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Many patients with advanced melanoma are resistant to immune checkpoint inhibition. In the ILLUMINATE-204 phase I/II trial, we assessed intratumoral tilsotolimod, an investigational Toll-like receptor 9 agonist, with systemic ipilimumab in patients with anti–PD-1– resistant advanced melanoma. In all patients, 48.4% experienced grade 3/4 treatment-emergent adverse events. The overall response rate at the recommended phase II dose of 8 mg was 22.4%, and an additional 49% of patients had stable disease. Responses in noninjected lesions and in patients expected to be resistant to ipilimumab monotherapy were observed. Rapid induction of a local IFNα gene signature, dendritic cell maturation and enhanced markers of antigen presentation, and T-cell clonal expansion correlated with clinical response. A phase III clinical trial with this combination (NCT03445533) is ongoing.Significance:Despite recent developments in advanced melanoma therapies, most patients do not experience durable responses. Intratumoral tilsotolimod injection elicits a rapid, local type 1 IFN response and, in combination with ipilimumab, activates T cells to promote clinical activity, including in distant lesions and patients not expected to respond to ipilimumab alone.This article is highlighted in the In This Issue feature, p. 1861

This single-arm, phase I dose-escalation trial (NCT02983045) evaluated bempegaldesleukin (NKTR-214/BEMPEG), a CD122-preferential IL2 pathway agonist, plus nivolumab in 38 patients with selected immunotherapy-naïve advanced solid tumors (melanoma, renal cell carcinoma, and non–small cell lung cancer). Three dose-limiting toxicities were reported in 2 of 17 patients during dose escalation [hypotension (n = 1), hyperglycemia (n = 1), metabolic acidosis (n = 1)]. The most common treatment-related adverse events (TRAE) were flu-like symptoms (86.8%), rash (78.9%), fatigue (73.7%), and pruritus (52.6%). Eight patients (21.1%) experienced grade 3/4 TRAEs; there were no treatment-related deaths. Total objective response rate across tumor types and dose cohorts was 59.5% (22/37), with 7 complete responses (18.9%). Cellular and gene expression analysis of longitudinal tumor biopsies revealed increased infiltration, activation, and cytotoxicity of CD8+ T cells, without regulatory T-cell enhancement. At the recommended phase II dose, BEMPEG 0.006 mg/kg plus nivolumab 360 mg every 3 weeks, the combination was well tolerated and demonstrated encouraging clinical activity irrespective of baseline PD-L1 status.Significance:These data show that BEMPEG can be successfully combined with a checkpoint inhibitor as dual immunotherapy for a range of advanced solid tumors. Efficacy was observed regardless of baseline PD-L1 status and baseline levels of tumor-infiltrating lymphocytes, suggesting therapeutic potential for patients with poor prognostic risk factors for response to PD-1/PD-L1 blockade.See related commentary by Rouanne et al., p. 1097.This article is highlighted in the In This Issue feature, p. 1079

NKTR-214 (bempegaldesleukin) is a novel IL2 pathway agonist, designed to provide sustained signaling through heterodimeric IL2 receptor βγ to drive increased proliferation and activation of CD8+ T and natural killer cells without unwanted expansion of T regulatory cells (Treg) in the tumor microenvironment. In this first-in-human multicenter phase I study, NKTR-214 administered as an outpatient regimen was well tolerated and showed clinical activity including tumor shrinkage and durable disease stabilization in heavily pretreated patients. Immune activation and increased numbers of immune cells were observed in the periphery across all doses and cycles with no loss of NKTR-214 activity with repeated administration. On-treatment tumor biopsies demonstrated that NKTR-214 promoted immune cell increase with limited increase of Tregs. Transcriptional analysis of tumor biopsies showed that NKTR-214 engaged the IL2 receptor pathway and significantly increased genes associated with an effector phenotype. Based on safety and pharmacodynamic markers, the recommended phase II dose was determined to be 0.006 mg/kg every three weeks.Significance:We believe that IL2- and IL2 pathway–targeted agents such as NKTR-214 are key components to an optimal immunotherapy treatment algorithm. Based on its biological activity and tolerability, NKTR-214 is being studied with approved immuno-oncology agents including checkpoint inhibitors.See related commentary by Sullivan, p. 694.This article is highlighted in the In This Issue feature, p. 681

Consolidated supplementary appendix: tables, figures, text plus legends

Supplementary Data from Tilsotolimod with Ipilimumab Drives Tumor Responses in Anti–PD-1 Refractory Melanoma

Supplementary Figures, Tables, and Methods

Supplementary Table from Tilsotolimod with Ipilimumab Drives Tumor Responses in Anti–PD-1 Refractory Melanoma

Many patients with advanced melanoma are resistant to immune checkpoint inhibition. In the ILLUMINATE-204 phase I/II trial, we assessed intratumoral tilsotolimod, an investigational Toll-like receptor 9 agonist, with systemic ipilimumab in patients with anti–PD-1– resistant advanced melanoma. In all patients, 48.4% experienced grade 3/4 treatment-emergent adverse events. The overall response rate at the recommended phase II dose of 8 mg was 22.4%, and an additional 49% of patients had stable disease. Responses in noninjected lesions and in patients expected to be resistant to ipilimumab monotherapy were observed. Rapid induction of a local IFNα gene signature, dendritic cell maturation and enhanced markers of antigen presentation, and T-cell clonal expansion correlated with clinical response. A phase III clinical trial with this combination (NCT03445533) is ongoing. Significance: Despite recent developments in advanced melanoma therapies, most patients do not experience durable responses. Intratumoral tilsotolimod injection elicits a rapid, local type 1 IFN response and, in combination with ipilimumab, activates T cells to promote clinical activity, including in distant lesions and patients not expected to respond to ipilimumab alone. This article is highlighted in the In This Issue feature, p. 1861

