Biomarker Analysis of a Phase Ia/Ib Open-Label, Multicentre Study of Tiragolumab or Tiragolumab + Rituximab in Patients with Relapsed or Refractory (R/R) B-Cell Non-Hodgkin Lymphoma (NHL)

Background: NHL is the most common hematologic malignancy in adults, with follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) being the most common subtypes. Despite therapeutic advances, most patients will experience relapse. New treatments are therefore needed to improve the outcome of patients with R/R NHL. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a well-known immune inhibitory receptor expressed on the surface of activated T cell and natural killer (NK)-cell subsets. TIGIT is expressed at higher levels than other checkpoints in intratumoral T cells in NHL and is highly correlated with PD-1 expression and T cell infiltration. This phase Ia/Ib trial (NCT04045028) evaluated the safety and pharmacokinetics of the anti-TIGIT agent, tiragolumab, alone or in combination with rituximab. Methods: Patients were recruited with histologically confirmed B-cell NHL whose disease has relapsed or failed to respond to ≥2 prior systemic treatment regimens, had ECOG PS 0-1, adequate hematologic and end organ function, and no history of CNS lymphoma. Patients received tiragolumab 600 mg IV Q3W with or without rituximab 375 mg/m 2 IV for the initial dose and 1400 mg SC rituximab/23400 U rHuPH20 QW for 8 doses. Here, we evaluate biomarker data collected from patients with R/R NHL dosed with tiragolumab as a single agent or in combination with rituximab via flow cytometry and IHC. Results: At data cut-off (July 2021), biomarker data had been collected from 14 patients with NHL. Baseline CD8 T cell density within the tumor region evaluated via IHC for these patients was between 500-6000 per mmA 2. In the peripheral blood of the 7 patients dosed with the combination of tiragolumab and rituximab, CD8 T cell expansion observed via absolute counts by flow cytometry was seen in 2 patients. Among the 7 patients, NK/NKT CD25 expression remained unchanged and a modest increase in NK CD69 expression was sustained above baseline in 1 patient. Overall, transient NK cell activation via increased CD69 expression was observed in 2 patients, which would be expected from the addition of rituximab. Increased PD-L1 expression was observed on multiple lymphocyte subsets in 4 of 7 patients in this cohort. Of the 7 patients who received single agent tiragolumab, trends in increased CD69 expression on NKs were observed in 4 patients and NK/NKT CD25 expression in 3 patients. A modest CD8 T cell activation, via increased CD69 expression, was observed in 2 patients, though T cell counts remained unchanged. At baseline, TIGIT was abundantly expressed on peripheral blood CD8 T cells, while co-expression of exhaustion markers on CD8 T cells was less widely observed. Although one patient experienced a sustained response, no other patients achieved clinical benefit. This heavily pretreated 65-year-old female patient with FL had an objective partial response (best overall response), determined via Lugano criteria, with a response duration on single agent tiragolumab for 11 months. The patient had a two-fold upregulated CD69 expression on NKs and sixty-three-fold CD25 upregulated expression on NK/NKTs, as well as increased frequencies of PD-L1+ on immune cells over course of treatment. In this patient, relatively higher TIGIT and lower expression of exhaustion markers on CD8 T cells were observed at baseline and over treatment compared to other patients analyzed. Conclusions: In this study, tiragolumab as a single agent and in combination with rituximab was seen to result in increased PD-L1 expression on multiple lymphocyte subsets (including B cells, CD4/CD8 T cells, and NKs), which support the combination of tiragolumab with PD-L1/PD-1 inhibitors. Increases in NK/NKT CD25 expression could suggest a tiragolumab-mediated increase in proliferative potential but further investigations are needed to confirm. A patient with R/R FL in this study was observed to have the first documented objective response to single agent tiragolumab in this disease indication, suggesting biomarker-driven combination strategies may be important in this population. Disclosures Ruppert: Genentech, Inc.: Current Employment. Cuchelkar: Genentech, Inc.: Current Employment. Meng: Genentech, Inc.: Current Employment. Cho: Genentech, Inc.: Current Employment; F. Hoffmann La Roche, Ltd: Current holder of individual stocks in a privately-held company. Lear: Genentech, Inc.: Current Employment. Wong: Genentech, Inc.: Current Employment. Raval: Genentech, Inc.: Ended employment in the past 24 months; Arcus Bioscience: Current Employment, Current holder of individual stocks in a privately-held company. Nouet: F. Hoffmann La Roche, Ltd: Current Employment.

