Basophils from allergic patients are neither hyperresponsive to activation signals nor hyporesponsive to inhibition signals.

BACKGROUND: Basophil activation contributes to inflammatory reactions, especially in allergy. It is controlled, both positively and negatively, by several mechanisms. High-affinity IgE receptors (FcεRI) generate a mixture of activation and inhibition signals when aggregated, the ratio of which depends on the concentration of allergen recognized by receptor-bound IgE. Low-affinity IgG receptors (FcγRIIA/B) generate inhibition signals when coengaged with FcεRI by allergen-antibody immune complexes. Commensal and probiotic bacteria, such as Lactobacillus paracasei, generate inhibition signals through still unclear mechanisms. OBJECTIVE: We sought to investigate whether mechanisms that control, both positively and negatively, basophil activation, which were unraveled and studied in basophils from healthy donors, are functional in allergic patients. METHODS: FcεRI and FcγRIIA/B expression, FcεRI-dependent activation, FcεRI-dependent inhibition, and FcγRIIB-dependent inhibition were examined in blood basophils incubated overnight with or without L paracasei and challenged under 10 experimental conditions. Basophils from healthy donors were compared with basophils from patients who consulted an allergology outpatient clinic over a period of 3 months with respiratory allergy, anaphylaxis antecedents, chronic urticaria, and/or atopic dermatitis. RESULTS: Patients' basophils expressed neither more FcεRI nor less FcγRIIB than basophils from healthy donors. They were neither hyperreactive to positive regulation nor hyporeactive to negative regulation, irrespective of the receptors or mechanisms involved and the allergic manifestations of the patients. CONCLUSION: Regulatory mechanisms that control basophil activation are fully functional in allergic patients. Intrinsic defects in these mechanisms do not explain allergic manifestations. Based on these mechanisms, immune checkpoint modifiers can be developed as novel therapeutic tools for allergy.

Deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses.

Multicolor flow cytometry is a technology of choice for phenotyping of immune cells, and it can be used routinely for the follow up of patients in clinical trials. But it is challenging to define combinations of conjugated antibodies that efficiently allow the detailed analysis of major immune cell subsets and the identification of rare cell populations. In a collaborative work among the Immunology, Immunopathology, Immunotherapy (I3 ) laboratory, and the laboratory of immunomonitoring in oncology (L.I.O), we developed and validated 12 different 10-color flow cytometry panels that allow the deep immunophenotyping of cells from whole blood for the follow up of autoimmune and cancer patients. Here, we describe these optimized flow cytometry panels, showing that they provide the advanced analysis of T cells (including regulatory T cells), B cells, NK cells, MAIT cells, myeloid cells, monocytes, and dendritic cells. Most of the panels have been dried to improve standardization of the labeling and the entire procedure can be performed on less than 2 ml of whole blood. These deep immunophenotyping flow cytometry panels constitute a powerful tool for the monitoring of immune blood cells and will hopefully lead to the discovery of new biomarkers and potential therapeutic targets in autoimmune and cancer clinical trials. © 2018 International Society for Advancement of Cytometry.

IL-15 Trans-Signaling with the Superagonist RLI Promotes Effector/Memory CD8+ T Cell Responses and Enhances Antitumor Activity of PD-1 Antagonists.