Agonistic CD40 antibodies have shown promise when used in combination with checkpoint inhibitors in clinical trials for the treatment of malignancies. However, the mechanisms driving antitumor immune responses in patients are not well understood. The aim of this study was to use a preclinical melanoma model to evaluate the impact of intratumoral anti-CD40 administration on treatment efficacy and the tumor immune landscape in the context of systemic anti-PD1 therapy. Mice bearing 8-day established B16 melanoma tumors were injected with CD40 agonist intratumorally, either alone or in combination with anti-PD1 Ab, every 3 days for a total 4 doses. All mice treated with the combination therapy exhibited tumor growth arrest while progressive tumor growth was observed in control mice and mice treated with anti-PD1 alone. At day 15, tumor weights were 7- and 3-fold reduced in mice treated with the combination therapy as compared to control IgG or PD1 monotherapy, respectively. CyTOF analysis showed a 4-fold increase in the frequency of tumor-infiltrating immune cells in mice treated with either CD40 agonist alone or the combination therapy. Interestingly, CD40 agonistic Ab selectively expanded CD8+ T cells and the combination therapy exhibited a more pronounced effect compared to treatment with anti-PD1 Ab alone. Moreover, CD39+ CD8 T cells, representing tumor antigen-specific cytotoxic T cells, were 14- and 3-fold higher in tumors from mice treated with the combination therapy as compared to control or PD1-treated mice, respectively. This effect correlated with increases in the frequency of antigen-presenting cells, including cDC1 (6-fold) and B cells expressing CD40 (3-fold) in response to the combination therapy. In addition, the combination therapy significantly decreased myeloid cell population resulting in an 8-fold reduction in the ratio of myeloid cells to T cells. Amongst myeloid cells, the density of the monocytic population was diminished, with a selective 10-fold reduction of CD206+ M2-macrophages in tumors from mice treated with both Abs. Our findings provide evidence that combining systemic anti-PD1 therapy with intratumoral CD40 agonist enhanced antitumor immune responses by selectively expanding tumor antigen-specific effector CD8+ T cells, which was associated with increased infiltration of antigen-presenting cells and attenuation of immunosuppressive myeloid cells. Citation Format: Barbara Pazdrak, Heather M. Sonnemann, Salah-Eddine Bentebibel, Barbara M. Nassif, Greg Lizee, Adi Diab. Intratumoral CD40 agonist enhances the antitumor effect of anti-PD1 immunotherapy by activation of antigen-presenting cells and selective expansion of effector CD8+ T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5079.

BackgroundTertiary lymphoid structures (TLSs) have been previously associated with ICI induced response in patients with cancer, but a commensurate observation has not been made in ICI associated immune related adverse events (irAEs). Acute interstitial nephritis (AIN) is the predominant lesion reported in patients with renal irAEs, but various etiologies can also trigger the development of AIN including non-ICI drugs (e.g. non-steroidal anti-inflammatory drugs, antibiotics, proton pump inhibitors, etc.), and it is unknown whether these mechanisms are similar. With increasing indications for ICIs in cancer therapy, there is a critical need to define immune pathways driving the emergence of irAEs. To address this critical knowledge gap, we performed gene expression profiling on ICI-AIN, drug-AIN, and control (non-AIN) kidney biopsy specimens.MethodsTotal RNA extracted from ICI-AIN (n = 6), drug-AIN (n = 4), and control (n = 4) fixed formalin paraffin embedded archival kidney biopsy samples was analyzed by Nanostring nCounter PanCancer Immune Profiling Panel using NanoString nCounter FLEX Analysis System.ResultsThree comparisons were conducted: ICI-AIN vs control, drug-AIN vs control, and ICI-AIN vs drug-AIN. A total of 147 genes were differentially expressed in ICI-AIN vs control and the most differentially expressed genes were CXCL 9, 10, and 11. Similarly, cell marker gene expression signatures (GES) revealed significant upregulation of T and B cell markers in ICI-AIN vs control (P < 0.01) and ICI-AIN vs drug-AIN (T cell P < 0.05; B cell P < 0.01). Differences in T and B cell score were not detected in drug-AIN vs control. Since irAEs have been associated with anti-tumor efficacy, we investigated whether a TLS signature could be detected in ICI-AIN using a four GES (CD79A, MS4A1, LAMP3 and POU2AF1). The ICI-AIN group had significantly higher TLS score compared to both control and drug-AIN groups. Since several TLS signatures have been reported, we also calculated a 12-chemokine TLS GES which was also found to be statistically significant (P < 0.05). Th1 and Th17 cells have been associated with the formation of TLS, differential upregulation of Th1 associated genes but not Th17 associated genes were detected. Furthermore, differential expression IFN-y and TNF signature was also observed in ICI-AIN group.ConclusionsThis study is the first to demonstrate the presence of TLS immune signature in irAEs. Further investigations into the prognostic significance and strategies to uncouple ICI-associated anti-tumor benefits from ICI-induced irAEs should be explored.Ethics ApprovalThe study was approved by The University of Texas MD Anderson Cancer Center intuition's Ethics Board, approval number PA16-1016

Memory T follicular helper (TFH) cells play an essential role to induce secondary antibody response by providing help to memory and naïve B cells. Here, we show that the transcription factor Tox2 is vital for the maintenance of TFH cells in germinal centers (GCs) and the generation of memory TFH cells. High Tox2 expression was almost exclusive to GC TFH cells among human tonsillar and blood CD4+ T cell subsets. Tox2 overexpression maintained the expression of TFH-associated genes in T cell receptor–stimulated human GC TFH cells and inhibited their spontaneous conversion into TH1-like cells. Tox2-deficient mice displayed impaired secondary TFH cell expansion upon reimmunization with an antigen and upon secondary infection with a heterologous influenza virus. Collectively, our study shows that Tox2 is highly integrated into establishment of durable GC TFH cell responses and development of memory TFH cells in mice and humans., 効率よい抗体反応の形成に必要なTリンパ球因子の発見. 京都大学プレスリリース. 2021-10-15., Molecules for building stronger antibodies. 京都大学プレスリリース. 2021-10-15.