857 Selective Treg depletion in solid tumors with ALD2510, a novel humanized CD25-specific, IL-2 sparing monoclonal antibody

BackgroundRegulatory T cells (Tregs) inhibit immune responses in solid cancers using cell-cell contacts and anti-inflammatory cytokine release. Also, due to high and constitutive levels of IL2Ralpha chain (CD25) expression, Tumor infiltrating (TIL)-Tregs cells preferably consume local Interleukin-2 (IL2), thus depriving conventional T cells from IL2-induced activation and proliferation. Therefore, the selective depletion of TIL-Tregs using therapeutic antibodies targeting CD25 represents a promising strategy to unleash tumor-specific immune responses in solid cancers.MethodsCD25 expression was evaluated by flow and mass cytometry on T -cell subsets from tumor biopsies collected in patients with various solid cancers (Breast, Endometrial and Cervix). ALD2510 potency was demonstrated in vitro and in vivo in human CD25 Knock-In huGEMM (huCD25-KI) MC38-bearing mice and in CD34+ humanized NSG mice grafted with human cancer cell lines (MDA-MB-231 and HT29).ResultsIn tumor biopsies, CD25 is highly and homogeneously expressed by TIL-Tregs, while being much less expressed by only a fraction of conventional CD4+ T cells and barely expressed by TIL-CD8+ cells. This confirms CD25 as the most selective marker to target TIL-Tregs in cancer patients.In vitro, ALD2510 shows potent ADCC and ADCP as well as strong Treg depletion capacity. Importantly, CD8+ and CD4+ conventional T cells are not impacted by ALD2510 even after activation confirming ALD2510 ability to selectively deplete Tregs. Accordingly, ALD2510 neither blocks IL-2 binding to CD25 nor inhibits IL-2 induced proliferation of activated T cells. In CD34+-humanized mice, ALD2510 efficiently depletes human Tregs but spares conventional T cells. Also, in the MC38 model in huCD25-KI mice, ALD2510 shows a strong anti-tumor activity as a single agent with 60% overall tumor growth inhibition together with massive Treg depletion 7 days after a single administration. In addition, combination of ALD2510 with anti-PD1 leads to complete tumor regression and strong activation of conventional T cells. Importantly, Basiliximab, a CD25-specific IL-2 blocking antibody, although efficient at depleting Treg cells, did not impact tumor growth, thus demonstrating that the IL-2 sparing feature of ALD2510 is critical to elicit anti-tumour response in vivo.ConclusionsThis preclinical data package supports CD25 as a potent and selective Treg marker allowing Tregs depletion while sparing conventional T cells. In this context, ALD2510, a novel humanized CD25-specific and IL-2 sparing antibody presents all the required attributes for selective and efficient TIL-Tregs depletion, making it a promising drug candidate to treat a broad range of solid tumor patients.Ethics ApprovalThe studies involving human material were approved by the ethical committee “Comité de Protection des Personnes Sud Méditerranée » under approval numbers 1362 and 1048. All participants gave informed consent before taking part.

