 or higher immune-related AE. Two patients were lost to follow-up in complete remission at 12 months. The PFS at 18 months for the 28 evaluable patients was 82%, meeting the primary end point. The 18-month overall survival was 100%. In conclusion, pembrolizumab was successfully administered as post-ASCT consolidation in patients with RR cHL, and resulted in a promising PFS in a high-risk patient cohort, supporting the testing of this strategy in a randomized trial. This trial was registered at www.clinicaltrials.gov as #NCT02362997.

Abstract CT120: A randomized, open-label, open-platform, Phase II study evaluating the efficacy and safety of novel spartalizumab (PDR001) combinations in previously treated unresectable or metastatic melanoma (PLATforM)

Abstract Background: Significant advancements, including development of immune checkpoint inhibitors and targeted therapies, have transformed outcomes in patients (pts) with unresectable or metastatic melanoma. However, pts who do not respond or who progress while receiving these regimens have limited options. Spartalizumab (PDR001) is a high-affinity, humanized monoclonal antibody blocking the programmed cell death 1 (PD-1) receptor. This study is evaluating combinations of spartalizumab with novel compounds to restore antitumor T-cell activity in pts with melanoma progressing after prior PD-1 blockade therapy. Methods: This randomized, open-label, 2-part, multicenter, open-platform, Phase II study (NCT03484923; PLATforM) will evaluate the safety and efficacy of spartalizumab combination treatment in pts with unresectable or metastatic melanoma progressing after prior anti-PD-1/L1 therapy and, if the tumor harbors a BRAF V600 mutation, a BRAF inhibitor. The primary endpoint is objective response rate per RECIST v1.1, with duration of response and assessment of paired tumor biopsies for biomarkers of antitumor T-cell activity as part of the secondary endpoints. Part 1, “selection”, will begin with 3 combination arms: (1) spartalizumab + LAG525 (LAG-3 antibody), (2) spartalizumab + capmatinib (c-MET inhibitor), and (3) spartalizumab + canakinumab (IL-1β antagonist). The PLATforM study will use an adaptive design during the selection part that allows for dropping arms for futility, adding new arms, and selecting 1 or multiple arms for further expansion. Bayesian methodology will be used with specific probability criteria for futility and efficacy assessments at each interim analysis. Pts (≈ 60-85) will be stratified by baseline lactate dehydrogenase level and randomized equally to all open arms during the selection part. Part 2, “expansion”, will further investigate the efficacy and safety of treatment combination(s) selected during part 1. The sample size for part 2 will be adaptive and based on predictive power calculations considering the results from part 1. Previously presented at ESMO 2018, FPN 1304TiP, Weber et al. Reused with permission. Citation Format: Caroline Robert, Reinhard Dummer, Ana Arance, Antoni Ribas, Jeffrey Weber, Georgina V. Long, John Haanen, Paul Nathan, Paolo A. Ascierto, Eduard Gasal, Anthony D'Amelio, Severine Bettinger, Aislyn D. Boran, Dirk Schadendorf. A randomized, open-label, open-platform, Phase II study evaluating the efficacy and safety of novel spartalizumab (PDR001) combinations in previously treated unresectable or metastatic melanoma (PLATforM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT120.

Abstract 573: Mutations found by targeted next generation sequencing is associated with intra-tumor immune profile and may predict response to anti-PD1 therapy in lung adenocarcinoma

Abstract Purpose: the immune environment of cancer is the target of immunotherapies with programmed cell death 1 (PD-1) therapeutic antibodies. However, factors that can robustly predict long-lasting responses are still needed. Previous studies showed a role of cancer cells on immune response setting, but the impact of somatic mutations on the composition of the immune stroma is not fully characterized. Experimental Design: We characterized the immune profile of 221 lung adenocarcinomas using immunohistochemistry (CD8 T cells, Macrophages, neutrophils, PD-L1) and compare to tumor mutational status using targeted next-generation sequencing. The response to PD-1 blockers was analyzed from a prospective cohort of 31 lung carcinomas according to tumor mutational profiles and to tumor cell PD-L1 expression. Results: We report that distinct combinations of STK11, EGFR and TP53 mutations, allows the identification of immune cells combination and prediction of PD-L1 expression on tumor cells. Lung adenocarcinomas with the highest CD8 T cell density and PD-L1 expression were those with TP53 mutations without co-occurring STK11or EGFR alterations (TP53-mut/STK11-EGFR-WT), and independently of KRAS status. Interestingly in TP53-mut/STK11-EGFR-WT tumors, pathways related to T cell chemotaxis, immune cell cytotoxicity, and antigen processing were up-regulated. In accordance with these results we found a prolonged progression-free survival (PFS: HR=0.32; 95% CI, 0.16-0.63, p&amp;lt;0.001) in anti-PD-1 treated patients harboring TP53-mut/STK11-EGFR-WT tumors. This clinical benefit was even more remarkable in patients with associated strong PD-L1 expression. Conclusions: Our study reveals that mutations in lung adenocarcinoma impact intra-tumor immune profile and predict response to PD-1 blockade. Citation Format: Audrey Lupo, Jerome Biton, Marco Alifano, Diane Damotte, Jennifer Arrondeau, Pascaline Boudou, François Goldwasser, Karen Leroy, Marie Wislez, Pierre Laurent-Puig, Isabelle Cremer, Helène Blons, Ronald Herbst. Mutations found by targeted next generation sequencing is associated with intra-tumor immune profile and may predict response to anti-PD1 therapy in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 573.