Tumors with the help of the surrounding environment facilitate the immune suppression in patients, and immunotherapy can counteract this inhibition. Among immunotherapeutic strategies, the immunostimulatory cytokine IL-15 could represent a serious candidate for the reactivation of antitumor immunity. However, exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Rα frequently downregulated in cancer patients. In this work, we studied the antitumor activity of the IL-15 superagonist receptor-linker-IL-15 (RLI), designed to bypass the need of endogenous IL-15Rα. RLI consists of human IL-15 covalently linked to the human IL-15Rα sushi(+) domain. In a mouse model of colorectal carcinoma, RLI as a stand-alone treatment could limit tumor outgrowth only when initiated at an early time of tumor development. At a later time, RLI was not effective, coinciding with the strong accumulation of terminally exhausted programmed cell death-1 (PD-1)(high) T cell Ig mucin-3(+) CD8(+) T cells, suggesting that RLI was not able to reactivate terminally exhausted CD8(+) T cells. Combination with PD-1 blocking Ab showed synergistic activity with RLI, but not with IL-15. RLI could induce a greater accumulation of memory CD8(+) T cells and a stronger effector function in comparison with IL-15. Ex vivo stimulation of tumor-infiltrated lymphocytes from 16 patients with renal cell carcinoma demonstrated 56% of a strong tumor-infiltrated lymphocyte reactivation with the combination anti-PD-1/RLI compared with 43 and 6% with RLI or anti-PD-1, respectively. Altogether, this work provides evidence that the sushi-IL-15Rα/IL-15 fusion protein RLI enhances antitumor activity of anti-PD-1 treatment and is a promising approach to stimulate host immunity.

Semi-automated and standardized cytometric procedures for multi-panel and multi-parametric whole blood immunophenotyping.

Immunophenotyping by multi-parametric flow cytometry is the cornerstone technology for enumeration and characterization of immune cell populations in health and disease. Standardized procedures are essential to allow for inter-individual comparisons in the context of population based or clinical studies. Herein we report the approach taken by the Milieu Intérieur Consortium, highlighting the standardized and automated procedures used for immunophenotyping of human whole blood samples. We optimized eight-color antibody panels and procedures for staining and lysis of whole blood samples, and implemented pre-analytic steps with a semi-automated workflow using a robotic system. We report on four panels that were designed to enumerate and phenotype major immune cell populations (PMN, T, B, NK cells, monocytes and DC). This work establishes a foundation for defining reference values in healthy donors. Our approach provides robust protocols for affordable, semi-automated eight-color cytometric immunophenotyping that can be used in population-based studies and clinical trial settings.

Fcγ receptors inhibit mouse and human basophil activation.

Besides high-affinity IgE receptors (FcεRI), human basophils express activating (FcγRIIA) and inhibitory (FcγRIIB) low-affinity IgG receptors. IgG receptors (FcγR) were also found on mouse basophils, but not identified. We investigated in this study FcγR and the biological consequences of their engagement in basophils of the two species. We found the following: (1) that mouse basophils also express activating (FcγRIIIA) and inhibitory (FcγRIIB) low-affinity FcγR; (2) that activating FcγR can activate both human and mouse basophils, albeit with different efficacies; (3) that negative signals triggered by inhibitory FcγR are dominant over positive signals triggered by activating FcγR, thus preventing both human and mouse basophils from being activated by IgG immune complexes; (4) that the coengagement of FcεRI with inhibitory and activating FcγR results in a FcγRIIB-dependent inhibition of IgE-induced responses of both human and mouse basophils; (5) that FcγRIIB has a similar dominant inhibitory effect in basophils from virtually all normal donors; and (6) that IL-3 upregulates the expression of both activating and inhibitory FcγR on human basophils from normal donors, but further enhances FcγRIIB-dependent inhibition. FcγR therefore function as a regulatory module, made of two subunits with antagonistic properties, that prevents IgG-induced and controls IgE-induced basophil activation in both mice and humans.

B cells and the coordination of immune checkpoint inhibitor response in patients with solid tumors.

Immunotherapy profoundly changed the landscape of cancer therapy by providing long-lasting responses in subsets of patients and is now the standard of care in several solid tumor types. However, immunotherapy activity beyond conventional immune checkpoint inhibition is plateauing, and biomarkers are overall lacking to guide treatment selection. Most studies have focused on T cell engagement and response, but there is a growing evidence that B cells may be key players in the establishment of an organized immune response, notably through tertiary lymphoid structures. Mechanisms of B cell response include antibody-dependent cellular cytotoxicity and phagocytosis, promotion of CD4+ and CD8+ T cell activation, maintenance of antitumor immune memory. In several solid tumor types, higher levels of B cells, specific B cell subpopulations, or the presence of tertiary lymphoid structures have been associated with improved outcomes on immune checkpoint inhibitors. The fate of B cell subpopulations may be widely influenced by the cytokine milieu, with versatile roles for B-specific cytokines B cell activating factor and B cell attracting chemokine-1/CXCL13, and a master regulatory role for IL-10. Roles of B cell-specific immune checkpoints such as TIM-1 are emerging and could represent potential therapeutic targets. Overall, the expanding field of B cells in solid tumors of holds promise for the improvement of current immunotherapy strategies and patient selection.