Central Nervous System-Infiltrating T Lymphocytes in Stroke Are Activated via Their TCR (T-Cell Receptor) but Lack CD25 Expression

Background and Purpose: T lymphocytes contribute to secondary brain damage after stroke. It has not been fully investigated whether this contribution is caused by antigen-specific or antigen-nonspecific activation of T lymphocytes. Lymphocytes from Nur77 GFP transgenic mice express a fluorescent protein upon activation via the TCR (T-cell receptor), allowing the differentiation of activation mode in a natural repertoire of immune cells and antigens. Methods: Middle cerebral artery occlusion or sham surgery was performed, and T-lymphocyte activation was analyzed by flow cytometry in the brain, spleen, and blood 16 hours, 2 days, 3 days, 4 days, and 7 days after surgery. Results: Ipsilateral hemispheric T-lymphocyte invasion peaked on day 4 poststroke. Here, we observed PD-1 (programmed cell death protein 1) expression on almost all invading T lymphocytes, while CD25 expression was low. CD25+, CD69+, or PD-1+ T lymphocytes predominantly displayed antigen-specific activation; the opposite was observed for T lymphocytes isolated from the blood. A mixed activation that favored antigen-specific activation was observed in the spleen. PD-1 was upregulated within the brain, whereas CD25 was not. Antigen-specific T lymphocytes home to the brain, while antigen- nonspecifically activated cells remain within the blood. Conclusions: Our data clearly demonstrate antigen-specific activation of T lymphocytes infiltrating ischemic brain lesions in stroke. The high expression of inhibitory PD-1 and low expression of CD25 on activated T lymphocytes in the brain most likely reflect immunosuppressive mechanisms.

Duplication of the IL2RA locus causes excessive IL-2 signaling and may predispose to very early onset colitis

Single genetic mutations predispose to very early onset inflammatory bowel disease (VEO-IBD). Here, we identify a de novo duplication of the 10p15.1 chromosomal region, including the IL2RA locus, in a 2-year-old girl with treatment-resistant pancolitis that was brought into remission by colectomy. Strikingly, after colectomy while the patient was in clinical remission and without medication, the peripheral blood CD4:CD8 ratio was constitutively high and CD25 expression was increased on circulating effector memory, Foxp3+, and Foxp3neg CD4+ T cells compared to healthy controls. This high CD25 expression increased IL-2 signaling, potentiating CD4+ T-cell-derived IFNγ secretion after T-cell receptor (TCR) stimulation. Restoring CD25 expression using the JAK1/3-inhibitor tofacitinib controlled TCR-induced IFNγ secretion in vitro. As diseased colonic tissue, but not the unaffected duodenum, contained mainly CD4+ T cells with a prominent IFNγ-signature, we hypothesize that local microbial stimulation may have initiated colonic disease. Overall, we identify that duplication of the IL2RA locus can associate with VEO-IBD and suggest that increased IL-2 signaling predisposes to colonic intestinal inflammation.

Synergistic Activation of Bovine CD4+ T Cells by Neutrophils and IL-12

CD4+ T cell activation requires inflammatory cytokines to provide a third signal (3SI), such as interleukin-12 (IL-12). We recently reported that bovine neutrophils can enhance the activation of bovine CD4+ T cells. To explore the interactions between neutrophils and third signal cytokines in bovine CD4+ T cell activation, naïve CD4+ T cells were isolated from cattle lymph nodes and stimulated for 3.5 days with anti-bovine CD3 (first signal; 1SI), anti-bovine CD28 (second signal; 2SI), and recombinant human IL-12 (3SI) in the presence or absence of neutrophils harvested from the same animals. Indeed, the strongest activation was achieved in the presence of all three signals, as demonstrated by CD25 upregulation, IFNγ production in CD4+ T cells, and secretion of IFNγ and IL-2 in cell supernatants. More importantly, 1SI plus neutrophils led to enhanced CD25 expression that was further increased by IL-12, suggesting synergistic action by IL-12 and neutrophils. Consistently, neutrophils significantly increased IFNγ production in 1SI plus IL-12-stimulated CD4+ T cells. Our data suggest the synergy of neutrophils and IL-12 as a novel regulator on bovine CD4+ T cell activation in addition to three signals. This knowledge could assist the development of immune interventions for the control of infectious diseases in cattle.