Immune checkpoint blockade (ICB) therapy frequently induces immune-related adverse events. To elucidate the underlying immunobiology, we performed a deep immune analysis of intestinal, colitis, and tumor tissue from ICB-treated patients with parallel studies in preclinical models. Expression of interleukin-6 (IL-6), neutrophil, and chemotactic markers was higher in colitis than in normal intestinal tissue; T helper 17 (Th17) cells were more prevalent in immune-related enterocolitis (irEC) than T helper 1 (Th1). Anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) induced stronger Th17 memory in colitis than anti-program death 1 (anti-PD-1). In murine models, IL-6 blockade associated with improved tumor control and a higher density of CD4

Durable remission rates with immune checkpoint inhibitor (ICI) monotherapy remain low. Combination ICI therapies could overcome immune resistance but are associated with higher rates of immune-related adverse events (irAEs). To elucidate the underlying irAE immunobiology, we profiled human intestinal tissue, enterocolitis (irEC), and tumor tissue from ICI-treated patients using NanoString PanCancer Immune Panel (NanoPCIP) and multiplex immunohistochemistry (mIHC); hypotheses generated from these translational results for potential irAE treatments were tested in parallel preclinical mouse model studies. In 23 patients identified with both normal intestinal and irEC tissue, we found genes encoding interleukin-6 (IL-6), Th17 differentiating cytokine, and neutrophil and monocyte chemotactic molecules were the highest significantly upregulated in irEC compared to patient-matched normal intestine. Using NanoPCIP cell scoring (23 patients) and mIHC (27 patients), Th17 cells were significantly higher in irEC than Th1 cells. T-cell subsets were similar between anti-CTLA-4 based regimen versus anti-PD-1monotherapy induced irEC, except Th17 memory cells were significantly higher CTLA-4i-induced irEC. We compared the degree of differential gene expression in irEC (normal intestine versus irEC) to the differential expression in tumors (baseline versus on-treatment tumors). The genes highest upregulated in irEC (including Th17 differentiating cytokines IL-6 and IL-1B) were not upregulated in responding tumors from patients receiving ICI. NanoPCIP Th17 cell score was significantly more upregulated than Th1 cells and neutrophils more upregulated than CD8 T cells in the irEC analysis, but not in the responding tumor analysis where IL-12 and CD8 cell score was significantly upregualted . Based on these discoveries, we used C57BL/6 and Balb/c mouse strains to address whether the antitumor effect of ICIs can be enhanced by IL-6 blockade while also improving irAEs. We found that IL-6 blockade + ICI was associated with improved tumor control and a higher density of CD4/CD8 effector T-cells, with reduced Th17, macrophages, and MDSCs compared to ICI alone. Furthermore, in the experimental autoimmune encephalomyelitis (EAE) mouse model, the pathogenesis of which is well documented to be Th17 mediated (Robinson et al., 2014), we demonstrated that combined IL-6 blockade and ICI enhanced tumor rejection while simultaneously mitigating EAE symptoms versus ICI alone. Based on the totality of the basic science, translational, and clinical data we hypothesize that IL-6 blockade combined with ICIs could de-couple autoimmunity from antitumor immunity. We are currently conducting a prospective phase 2 trials of combination ipilimumab (3mg/kg) + Nivolumab (1mg/kg) + Tocilizumab in patients with metastatic melanoma, EGFR mutant NSCLC, and urothelial carcinoma ( NCT04940299). Citation Format: Daniel H. Johnson, Yared Hailemichael, Salah-Eddine Bentebibel, Noha Abdel-Wahab, Sungnam Cho, Wai C. Foo, Khalida Wani, Stephanie S. Watowich, Suhendan Ekmekcioglu, Adi Diab. Th17 inhibition with interleukin 6 blockade decouples immunotoxicity from tumor immunity [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 4177.