Abstract LB-014: Antibody targeting soluble NKG2D ligand sMIC sensitizes metastatic prostate tumor and other MIC+ tumors to PD1/PD-L1 blockade therapy in pre-clinical models

Abstract Targeting programmed cell death-1 (PD-1) has produced a survival benefit for patients diagnosed with many types of malignancies. However, the clinical survival benefit in metastatic prostate cancer is only marginal to-date. Currently, the understanding of why metastatic prostate cancer is resistant to PD-1 blockade therapy remains to be defined. Among the many factors that negatively impact response to PD-1 blockade treatment, tumor-derived soluble NKG2D ligands have been reported to hinder the clinical response to immune checkpoint blockade in melanoma patients. Metastatic prostate cancer cells release high levels of the circulating soluble NKG2D ligand, soluble MHC I Chain related molecule (sMIC), a molecule that is absent in classical rodent tumor model. Here, we tested the hypothesis that targeting sMIC sensitizes metastatic prostate tumors to PD-1 blockade therapy. Using a well-described double transgenic TRAMP/MIC mouse model that closely resembles the onco-immune dynamics of human metastatic prostate cancer, we demonstrate that an antibody targeting sMIC significantly enhances the response of prostate tumors to PD-1/PD-L1 blockade therapy. Mechanistically, targeting sMIC enriches NK cell and CD8 T cell infiltration into tumors, equips CD8 T cells with enhanced NKG2D and CD28 dual co-stimulation, and increases the expression of PD-L1+ on prostate tumor cells. Combination of the antibody targeting sMIC and a PD-1/PD-L1 blockade cooperatively augments intra-tumoral NK and antigen-specific CD8 T cell anti-tumor immunity. These findings were validated in syngeneic prostate tumor transplant models and other models of other cancer types. Our pre-clinical study provides proof-of-concept and uncovered novel mechanisms of a viable combination immunotherapy for metastatic prostate cancer and other MIC+ cancers. Citation Format: Jennifer D. Wu, Jinyu Zhang, Pablo Larrocha, Payal Dhar. Antibody targeting soluble NKG2D ligand sMIC sensitizes metastatic prostate tumor and other MIC+ tumors to PD1/PD-L1 blockade therapy in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-014.

Epigenetic signature of PD-1+ TCF1+ CD8 T cells that act as resource cells during chronic viral infection and respond to PD-1 blockade

We have recently defined a novel population of PD-1 (programmed cell death 1)+ TCF1 (T cell factor 1)+ virus-specific CD8 T cells that function as resource cells during chronic LCMV infection and provide the proliferative burst seen after PD-1 blockade. Such CD8 T cells have been found in other chronic infections and also in cancer in mice and humans. These CD8 T cells exhibit stem-like properties undergoing self-renewal and also differentiating into the terminally exhausted CD8 T cells. Here we compared the epigenetic signature of stem-like CD8 T cells with exhausted CD8 T cells. ATAC-seq analysis showed that stem-like CD8 T cells had a unique signature implicating activity of HMG (TCF) and RHD (NF-κB) transcription factor family members in contrast to higher accessibility to ETS and RUNX motifs in exhausted CD8 T cells. In addition, regulatory regions of the transcription factors Tcf7 and Id3 were more accessible in stem-like cells whereas Prdm1 and Id2 were more accessible in exhausted CD8 T cells. We also compared the epigenetic signatures of the 2 CD8 T cell subsets from chronically infected mice with effector and memory CD8 T cells generated after an acute LCMV infection. Both CD8 T cell subsets generated during chronic infection were strikingly different from CD8 T cell subsets from acute infection. Interestingly, the stem-like CD8 T cell subset from chronic infection, despite sharing key functional properties with memory CD8 T cells, had a very distinct epigenetic program. These results show that the chronic stem-like CD8 T cell program represents a specific adaptation of the T cell response to persistent antigenic stimulation.

CD4+ T-cell immunity predicts long-lasting antitumor immunity after PD-1 blockade therapy.