Peripheral Blood from Rheumatoid Arthritis Patients Shows Decreased Treg CD25 Expression and Reduced Frequency of Effector Treg Subpopulation

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by immune cell infiltration of the synovium, leading to the loss of cartilage, bone, and joint function. Although regulatory T (Treg) cells are thought to modulate the initiation and progression of RA, a consensus has yet to be reached regarding the function and composition of Treg cells in RA patients. To address these discrepancies, we analyzed not only the total Treg frequency but also that of Treg subpopulations in the peripheral blood of RA patients and healthy controls by flow cytometry. We found that the total Treg population was not significantly different between RA and control subjects. However, the effector Treg cell subgroup, defined as CD45RA−CD25hi, showed markedly decreased frequency in RA patients. In addition, the total Treg population from RA patients showed a significant decline in the expression of CD25. Both the naïve and effector Treg subgroups also showed marked reduction of CD25 expression in RA patients compared to controls. These data suggest that the decreased frequency of effector Treg cells and overall reduction of CD25 expression in Treg cells in the peripheral blood may be evidence of altered Treg homeostasis associated with RA pathogenesis.

Non-deletional CD8+ T cell self-tolerance permits responsiveness but limits tissue damage

Self-specific CD8+ T cells often escape clonal deletion, but the properties and capabilities of such cells in a physiological setting are unclear. We characterized polyclonal CD8+ T cells specific for the melanocyte antigen tyrosinase-related protein 2 (Trp2) in mice that express or lack this enzyme due to deficiency in Dct, which encodes Trp2. The size, phenotype, and gene expression profile of the pre-immune Trp2/Kb-specific pool were similar in wild-type (WT) and Dct-deficient (Dct-/-) mice. Despite comparable initial responses to Trp2 immunization, WT Trp2/Kb-specific cells showed blunted expansion, and scRNAseq revealed WT cells less readily differentiated into a CD25+ proliferative population. Functional self-tolerance clearly emerged when assessing immunopathology: adoptively transferred WT Trp2/Kb-specific cells mediated vitiligo much less efficiently. Hence, CD8+ T cell self-specificity is poorly predicted by precursor frequency, phenotype or even initial responsiveness, while deficient activation-induced CD25 expression and other gene expression characteristics may help to identify functionally tolerant cells.

Methanolic Extract of Teucrium Polium Exerts Immunomodulatory Properties in Human Peripheral Blood Mononuclear Cells

Teucrium polium has been used in traditional medicine around the world for centuries in treatment of various conditions and diseases. Many studies have confirmed pharmacological effects of its extracts, although the immunomodulatory effect has not been investigated. Therefore, the aim of our study was to examine the immunomodulatory effect of methanolic extract of T. polium (TPE) on peripheral blood mononuclear cells (PBMCs) derived from healthy donors and patients with HCV infection. We analyzed the effect of the extract on PBMCs viability using the MTT test. The cell death type was determined using Annexin V-FITC/7-AAD staining. Immunophenotyping using anti-CD8 FITC, anti-CD4 PE, anti-CD3 ECD, anti-CD20 PC5, anti-CD14 FITC and anti-CD25 PC7 was performed by flow cytometry. Results of the MTT test indicate that TPE stimulates proliferation of healthy PBMCs, while the HCV PBMCs viability was slightly reduced.The percentage of apoptotic HCV PBMCs was higher after TPE treatment compared to the control. The proportion of CD25-expressing cells was higher among the untreated HCV PBMCs than in the untreated healthy PBMCs. TPE treatment significantly and gradually increased CD25 expression in healthy PBMCs, whereas CD25 expression on HCV PBMCs increased only at the highest TPE concentration. The upregulation of double-positive CD3+CD25+, CD20+CD25+ and CD14+CD25+ cells was significant in TPE treated healthy PBMCs, while only the highest concentration was effective on HCV PBMCs. In summary, TPE exerts a strong immunomodulatory effect on healthy PBMCs and, only at the highest concentration, on HCV PBMNCs.