The use of immune-checkpoint inhibitors (CPI) has become an important modality in the treatment of metastatic melanoma (MM). However, most patients (pts) do not experience durable responses and new treatment options are needed to improve clinical outcomes. Our pre-clinical studies have demonstrated that intratumoral CD40 activation synergizes with anti-PD-1 based therapy and induces systemic and distant anti-tumor effects. In this ongoing phase I/II study, we assessed intratumoral sotigalimab (APX005M), a CD40 agonist antibody, in combination with systemic pembrolizumab in CPI treatment naïve, unresectable stage III or IV MM. A total of 40 participants will be enrolled. As of December 15, 2021, 30 pts were enrolled. Pts received sotigalimab every 3 weeks for a total of 4 doses. The dose escalation portion of the trial has been completed, with 14 pts enrolled in 5 dosing cohorts of sotigalimab at 0.1, 0.5, 1, 3 and 10 mg. The primary objectives include safety and tolerability, determination of the recommended phase 2 dose (RP2D), and assessment of the overall response rate (ORR) by RECIST v1.1. Biomarker analyses of blood and tumor samples were performed to measure immune activation using immunophenotyping including imaging mass cytometry, TCR sequencing, and a cross-cohort comparison of gene expression data (sotigalimab plus pembrolizumab versus anti-PD1 monotherapy). The combination therapy has been well-tolerated, and there were no study discontinuations or death due to treatment-related adverse events (TRAEs). Most common TRAEs were injection-site reactions; six pts experienced grade-3 immune-related adverse events. Efficacy analysis of 30 pts with post-baseline disease evaluations demonstrated an ORR of 50% (5 CR and 10 PR) in distant lesions and a disease control rate of 67%. The ORR at the RP2D of 10 mg is 55% (12/22). Responses were observed in PD-L1 negative pts and those with elevated LDH. Comprehensive transcriptome and immune cell profiling of peripheral blood mononuclear cells and tumor biopsies obtained from local lesions at baseline and 24 hours post sotigalimab injection demonstrate that sotigalimab effectively engaged CD40 pathway. In comparison to anti-PD1 monotherapy, the combination therapy significantly increased expression of genes associated with antigen presentation and effector T cells in local lesions accompanied by an increase in T cell activation genes at distant lesions. Additionally, T cell repertoire analysis demonstrated a significant increase in T cell clonality with expansion of new clones shared between local and distant tumors. Importantly, these immunologic changes were correlated with clinical response. Collectively, this combination therapy is well tolerated and has a notable clinical response rate, accompanied by broad innate and adaptive immune activation at both local and distant lesions. Citation Format: Salah-Eddine Bentebibel, Daniel Johnson, Rodabe Amariae, Daniel McGrail, Srisuda Lecagoonporn, Cara Haymaker, Dzifa Duose, Khalida Wani, Houssein Safa, Isabella Claudia Glitza, Sapna Pradyuman Patel, Michael K. Wong, Hussein Tawbi, Jared Burks, Xiaodong Yang, Patrick Hwu, Cassian Yee, Michael A. Davies, Ravi Murthy, Chantale Bernatchez, Suhendan Ekmekcioglu, Adi Diab, Gregory Lizée. Intratumoral CD40 agonist sotigalimab with pembrolizumab induces broad innate and adaptive immune activation in local and distant tumors in metastatic melanoma [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 CT039.

TPS9600 Background: Immune checkpoint inhibitors (ICIs) are approved for multiple malignancies, however, durable remission rates with ICI monotherapy remains low. Combined treatment with anti-CTLA-4 and anti-PD1 has shown higher response rates in several cancers but is associated with up to 60% grade 3/4 immune-related adverse events (irAEs) leading to frequent treatment discontinuation. The need for corticosteroids to control irAEs may further diminish anti-tumor activity. A multi-disciplinary approach using clinical, preclinical, and translational analyses implicated the IL-6/Th17 axis in both ICI-related autoimmunity and resistance. Further, preliminary data showed that targeting interleukin 6 (IL-6) could be an effective approach to reduce irAEs while maintaining and possibly boosting the antitumor immune response. Methods: We are conducting a phase II, open-label, single center study to evaluate the use of combination treatment with tocilizumab (toci; anti-IL6), ipilimumab (ipi; anti-CTLA4) and nivolumab (nivo; anti-PD1) as a front-line therapy for patients (pts) with treatment-naïve advanced cutaneous melanoma (cohort 1), urothelial carcinoma (cohort 2), and EGFR mutant non-small cell lung cancer after tyrosine kinase inhibitors failure (cohort 3) (NCT04940299). Ten pts per disease site will be enrolled, plus an additional 25 melanoma pts in an expansion cohort. Key inclusion criteria are age ≥18 years (yrs) and histologically confirmed locally advanced or metastatic disease, with specific eligibility criteria defined for each cohort. Patients with interstitial lung diseases, autoimmune diseases, infection, or conditions requiring immunosuppressive therapies are not eligible, but stable asymptomatic brain mets are allowed. Ipi/Nivo dosing is as per approved disease indications: in cohort 1 &2, ipi 3 mg/kg + nivo 1 mg/kg is administered intravenously (IV) every 3 weeks (wks) for 4 doses then nivo 480 mg/4 wks up to 2 yrs. In cohort 3, IV ipi 1 mg/kg/6 wks + nivo 3 mg/kg/2 wks is administered up to 2 yrs. In all 3 cohorts, subcutaneous (SQ) toci 162 mg/2wks is administered up to 12 wks. Imaging is every 12 wks up to 2 yrs or until dose-limiting toxicities or progression. The primary outcome is safety/tolerability of the triple therapy. The secondary outcomes are antitumor efficacy and overall survival. Additionally, tumor and blood samples are being collected for longitudinal immune analysis, including gene expression and multiplex histochemistry to identify predictive biomarkers of response, resistance, and toxicity. The trial opened in October 2021 and has enrolled 14 patients to date. Clinical trial information: NCT04940299.