2545 Background: Patients treated with programmed cell death 1 (PD-1)-blockade therapy fall into 3 distinct subgroups: non-responders presenting early disease progression, long survivors who achieve durable disease control, and the remaining short-term responders. We reported that the prediction formula comprised of the percentages of CD62L-downregulated (CD62Llow) and CD25+FOXP3+CD4+T cells in the peripheral blood predicted non-responders of non-small cell lung cancer patients (n = 50) scheduled to receive anti-PD-1-antibody (nivolumab) therapy in the 2017 ASCO meeting. In this study, we included 171 patients with NSCLC who were scheduled for nivolumab treatment after obtaining written informed consent. Peripheral blood mononuclear cells (PBMC) were examined before and after Nivolumab therapy up to 2 years to investigate the differences between long survivors and short-term responders. Methods: The patients received Nivolumab at a dose of 3 mg per kilogram of body weight every 2 weeks. Tumor response was assessed with the use of the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, at week 8 and every 8 weeks thereafter. PBMCs were analyzed with a 18-color microfluorometer, LSR Fortessa and a masscytometer, CyTOF. Results: The responder-type patient group whose prediction formula values were greater than 192 showed significantly longer PFS ( P&lt; 0.0001) and OS ( P&lt; 0.0001). The long survivors who consisted of tail plateau of PFS exhibited significantly more CD62LlowCD4+T cells than the short-term responders as pre-existing immunity. The remaining responders kept significantly higher percentages of CD62LlowCD4+T cells ( P= 0.0088) and prediction formula values ( P= 0.017) than the patients with acquired resistance. Conclusions: The pre-existing CD4+T cell balance between primed effector and regulatory T cells correlated with anti-PD-1 therapy response. Further, CD62Llowcell-dominant CD4+T cell immunity was required to maintain durable antitumor reactivity induced by anti-PD-1 antibody therapy. These results have important clinical implication, as they support anti-PD-1 therapy provision to all potentially responding patients and pave the way for new treatment strategies for patients with distinct CD4+T cell immune statuses. Clinical trial information: UMIN000020719.

Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy

Despite compelling rates of durable clinical responses to programmed cell death-1 (PD-1) blockade, advances are needed to extend these benefits to resistant tumors. We found that tumor-bearing mice deficient in the chemokine receptor CXCR3 responded poorly to anti-PD-1 treatment. CXCR3 and its ligand CXCL9 were critical for a productive CD8+ Tcell response in tumor-bearing mice treated with anti-PD-1 but were not required for the infiltration of CD8+ Tcells into tumors. The anti-PD-1-induced anti-tumor response was facilitated by CXCL9 production from intratumoral CD103+ dendritic cells, suggesting that CXCR3 facilitates dendritic cell-T cell interactions within the tumor microenvironment. CXCR3 ligands in murine tumors and in plasma of melanoma patients were an indicator of clinical response to anti-PD-1, and their induction in non-responsive murine tumors promoted responsiveness to anti-PD-1. Our data suggest that the CXCR3 chemokine system is a biomarker for sensitivity to PD-1 blockade and that augmenting the intratumoral function of this chemokine system could improve clinical outcomes.

PD-L1 Testing in Non-small Cell Lung Cancer: Past, Present, and Future.

Blockade of the programmed cell death-1 (PD-1) axis has already been established as an effective treatment of non-small cell lung cancer. Immunohistochemistry (IHC) for programmed death-ligand 1 (PD-L1) protein is the only available biomarker that can guide treatment with immune checkpoint inhibitors in non-small cell lung cancer. Because each PD-1/PD-L1 blockade was approved together with a specific PD-L1 IHC assay used in the clinical trials, pathologists have been challenged with performing various assays with a limited sample. To provide a more unified understanding of this, several cross-validation studies between platforms have been performed and showed consistent results. However, the interchangeability of assays may be limited in practice because of the risk of misclassification of patients for the treatment. Furthermore, several issues, including the temporal and spatial heterogeneity of PD-L1 expression in the tumor, and the potential for cytology specimens to be used as an alternative to tissue samples for PD-L1 testing, have still not been resolved. In the future, one of the main aims of immunotherapy research should be to find a novel predictive biomarker for PD-1 blockade therapy and a way to combine it with PD-L1 IHC and other tests.