A strain of Lactobacillus casei inhibits the effector phase of immune inflammation.

Some nonpathogenic bacteria were found to have protective effects in mouse models of allergic and autoimmune diseases. These "probiotics" are thought to interact with dendritic cells during Ag presentation, at the initiation of adaptive immune responses. Many other myeloid cells are the effector cells of immune responses. They are responsible for inflammation that accounts for symptoms in allergic and autoimmune diseases. We investigated in this study whether probiotics might affect allergic and autoimmune inflammation by acting at the effector phase of adaptive immune responses. The effects of one strain of Lactobacillus casei were investigated in vivo on IgE-induced passive systemic anaphylaxis and IgG-induced passive arthritis, two murine models of acute allergic and autoimmune inflammation, respectively, which bypass the induction phase of immune responses, in vitro on IgE- and IgG-induced mouse mast cell activation and ex vivo on IgE-dependent human basophil activation. L. casei protected from anaphylaxis and arthritis, and inhibited mouse mast cell and human basophil activation. Inhibition required contact between mast cells and bacteria, was reversible, and selectively affected the Lyn/Syk/linker for activation of T cells pathway induced on engagement of IgE receptors, leading to decreased MAPK activation, Ca(2+) mobilization, degranulation, and cytokine secretion. Also, adoptive anaphylaxis induced on Ag challenge in mice injected with IgE-sensitized mast cells was abrogated in mice injected with IgE-sensitized mast cells exposed to bacteria. These results demonstrate that probiotics can influence the effector phase of adaptive immunity in allergic and autoimmune diseases. They might, therefore, prevent inflammation in patients who have already synthesized specific IgE or autoantibodies.

Human virome profiling identified CMV as the major viral driver of a high accumulation of senescent CD8+ T cells in patients with advanced NSCLC.

Circulating senescent CD8+ T (T8sen) cells are characterized by a lack of proliferative capacities but retain cytotoxic activity and have been associated to resistance to immunotherapy in patients with advanced non-small cell lung cancer (aNSCLC). We aimed to better characterize T8sen and to determine which factors were associated with their accumulation in patients with aNSCLC. Circulating T8sen cells were characterized by a higher expression of SA-ßgal and the transcription factor T-bet, confirming their senescent status. Using whole virome profiling, cytomegalovirus (CMV) was the only virus associated with T8sen. CMV was necessary but not sufficient to explain high accumulation of T8sen (T8senhigh status). In CMV+ patients, the proportion of T8sen cells increased with cancer progression. Last, CMV-induced T8senhigh phenotype but not CMV seropositivity itself was associated with worse progression-free and overall survival in patients treated with anti-PD-(L)1 therapy but not with chemotherapy. Overall, CMV is the unique viral driver of T8sen-driven resistance to anti-PD-(L)1 antibodies in patients with aNSCLC.

Genomic Instability and Protumoral Inflammation Are Associated with Primary Resistance to Anti-PD-1 + Antiangiogenesis in Malignant Pleural Mesothelioma.