TPS12147 Background: Adjuvant therapy with immune checkpoint inhibitors (ICIs) is approved for melanoma, but immune-related adverse events (irAEs) remain a challenge. Although acute toxicities are well defined, long-term AEs and impact on quality of life (QOL) are undetermined. Available data derived from clinical trials involve highly selected populations and do not reflect real world experience. Additionally, trials measure outcomes only at predetermined endpoints, and symptoms may vary throughout the course of therapy. Moreover, the pathogenesis of irAEs and symptoms remains poorly understood. We hypothesize that AEs and sustained inflammation induced by adjuvant ICIs increase symptom burden and negatively impact function and QOL in a subset of patients (pts), and elevated expression of pro-inflammatory cytokines and T cell signatures during therapy correlate with toxicity and symptom burden. Our preliminary data identified i) interleukin-6/Th-17 pathway as a possible mediator of irAEs, ii) immune reactivity and increases in inflammatory cytokines are associated with symptom burden in cancer survivors, and iii) prioritized 30 genetic markers conferring risk for irAEs in ICI-treated melanoma pts. Methods: This is a prospective longitudinal cohort study to evaluate potential toxicity/symptom burden and immune correlates in melanoma pts receiving adjuvant ICIs (NCT04990726). A total of 240 pts will be enrolled. Eligibility criteria: age ≥18 years (yrs), surgically resected stage II, III, or IV melanoma, initiating adjuvant nivolumab or pembrolizumab, no prior systemic therapy for melanoma, and no prior autoimmune diseases. Patients will be assessed at baseline (before ICI infusion) and every 3 months (mos) up to 2 yrs or until attrition or death. The primary endpoint is the incidence rate of any irAEs at 12 mos. Demographics, personal/family history, comorbidities, tumor history/stage, prior therapies, performance status, concurrent medications, and other factors that play a role in pts perceptions of disease are collected. At each visit, pts undergo a clinical evaluation to assess potential irAEs, new comorbidities, and tumor recurrence. Patient-reported outcomes of fatigue, depression, sleep disturbance, and QOL are collected at each visit to assess changes from baseline up to 2 yrs. In addition to standard methods of data collection at pre-specified times, we leverage mobile technology to capture symptoms and AEs in real time. Longitudinal blood samples will characterize pts immune signatures from baseline up to 2 yrs to evaluate their association with irAEs, symptom burden, and QOL, and to compare the genotype of pts with and without irAEs. To characterize the effect of adjuvant ICI on bone health, eligible pts are evaluated by whole body dual-energy X-ray absorptiometry at baseline and at 12 mos as an exploratory aim. The study is currently active, and 27 pts are enrolled. Clinical trial information: NCT04990726.

Immune checkpoint blockade (ICB) for cancer is associated with high response rates but also high rates of adverse events. To elucidate the underlying immunobiology, we profiled gene expression in intestinal, colitis, and tumor tissue from ICB-treated patients, with parallel studies in preclinical models, and validated our findings in a review of clinical cohort treated with interleukin-6 blockade. Expression of interleukin-6, neutrophil and chemotactic markers was higher in colitis than in normal intestinal tissue. The genes upregulated in colitis were not upregulated in responding tumors from patients receiving ICB. In murine models, interleukin-6 blockade was associated with improved tumor control and a higher density of CD4/CD8 effector T-cells, with reduced Th17, macrophages, and myeloid cells. In an experimental autoimmune encephalomyelitis (EAE) model with tumors, combined interleukin-6 blockade and ICB enhanced tumor rejection while simultaneously mitigating EAE symptoms versus ICB alone. Interleukin-6 blockade with ICB could de-couple autoimmunity from antitumor immunity.

This single-arm, phase I dose-escalation trial (NCT02983045) evaluated bempegaldesleukin (NKTR-214/BEMPEG), a CD122-preferential IL2 pathway agonist, plus nivolumab in 38 patients with selected immunotherapy-naïve advanced solid tumors (melanoma, renal cell carcinoma, and non–small cell lung cancer). Three dose-limiting toxicities were reported in 2 of 17 patients during dose escalation [hypotension (n = 1), hyperglycemia (n = 1), metabolic acidosis (n = 1)]. The most common treatment-related adverse events (TRAE) were flu-like symptoms (86.8%), rash (78.9%), fatigue (73.7%), and pruritus (52.6%). Eight patients (21.1%) experienced grade 3/4 TRAEs; there were no treatment-related deaths. Total objective response rate across tumor types and dose cohorts was 59.5% (22/37), with 7 complete responses (18.9%). Cellular and gene expression analysis of longitudinal tumor biopsies revealed increased infiltration, activation, and cytotoxicity of CD8+ T cells, without regulatory T-cell enhancement. At the recommended phase II dose, BEMPEG 0.006 mg/kg plus nivolumab 360 mg every 3 weeks, the combination was well tolerated and demonstrated encouraging clinical activity irrespective of baseline PD-L1 status. Significance: These data show that BEMPEG can be successfully combined with a checkpoint inhibitor as dual immunotherapy for a range of advanced solid tumors. Efficacy was observed regardless of baseline PD-L1 status and baseline levels of tumor-infiltrating lymphocytes, suggesting therapeutic potential for patients with poor prognostic risk factors for response to PD-1/PD-L1 blockade. See related commentary by Rouanne et al., p. 1097. This article is highlighted in the In This Issue feature, p. 1079

e15242 Background: The Cancer Immune Monitoring and Analysis Centers – Cancer Immunology Data Commons (CIMAC-CIDC) network is a National Cancer Institute-funded initiative to identify biomarkers of mechanisms and response to cancer immunotherapy clinical trials, using state-of-the-art assay technologies. A primary platform for CIMAC-CIDC biomarker studies is CyTOF mass cytometry, which is performed at all four CIMAC laboratories. Methods: To test the ability to generate comparable data across labs, a cross-site harmonization effort was undertaken. We first harmonized SOPs between centers. Because of a new acquisition protocol introduced by the vendor (Fluidigm), we also tested this protocol across sites before finalizing the harmonized SOP. We then performed a cross-site assay harmonization experiment, using 5 shared cryopreserved PBMC samples and one lyophilized control cell preparation, along with a shared lyophilized antibody cocktail consisting of 14 markers, as validated in the HIPC consortium, plus CD45. These reagents and samples were distributed to the four sites, and FCS files were centrally analyzed by both manual gating and automated methods (Astrolabe). Results: Average CVs across sites for each cell population were reported and compared to a previous multisite CyTOF study. Once a cell recovery issue at two sites was resolved, this experiment resulted in inter-site reproducibility of under 20% CV for most cell subsets, very similar to the previous study. Conclusions: These results emphasize the ability to reproduce CyTOF across sites, and also highlights procedures, such as use of spike-in control samples, useful for tracking variability in this assay.