PD-1 is negatively regulated by LSD1 through interactions with Blimp-1 during acute viral infection

Abstract Prolonged exposure to antigen, such as during chronic viral infections and cancer, leads to sustained TCR signaling and ultimately T cell exhaustion, which is in part mediated by up regulation of Programmed cell death 1 (PD-1). Antibody blockade of PD-1 does not work in all instances and can lead to autoimmunity, thus identifying novel tools to manipulate PD-1 are needed. Therefore, we sought to understand the genetic and epigenetic mechanisms that regulate PD-1 during acute and chronic antigen conditions in order to identify additional therapeutic targets to modulate PD-1. We determined the chromatin status at the PD-1 locus and found that activating H3K4me1 and H3K4me2 epigenetic modifications were still present at day 8 during a chronic but not acute LCMV infection. This suggested a failure to remove the active histone marks in chronic infections. Indeed, CD8 T cells lacking lysine-specific histone demethylase 1A (LSD1), a H3K4 demethylase, display significantly higher levels of PD-1 mRNA and protein during acute but not chronic infection. Molecular analysis revealed that Blimp-1 recruits LSD1 to the PD-1 locus to induce silencing during acute infection. Despite Blimp-1 binding to the PD-1 locus during both acute and chronic LCMV infection, it fails to recruit LSD1 during the chronic setting. Additionally, failure to remove H3K4 methylation resulted in less DNA methylation at known PD-1 control regions compared to wild-type cells, consistent with the differential expression of PD-1. Taken together, these results demonstrate that LSD1 downregulates PD-1 by interacting with Blimp-1 during acute, but not chronic LCMV infection, facilitating full epigenetic repression of the locus.

Sequential Blockade of PD-1 and PD-L1 Causes Fulminant Cardiotoxicity-From Case Report to Mouse Model Validation.

The combined administration of programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) inhibitors might be considered as a treatment for poorly responsive cancer. We report a patient with brain metastatic lung adenocarcinoma in whom fatal myocarditis developed after sequential use of PD-1 and PD-L1 inhibitors. This finding was validated in syngeneic tumor-bearing mice. The mice bearing lung metastases of CT26 colon cancer cells treated with PD-1 and/or PD-L1 inhibitors showed that the combination of anti-PD-1 and anti-PD-L1, either sequentially or simultaneously administered, caused myocarditis lesions with myocyte injury and patchy mononuclear infiltrates in the myocardium. A significant increase of infiltrating neutrophils in myocytes was noted only in mice with sequential blockade, implying a role for the pathogenesis of myocarditis. Among circulating leukocytes, concurrent and subsequent treatment of PD-1 and PD-L1 inhibitors led to sustained suppression of neutrophils. Among tumor-infiltrating leukocytes, combinatorial blockade increased CD8+ T cells and NKG2D+ T cells, and reduced tumor-associated macrophages, neutrophils, and natural killer (NK) cells in the lung metastatic microenvironment. The combinatorial treatments exhibited better control and anti-PD-L1 followed by anti-PD-1 was the most effective. In conclusion, the combinatory use of PD-1 and PD-L1 blockade, either sequentially or concurrently, may cause fulminant cardiotoxicity, although it gives better tumor control, and such usage should be cautionary.

Potential markers of response and resistance to programmed cell death-1 blockade in first-line therapy of cisplatin-inilegible advanced urothelial cancer.

449 Background: Immune checkpoint inhibitors (ICIs) have demonstrated clinical benefit in advanced urothelial cancer (UC) patients. An alert was issued about a decrease in survival for bladder cancer patients with PD-L1-low status receiving immunotherapy versus chemotherapy as first-line therapy. We studied a small series of UC patients treated with first-line PD-1 checkpoint inhibition in order to analyze their characteristics and patterns of response to therapy. Methods: Eleven UC samples were obtained from patients before undergoing therapy with ICIs. Patients were classified according to their benefit from therapy in responders (n=5) and non-responders (n=6). Genomic and immunohistochemistry analyses were performed. Results: Both luminal and basal UC subtypes showed benefit from ICIs. Tumors from non-responders showed increased mutations in chromatin remodelling genes and the amplification of 3q26-28 region. Transcriptome analyses showed that tumors from responders displayed a significant enrichment of genes associated with interferon γ and α response, TNFα via NFκB, genes upregulated by MYC or E2F1, genes involved in G2/M checkpoint and epithelial-mesenchymal transition compared to non-responders. Specific immune cell subsets were present in the tumor microenvironment of tumors from responders and non-responders. Immunohistochemistry showed that none of the immune cell markers analyzed individually was sufficient to discriminate between responders and non-responders. However, the increase in FOXP-3, PD-L1, PD-1, CD8, β2 microglobulin and CD68 and the decrease in CD4 and CD163 cells identified UC patients that responded to ICIs. Conclusions: Our findings confirm that the evaluation of pre-treatment UC tumor samples provides valuable information that could influence treatment decisions. Despite the clinical benefit of PD-1/PD-L1 inhibition in UC, only a fraction of patients benefit from therapy. Our data suggest that responders and non-responders display diverse genomic and transcriptome changes as well as specific immune cell subsets in the tumor microenvironment that can be identified by conventional IHC staining.