Cancer immunotherapy combinations have recently been shown to improve the overall survival of advanced mesotheliomas, especially for patients responding to those treatments. We aimed to characterize the biological correlates of malignant pleural mesotheliomas' primary resistance to immunotherapy and antiangiogenics by testing the combination of pembrolizumab, an anti-PD-1 antibody, and nintedanib, a pan-antiangiogenic tyrosine kinase inhibitor, in the multicenter PEMBIB trial (NCT02856425). Thirty patients with advanced malignant pleural mesothelioma were treated and explored. Unexpectedly, we found that refractory patients were actively recruiting CD3+CD8+ cytotoxic T cells in their tumors through CXCL9 tumor release upon treatment. However, these patients displayed high levels of somatic copy-number alterations in their tumors that correlated with high blood and tumor levels of IL6 and CXCL8. Those proinflammatory cytokines resulted in higher tumor secretion of VEGF and tumor enrichment in regulatory T cells. Advanced mesothelioma should further benefit from stratified combination therapies adapted to their tumor biology. SIGNIFICANCE: Sequential explorations of fresh tumor biopsies demonstrated that mesothelioma resistance to anti-PD-1 + antiangiogenics is not due to a lack of tumor T-cell infiltration but rather due to adaptive immunosuppressive pathways by tumors, involving molecules (e.g., IL6, CXCL8, VEGF, and CTLA4) that are amenable to targeted therapies. This article is highlighted in the In This Issue feature, p. 799.

Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity.

Effector cells derived from central memory CD8(+) T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve, rather than central memory T cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1(-) phenotype, naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8(+) T cells may allow superior efficacy upon adoptive transfer.

CD8+PD-1+ to CD4+PD-1+ ratio (PERLS) is associated with prognosis of patients with advanced NSCLC treated with PD-(L)1 blockers.

BACKGROUND: Programmed cell death protein-1 (PD-1) expression has been associated with activation and exhaustion of both the CD4 and CD8 populations in advanced non-small cell lung cancer (aNSCLC). Nevertheless, the impact of the balance between circulating CD8+PD-1+ and CD4+PD-1+ in patients treated with immune checkpoint blockers (ICB) is unknown. METHODS: The CD8+PD-1+ to CD4+PD-1+ ratio (PD-1-Expressing Ratio on Lymphocytes in a Systemic blood sample, or 'PERLS') was determined by cytometry in fresh whole blood from patients with aNSCLC before treatment with single-agent ICB targeting PD-1 or programmed cell death-ligand 1 (PD-L1 (discovery cohort). A PERLS cut-off was identified by log-rank maximization. Patients treated with ICB (validation cohort) or polychemotherapy (control cohort) were classified as PERLS+/- (above/below cut-off). Circulating immune cell phenotype and function were correlated with PERLS. A composite score (good, intermediate and poor) was determined using the combination of PERLS and senescent immune phenotype as previously described in aNSCLC. RESULTS: In the discovery cohort (N=75), the PERLS cut-off was 1.91, and 11% of patients were PERLS+. PERLS + correlated significantly with median progression-free survival (PFS) of 9.63 months (95% CI 7.82 to not reached (NR)) versus 2.69 months (95% CI 1.81 to 5.52; p=0.03). In an independent validation cohort (N=36), median PFS was NR (95% CI 7.9 to NR) versus 2.00 months (95% CI 1.3 to 4.5; p=0.04) for PERLS + and PERLS-, respectively; overall survival (OS) followed a similar but non-significant trend. In the pooled cohort (N=111), PERLS + correlated significantly with PFS and OS. PERLS did not correlate with outcome in the polychemotherapy cohort. PERLS did not correlate with clinical characteristics but was significantly associated with baseline circulating naïve CD4+ T cells and the increase of memory T cells post-ICB treatment. Accumulation of memory T cells during treatment was linked to CD4+ T cell polyfunctionality. The composite score was evaluated in the pooled cohort (N=68). The median OS for good, intermediate and poor composite scores was NR (95% CI NR to NR), 8.54 months (95% CI 4.96 to NR) and 2.42 months (95% CI 1.97 to 15.5; p=0.001), respectively. The median PFS was 12.60 months (95% CI 9.63 to NR), 2.58 months (95% CI 1.74 to 7.29) and 1.76 months (95% CI 1.31 to 4.57; p<0.0001), respectively. CONCLUSIONS: Elevated PERLS, determined from a blood sample before immunotherapy, was correlated with benefit from PD-(L)1 blockers in aNSCLC.

Gefitinib plus tremelimumab combination in refractory non-small cell lung cancer patients harbouring EGFR mutations: The GEFTREM phase I trial.