NKTR-214 (bempegaldesleukin) is a novel IL2 pathway agonist, designed to provide sustained signaling through heterodimeric IL2 receptor βγ to drive increased proliferation and activation of CD8+ T and natural killer cells without unwanted expansion of T regulatory cells (Treg) in the tumor microenvironment. In this first-in-human multicenter phase I study, NKTR-214 administered as an outpatient regimen was well tolerated and showed clinical activity including tumor shrinkage and durable disease stabilization in heavily pretreated patients. Immune activation and increased numbers of immune cells were observed in the periphery across all doses and cycles with no loss of NKTR-214 activity with repeated administration. On-treatment tumor biopsies demonstrated that NKTR-214 promoted immune cell increase with limited increase of Tregs. Transcriptional analysis of tumor biopsies showed that NKTR-214 engaged the IL2 receptor pathway and significantly increased genes associated with an effector phenotype. Based on safety and pharmacodynamic markers, the recommended phase II dose was determined to be 0.006 mg/kg every three weeks. Significance: We believe that IL2- and IL2 pathway–targeted agents such as NKTR-214 are key components to an optimal immunotherapy treatment algorithm. Based on its biological activity and tolerability, NKTR-214 is being studied with approved immuno-oncology agents including checkpoint inhibitors. See related commentary by Sullivan, p. 694. This article is highlighted in the In This Issue feature, p. 681

Our understanding of the origin and functions of human blood CXCR5(+) CD4(+) T cells found in human blood has changed dramatically in the past years. These cells are currently considered to represent a circulating memory compartment of T follicular helper (Tfh) lineage cells. Recent studies have shown that blood memory Tfh cells are composed of phenotypically and functionally distinct subsets. Here, we review the current understanding of human blood memory Tfh cells and the subsets within this compartment. We present a strategy to define these subsets based on cell surface profiles. Finally, we discuss how increased understanding of the biology of blood memory Tfh cells may contribute insight into the pathogenesis of autoimmune diseases and the mode of action of vaccines.

Understanding the developmental mechanisms of follicular helper T cells (TFH cells) in humans is relevant to the clinic. However, the factors that drive the differentiation of human CD4+ helper T cells into TFH cells remain largely undefined. Here we found that transforming growth factor-β (TGF-β) provided critical additional signals for the transcription factors STAT3 and STAT4 to promote initial TFH differentiation in humans. This mechanism did not appear to be shared by mouse helper T cells. Developing human TFH cells that expressed the transcriptional repressor Bcl-6 also expressed RORγt, a transcription factor typically expressed by the TH17 subset of helper T cells. Our study documents a mechanism by which TFH cells and TH17 cells emerge together in inflammatory environments in humans, as is often observed in many human autoimmune diseases.

2616 Background: A deep understanding of the immunobiology of checkpoint inhibitor (CPI) induced immune related toxicities, such as immune related enterocolitis (irEC), and how these compare to the immune signatures in tumors could lead to the development of strategies that de-couple autoimmunity from anti-tumor immunity. Methods: Total RNA from patient-matched irEC and normal colon FFPE tissue from patients [n = 12] receiving CPIs were profiled with the 770 gene NanoString nCounter PanCancer Immune Profiling Panel (NanoPCIP). The mean fold change in gene expression from normal vs. irEC inflamed colonic tissue and baseline vs. on-treatment tumor samples from patients responding or non-responding to ipilimumab based therapy were analyzed. C57BL/6 mice with B16.BL6 melanoma tumors were treated with systemic anti-IL-6 + anti-CTLA-4 vs. anti-CTLA4 alone vs. placebo and tumor size was measured. Results: In patients with irEC, the highest significantly upregulated differentially expressed gene (DEG) in inflamed colon tissue encoded for IL-6 (Fold change +24.1). None of the significant and highest upregulated DEGs in the colitis, including IL-6, were significantly upregulated in responding tumors. Interestingly, IL-6 was also the highest upregulated DEG in non-responding tumors numerically. When comparing mean fold changes across these analyses, the gene with the largest difference in upregulatation between colitis and responding tumors was IL-6; the other highest upregulated genes in colitis encoded for neutrophil and monocyte chemotactic molecules. In our mouse models, the addition of IL-6 blockade to anti-CTLA-4 therapy significantly improved tumor shrinkage compared to anti-CTLA-4 alone. Conclusions: Our data demonstrates that IL-6-mediated inflammation may be more prevalent in irEC and tumors not responding to CPIs than in tumors responding, and blocking IL-6 enhances CPI anti-melanoma activity. Targeting IL-6 may ameliorate irEC without hindering anti-tumor immunity.

Antibody responses represent a key immune protection mechanism. T follicular helper (Tfh) cells are the major CD4(+) T-cell subset that provides help to B cells to generate an antibody response. Tfh cells together with B cells form germinal centers (GCs), the site where high-affinity B cells are selected and differentiate into either memory B cells or long-lived plasma cells. We show here that interleukin-12 receptor β1 (IL-12Rβ1)-mediated signaling is important for in vivo Tfh response in humans. Although not prone to B cell-deficient-associated infections, subjects lacking functional IL-12Rβ1, a receptor for IL-12 and IL-23, displayed substantially less circulating memory Tfh and memory B cells than control subjects. GC formation in lymph nodes was also impaired in IL-12Rβ1-deficient subjects. Consistently, the avidity of tetanus toxoid-specific serum antibodies was substantially lower in these subjects than in age-matched controls. Tfh cells in tonsils from control individuals displayed the active form of signal transducer and activator of transcription 4 (STAT4), demonstrating that IL-12 is also acting on Tfh cells in GCs. Thus, our study shows that the IL-12-STAT4 axis is associated with the development and the functions of Tfh cells in vivo in humans.

The immune mechanism leading to the generation of protective antibody responses following influenza trivalent inactivated vaccine (TIV) vaccinations remains largely uncharacterized. We recently reported that TIV vaccination induced a transient increase of circulating ICOS+PD-1+CXCR3+ T follicular helper (cTfh) cells in blood, which positively correlated with the induction of protective antibody responses measured at day 28. However, whether and how these T cells directly contribute to antibody response remains unclear. In this study, we analyzed the changes after TIV vaccination in the amount and the avidity of the polyclonal antibodies specific for the HA1 subunit of the pandemic H1N1 virus, and analyzed the correlation with the increase of ICOS+PD-1+CXCR3+ cTfh cells. We found that both the amount and the avidity of specific antibodies rapidly increased during the first 7 days after TIV. Importantly, the increase of ICOS+PD-1+CXCR3+ cTfh cells strongly correlated with the increase in the avidity of antibodies, particularly in subjects who did not have high affinity antibodies at baseline. We propose that ICOS+PD-1+CXCR3+ Tfh cells directly contribute to the generation of high-avidity antibodies after TIV vaccinations by selectively interacting with high affinity B cells at extrafollicular sites.

TPS3133Background: Checkpoint blockade has become a major modality in the treatment of metastatic melanoma (MM). However, durable remission rates remain low. CD40 activation on antigen presenting c...

T follicular helper (Tfh) cells help development of antibody responses via interleukin-21 (IL-21). Here we show that activated human dendritic cells (DCs) induced naive CD4(+) T cells to become IL-21-producing Tfh-like cells through IL-12. CD4(+) T cells primed with IL-12 induced B cells to produce immunoglobulins in a fashion dependent on IL-21 and inducible costimulator (ICOS), thus sharing fundamental characteristics with Tfh cells. The induction of Tfh-like cells by activated DCs was inhibited by neutralizing IL-12. IL-12 induced two different IL-21-producers: IL-21(+)IFN-gamma(+)T-bet(+) Th1 cells and IL-21(+)IFN-gamma(-)T-bet(-) non-Th1 cells, in a manner dependent on signal transducer and activator of transcription 4 (STAT4). IL-12 also regulated IL-21 secretion by memory CD4(+) T cells. Thus, IL-12 produced by activated DCs regulates antibody responses via developing IL-21-producing Tfh-like cells and inducing IL-21 secretion from memory CD4(+) T cells. These data suggest that the developmental pathway of Tfh cells differs between mice and humans, which have considerable implications for vaccine development.

Human blood contains a memory counterpart of T follicular helper (T(FH)) cells. Blood T(FH) cells are composed of subsets that differ in phenotype and function. Recent studies show that analysis of blood circulating memory T(FH) cells can provide clues to understand the mode of actions of vaccines and the pathogenesis of human autoimmune diseases. We will describe here a detailed protocol to analyze the memory T(FH) subsets in human whole blood samples. We will also describe a protocol to assess the helper capacity of blood memory T(FH) subsets.

Human blood contains a memory counterpart of T follicular helper (T(FH)) cells. Blood T(FH) cells are composed of subsets that differ in phenotype and function. Recent studies show that analysis of blood circulating memory T(FH) cells can provide clues to understand the mode of actions of vaccines and the pathogenesis of human autoimmune diseases. We will describe here a detailed protocol to analyze the memory T(FH) subsets in human whole blood samples. We will also describe a protocol to assess the helper capacity of blood memory T(FH) subsets.

2545 Background: NKTR-214 is a CD122-biased agonist designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβɣ) to preferentially activate and expand effector CD8+ T and NK cells over T regulatory cells in the tumor microenvironment. Immune changes in the tumor microenvironment after NKTR-214 treatment was assessed in patients with locally advanced or metastatic solid tumors. Methods: NKTR-214 was administered IV in an outpatient setting q2w or q3w. Serial blood and tumor tissue samples were collected to measure immune activation using immunophenotyping including flow cytometry, immunohistochemistry (IHC), T cell clonality and gene expression analyses. Results: 26 patients (pts) have been treated with NKTR-214 at q3w, 4@0.003, 9@0.006, 6@0.009 and 1@0.012 mg/kg. Six pts received 0.006 mg/kg q2w. 58% of pts had prior immunotherapy. The most common Gr1-2 TRAEs were fatigue (73%) and pruritus (65%), and decreased appetite (46%). One pt experienced Gr3 syncope and hypotension at the highest dose tested and continued treatment at a lower dose. No drug-related AEs led to study discontinuation. No immune-related AEs or capillary leak syndrome were observed. 6 pts (23%) experienced tumor shrinkage from 10-30%. Three immunotherapy naïve pts receiving sequential anti-PD1 therapy, after ending treatment with NKTR-214, experienced significant tumor regression at first scan. In all pts evaluated, blood samples showed increases in newly proliferating (Ki67+) T and NK cells 8 days post dose. Flow cytometry and/or IHC revealed an up to 10-fold increase from baseline in tumor CD8+T and NK cells in the tumor microenvironment, with minimal changes to Tregs. PD-1 expression increased 2-fold in TILs. Gene expression analysis of tumor tissue showed increases in several immune checkpoint genes, cytotoxic markers (IFNg, PRF1, and GZMB), as well as a dynamic change in T cell clonality. Conclusions: Based on a favorable safety profile and strong correlative biomarker data, a phase 1/2 trial combining NKTR-214 and nivolumab is currently enrolling. Clinical trial information: NCT02869295.