INTRODUCTION: A phase I open-label multicentre study was initiated to evaluate the association of tremelimumab with gefitinib in EGFR-mutant NSCLC patients who progressed after first-generation EGFR-TKI. Here we provide the efficacy data from the entire cohort. MATERIAL AND METHODS: Patients with advanced EGFR-mutant NSCLC with progression after response to EGFR-TKI were enrolled. Study treatment was gefitinib 250 mg daily and tremelimumab at 3 dose levels: 3, 6 and 10 mg/kg IV Q4W for 6 cycles followed by Q12W until progression or unacceptable toxicity. The primary objective was safety and tolerability, and to establish a RP2D. RESULTS: Between January 2014 and July 2015, 27 patients (21 in the escalating dose cohort and 6 in expansion cohort) received at least one dose of tremelimumab. DLTs occurred in 4 patients: 1 at 3 mg/kg (one grade 3 diarrhoea), 1 at 6 mg/kg (one grade 3 diarrhoea) and 2 at 10 mg/kg (one grade 3 diarrhoea and one grade 3 AST/ALT increase) of tremelimumab. Grade 3 TRAE occurred in 22 patients (81%), most frequently diarrhoea (30%) and ALT/AST increase (15%). Stable disease was the best overall response in 72% patients, with median PFS of 2.2 months (95% CI, 1.8-4.2). All patients discontinued treatment, most frequently due to disease progression (63% of patients). CONCLUSION: The recommended dose of tremelimumab in combination with gefitinib in EGFR-mutant NSCLC patients was 3 mg/kg. The gastrointestinal toxicity and the limited efficacy data prevented further evaluation of this combination. (GEFTREM; clinical trial number NCT02040064).

Overcoming resistance to αPD-1 of MMR-deficient tumors with high tumor-induced neutrophils levels by combination of αCTLA-4 and αPD-1 blockers.

BACKGROUND: Clinical studies have highlighted the efficacy of anti-programmed death 1 (αPD-1) monoclonal antibodies in patients with DNA mismatch repair-deficient (MMRD) tumors. However, the responsiveness of MMRD cancers to αPD-1 therapy is highly heterogeneous, and the origins of this variability remain not fully understood. METHODS: 4T1 and CT26 mouse tumor cell lines were inactivated for the MMRD gene Msh2, leading to a massive accumulation of mutations after serial passages of cells. Insertions/deletion events and mutation load were evaluated by whole exome sequencing. Mice bearing highly mutated MMRD tumor or parental tumors were treated with αPD-1 and tumor volume was monitored. Immune cell type abundance was dynamically assessed in the tumor microenvironment and the blood by flow cytometry. Neutrophils were depleted in mice using αLY6G antibody, and regulatory T (Treg) cell population was reduced with αCD25 or anti-cytotoxic T-lymphocytes-associated protein 4 (αCTLA-4) antibodies. Patients with MMRD tumors treated with immune checkpoint blockade-based therapy were retrospectively identified and neutrophil-to-lymphocyte ratio (NLR) was evaluated and examined for correlation with clinical benefit. RESULTS: By recapitulating mismatch repair deficiency in different mouse tumor models, we revealed that elevated circulating tumor-induced neutrophils (TIN) in hypermutated MMRD tumors hampered response to αPD-1 monotherapy. Importantly, depletion of TIN using αLy-6G antibody reduced Treg cells and restored αPD-1 response. Conversely, targeting Treg cells by αCD25 or αCTLA-4 antibodies limited peripheral TIN accumulation and elicited response in αPD-1-resistant MMRD tumors, highlighting a crosstalk between TIN and Treg cells. Thus, αPD-1+αCTLA-4 combination overcomes TIN-induced resistance to αPD-1 in mice bearing MMRD tumors. Finally, in a cohort of human (high microsatellite instability)/MMRD tumors we revealed that early on-treatment change in the NLR ratio may predict resistance to αPD-1 therapy. CONCLUSIONS: TIN countered αPD-1 efficacy in MMRD tumors. Since αCTLA-4 could restrict TIN accumulation, αPD-1+αCTLA-4 combination overcomes αPD-1 resistance in hosts with hypermutated MMRD tumors displaying abnormal neutrophil accumulation.

Baseline circulating unswitched memory B cells and B-cell related soluble factors are associated with overall survival in patients with clear cell renal cell carcinoma treated with nivolumab within the NIVOREN GETUG-AFU 26 study.

BACKGROUND: The phase II NIVOREN GETUG-AFU 26 study reported safety and efficacy of nivolumab in patients with metastatic clear cell renal cell carcinoma (m-ccRCC) in a 'real-world setting'. We conducted a translational-research program to determine whether specific circulating immune-cell populations and/or soluble factors at baseline were predictive of clinical outcomes in patients with m-ccRCC treated with nivolumab within the NIVOREN study. METHODS: Absolute numbers of 106 circulating immune-cell populations were prospectively analyzed in patients treated at a single institution within the NIVOREN trial with available fresh-whole-blood, using dry formulation panels for multicolor flow cytometry. In addition, a panel of 14 predefined soluble factors was quantified for each baseline plasma sample using the Meso-Scale-Discovery immunoassay. The remaining patients with available plasma sample were used as a validation cohort for the soluble factor quantification analysis. Tumor immune microenvironment characterization of all patients included in the translational program of the study was available. The association of blood and tissue-based biomarkers, with overall survival (OS), progression-free survival (PFS) and response was analyzed. RESULTS: Among the 44 patients, baseline unswitched memory B cells (NSwM B cells) were enriched in responders (p=0.006) and associated with improved OS (HR=0.08, p=0.002) and PFS (HR=0.54, p=0.048). Responders were enriched in circulating T follicular helper (Tfh) (p=0.027) and tertiary lymphoid structures (TLS) (p=0.043). Circulating NSwM B cells positively correlated with Tfh (r=0.70, p<0.001). Circulating NSwM B cells correlated positively with TLS and CD20 +B cells at the tumor center (r=0.59, p=0.044, and r=0.52, p=0.033) and inversely correlated with BCA-1/CXCL13 and BAFF (r=-0.55 and r=-0.42, p<0.001). Tfh cells also inversely correlated with BCA-1/CXCL13 (r=-0.61, p<0.001). IL-6, BCA-1/CXCL13 and BAFF significantly associated with worse OS in the discovery (n=40) and validation cohorts (n=313). CONCLUSION: We report the first fresh blood immune-monitoring of patients with m-ccRCC treated with nivolumab. Baseline blood concentration of NSwM B cells was associated to response, PFS and OS in patients with m-ccRCC treated with nivolumab. BCA-1/CXCL13 and BAFF, inversely correlated to NSwM B cells, were both associated with worse OS in discovery and validation cohorts. Our data confirms a role for B cell subsets in the response to immune checkpoint blockade therapy in patients with m-ccRCC. Further studies are needed to confirm these findings.

Safety, recommended dose, efficacy and immune correlates for nintedanib in combination with pembrolizumab in patients with advanced cancers.