Seasonal influenza vaccine protects 60 to 90% of healthy young adults from influenza infection. The immunological events that lead to the induction of protective antibody responses remain poorly understood in humans. We identified the type of CD4+ T cells associated with protective antibody responses after seasonal influenza vaccinations. The administration of trivalent split-virus influenza vaccines induced a temporary increase of CD4+ T cells expressing ICOS, which peaked at day 7, as did plasmablasts. The induction of ICOS was largely restricted to CD4+ T cells co-expressing the chemokine receptors CXCR3 and CXCR5, a subpopulation of circulating memory T follicular helper cells. Up to 60% of these ICOS+CXCR3+CXCR5+CD4+ T cells were specific for influenza antigens and expressed interleukin-2 (IL-2), IL-10, IL-21, and interferon-γ upon antigen stimulation. The increase of ICOS+CXCR3+CXCR5+CD4+ T cells in blood correlated with the increase of preexisting antibody titers, but not with the induction of primary antibody responses. Consistently, purified ICOS+CXCR3+CXCR5+CD4+ T cells efficiently induced memory B cells, but not naïve B cells, to differentiate into plasma cells that produce influenza-specific antibodies ex vivo. Thus, the emergence of blood ICOS+CXCR3+CXCR5+CD4+ T cells correlates with the development of protective antibody responses generated by memory B cells upon seasonal influenza vaccination.

T follicular helper (Tfh) cells represent a Th subset engaged in the help of B-cell responses in germinal centers (GCs). Tfh cells abundantly express the transcription repressor B-cell lymphoma 6 (Bcl6), a factor that is necessary and sufficient for their development in vivo. Whether Tfh or Tfh-committed cells are involved in the help of B cells outside GCs remains unclear, particularly in humans. In this study, we identified a previously undefined BCL6 -expressing CD4 + T-cell subset in human tonsils. This subset expressed IL-7 receptor and chemokine receptor 5 (CXCR5) and inducible costimulator (ICOS) at low levels (CXCR5 lo ICOS lo ), and it was found exclusively outside GCs. CXCR5 lo ICOS lo CD4 + T cells secreted larger amounts of IL-21 and IL-10 than CXCR5 hi ICOS hi GC-Tfh cells upon activation, and they induced proliferation and differentiation of naïve B cells into Ig-producing cells more efficiently than GC-Tfh cells. However, this subset lacked the capacity to help GC-B cells because of the induction of apoptosis of GC-B cells through the FAS/FAS–ligand interaction. CXCR5 lo ICOS lo CD4 + T cells expressed equivalent amounts of BCL6 transcript with CXCR5 hi ICOS hi GC-Tfh cells, but they expressed less Bcl6 protein. Collectively, our study indicates that CXCR5 lo ICOS lo CD4 + T cells in human tonsils represent Tfh lineage-committed cells that provide help to naïve and memory B cells outside GCs.

Although a fraction of human blood memory CD4(+) T cells expresses chemokine (C-X-C motif) receptor 5 (CXCR5), their relationship to T follicular helper (Tfh) cells is not well established. Here we show that human blood CXCR5(+)CD4(+) T cells share functional properties with Tfh cells and appear to represent their circulating memory compartment. Blood CXCR5(+)CD4(+) T cells comprised three subsets: T helper 1 (Th1), Th2, and Th17 cells. Th2 and Th17 cells within CXCR5(+), but not within CXCR5(-), compartment efficiently induced naive B cells to produce immunoglobulins via interleukin-21 (IL-21). In contrast, Th1 cells from both CXCR5(+) and CXCR5(-) compartments lacked the capacity to help B cells. Patients with juvenile dermatomyositis, a systemic autoimmune disease, displayed a profound skewing of blood CXCR5(+) Th cell subsets toward Th2 and Th17 cells. Importantly, the skewing of subsets correlated with disease activity and frequency of blood plasmablasts. Collectively, our study suggests that an altered balance of Tfh cell subsets contributes to human autoimmunity.

T follicular helper cells (Tfh) represent a Th subset specialized for the help of antibody responses. In contrast to recent progress regarding the phenotype and developmental mechanism of Tfh cells, how Tfh cells regulate B cell responses is less well characterized. In particular, whether Tfh cells are involved in the differentiation of naïve B cells into antibody-secreting cells (ASCs) remains largely unknown. Here we show that human tonsillar Tfh cells comprise subsets specialized for the help of distinct B cell subsets. A subset of Tfh cells, here called Pre-Tfh cells, is specialized for the help of naïve B cells. In contrast, Tfh cells at germinal centers (GC-Tfh cells) are the only Tfh subset able to induce germinal center B (GC-B) cells to produce Igs. Despite the capacity to secrete high levels of Interleukin-21 (IL-21), Pre-Tfh cells failed to help GC-B cells, due to the induction of apoptosis of GC-B cells via FAS/FAS ligand interaction. While Pre-Tfh cells were found exclusively at extrafollicular sites, Pre-Tfh cells expressed Bcl6, a master transcription factor of Tfh cells, at equivalent levels with GC-Tfh cells. A comprehensive gene expression profiling strongly suggests that pre-Tfh cells are progenitors of GC-Tfh cells. Thus, the differentiation of functional human Tfh cells already occurs at outside of B cell follicles, and such extrafollicular Tfh cells are engaged in the differentiation of naïve B cells.