BACKGROUND: We aimed to determine the safety and efficacy of nintedanib, an oral anti-angiogenic tyrosine kinase inhibitor, in combination with pembrolizumab, an anti-PD1 immunotherapy, in patients with advanced solid tumors (PEMBIB trial; NCT02856425). METHODS: In this monocentric phase Ib dose escalation cohort, we evaluated escalating doses of nintedanib (Dose level 1 (DL1) = 150 mg bid [bis in die, as twice a day]; DL2 = 200 mg bid, oral delivery) in combination with pembrolizumab (200 mg Q3W, IV). Patients received a 1-week lead-in dose of nintedanib monotherapy prior starting pembrolizumab. The primary objective was to establish the maximum tolerated dose (MTD) of the combination based on dose limiting toxicity (DLT) occurrence during the first 4 weeks. Secondary objectives were to assess the anti-tumor efficacy and to identify the associated immune and angiogenic parameters in order to establish the recommended nintedanib dose for expansion cohorts. Flow cytometry (FC), Immuno-Histo-Chemistry (IHC) and electrochemiluminescence multi-arrays were prospectively performed on baseline & on-treatment tumor and blood samples to identify immune correlates of efficacy. RESULTS: A total of 12/13 patients enrolled were evaluable for DLT (1 patient withdrew consent prior receiving pembrolizumab). Three patients at 200 mg bid experienced a DLT (grade 3 liver enzymes increase). Four patients developed grade 1-2 immune related adverse events (irAE). Eight patients died because of cancer progression. Median follow-up was 23.7 months (95%CI: 5.55-40.5). Three patients developed a partial response (PR) (ORR = 25%) and five patients (42%) had durable clinical benefit (DCB), defined as PR or stable disease (SD) ≥ 6 months. At baseline, patients with DCB had higher plasma levels of Tie2, CXCL10, CCL22 and circulating CD4+ PD1+ OX40+ T cells than patients without DCB. Patients with DCB presented also with more DC-LAMP+ dendritic cells, CD3+ T cells and FOXP3+ Tregs in baseline tumor biopsies. For DCB patients, the nintedanib lead-in monotherapy resulted in higher blood CCL3, Tregs and CCR4+ CXCR3+ CXCR5- memory CD4 T cells. After the first pembrolizumab infusion, patients with DCB showed lower IL-6, IL-8, IL-27 plasma levels. CONCLUSION: Nintedanib 150 mg bid is the recommended dose for combination with pembrolizumab and is currently investigated in multiple expansion cohorts. Early tumoral and circulating immune factors were associated with cancer outcome under nintedanib & pembrolizumab therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02856425 . Registered August 4, 2016 - Prospectively registered.

Type 17 CD8+ T cells display enhanced antitumor immunity.

Interleukin-17 (IL-17)-secreting CD8(+) T cells have been described, but they have not been thoroughly studied and they do not have a known role in cancer immunotherapy. We skewed CD8(+) T cells to secrete IL-17 through priming in Th17-polarizing conditions. IL-17-producing CD8(+) T cells demonstrated reduced expression of Eomes and diminished cytolytic differentiation in vitro. However, after adoptive transfer, these cells converted to interferon-gamma-producing effector cells and mediated regression of large, established tumors. This improved antitumor immunity was associated with increased expression of IL-7R-alpha, decreased expression of killer cell lectin-like receptor G1, and enhanced persistence of the transferred cells. This report is the first description of a cancer therapy with IL-17-secreting CD8(+) T cells. These findings have implications for the improvement of CD8(+) T cell-based adoptive immunotherapy.

Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen.

Gene therapy of human cancer using genetically engineered lymphocytes is dependent on the identification of highly reactive T-cell receptors (TCRs) with antitumor activity. We immunized transgenic mice and also conducted high-throughput screening of human lymphocytes to generate TCRs highly reactive to melanoma/melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA(-) and CD45RO(+) after ex vivo expansion. After infusion, the persisting cells displayed a CD45RA(+) and CD45RO(-) phenotype. Gene-engineered cells persisted at high levels in the blood of all patients 1 month after treatment, responding patients with higher ex vivo antitumor reactivity than nonresponders. Objective cancer regressions were seen in 30% and 19% of patients who received the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear, and sometimes required local steroid administration to treat uveitis and hearing loss. Thus, T cells expressing highly reactive TCRs mediate cancer regression in humans and target rare cognate-antigen-containing cells throughout the body, a finding with important implications for the gene therapy of cancer. This trial was registered at www.ClinicalTrials.gov as NCI-07-C-0174 and NCI-07-C-0175.

Current and future biomarkers for outcomes with immunotherapy in non-small cell lung cancer.

Immune checkpoint inhibitors (ICI) have been validated as an effective new treatment strategy in several tumoral types including lung cancer. This remarkable shift in the therapeutic paradigm is in large part due to the duration of responses and long-term survival seen with ICI. However, despite this, the majority of cancer patients do not experience benefit from ICI. Even among patients who initially respond to ICI, disease progression may ultimately occur. Moreover, in some patients, these drugs may be associated with new patterns of progression such as pseudo-progression and hyper-progressive disease, and different toxicity profiles with immune-related adverse events. Therefore, the identification of predictive biomarkers may help to select those patients most likely to obtain a true benefit from these drugs, and avoid exposure to potential toxicity in patients who will not obtain clinical benefit, while also reducing the economic impact. In this review, we summarize current and promising potential predictive biomarkers of ICI in patients with non-small cell lung cancer (NSCLC), as well as pitfalls encountered with their use and areas of focus to optimize their routine clinical implementation.

Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation.

AMHRII, the anti-Müllerian hormone receptor, is selectively expressed in normal sexual organs but is also re-expressed in gynecologic cancers. Hence, we developed murlentamab, a humanized glyco-engineered anti-AMHRII monoclonal antibody currently in clinical trial. Low-fucosylated antibodies are known to increase the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) potency of effector cells, but some preliminary results suggest a more global murlentamab-dependent activation of the immune system. In this context, we demonstrate here that the murlentamab opsonization of AMHRII-expressing ovarian tumor cells, in the presence of unstimulated- or tumor-associated macrophage (TAM)-like macrophages, significantly promotes macrophage-mediated ADCC and shifts the whole microenvironment towards a pro-inflammatory and anti-tumoral status, thus triggering anti-tumor activity. We also report that murlentamab orients both unstimulated- and TAM-like macrophages to an M1-like phenotype characterized by a strong expression of co-stimulation markers, pro-inflammatory cytokines and chemokines, favoring T cell recruitment and activation. Moreover, we show that murlentamab treatment shifts CD4+ Th1/Th2 balance towards a Th1 response and activates CD8+ T cells. Altogether, these results suggest that murlentamab, through naïve macrophage orientation and TAM reprogrammation, stimulates the anti-tumor adaptive immune response. Those mechanisms might contribute to the sustained clinical benefit observed in advanced cancer patients treated with murlentamab. Finally, the enhanced murlentamab activity in combination with pembrolizumab opens new therapeutic perspectives.

Phase II and biomarker study of programmed cell death protein 1 inhibitor nivolumab and metronomic cyclophosphamide in paediatric relapsed/refractory solid tumours: Arm G of AcSé-ESMART, a trial of the European Innovative Therapies for Children With Cancer Consortium.

PURPOSE: AcSé-ESMART is a European multicentre, proof-of-concept multiarm phase I/II platform trial in paediatric patients with relapsed/refractory cancer. Arm G assessed the activity and safety of nivolumab in combination with metronomic cyclophosphamide +/- irradiation. EXPERIMENTAL DESIGN: Following a Phase II Simon two-stage design, nivolumab was administered intravenously at 3 mg/kg every 2 weeks of a 28-day cycle, oral cyclophosphamide at 25 mg/m2 twice a day, 1 week on/1 week off. The primary endpoint was objective response rate. Irradiation/radioablation of primary tumour or metastasis could be administered as per physician's choice. Biomarker evaluation was performed by tumour immunohistochemistry, whole exome and RNA sequencing, and immunophenotyping of peripheral blood by flow cytometry. RESULTS: Thirteen patients were treated with a median age of 15 years (range: 5.5-19.4). The main histologies were high-grade glioma, neuroblastoma, and desmoplastic small round cell tumour (DSRCT). The safety profile was similar to those of single-agent nivolumab, albeit haematologic toxicity, mainly lymphocytopenia, was commonly reported with the addition of cyclophosphamide +/- irradiation. Two patients with DSRCT and ependymoma presented unconfirmed partial response and prolonged disease stabilisation. Low mutational load with modest intratumour CD3+ T-cell infiltration and immunosuppressive tumour microenvironment were observed in the tumour samples. Under combined treatment, no positive modulation of circulating T cells was displayed, while derived neutrophil-to-lymphocyte ratio increased. CONCLUSIONS: Nivolumab in combination with cyclophosphamide was well tolerated but had limited activity in this paediatric setting. Metronomic cyclophosphamide did not modulate systemic immune response that could compensate limited T-cell infiltration and the immunosuppressive tumour microenvironment. CLINICALTRIALS. GOV IDENTIFIER: NCT2813135.