Lymphocyte Activation Gene-3 Protein Research Articles

Eftilagimod Alpha (a Soluble LAG-3 Protein) Combined With Pembrolizumab in Second-Line Metastatic NSCLC Refractory to Anti–Programmed Cell Death Protein 1/Programmed Death-Ligand 1-Based Therapy: Final Results from a Phase 2 Study

IntroductionEftilagimod alpha (efti), a soluble lymphocyte activation gene-3 protein, triggers antigen-presenting cell and T-cell (CD4+ and CD8+) activation and helps overcome resistance to programmed cell death protein 1 or programmed cell death-ligand 1 (PD-(L)1) inhibitors. We assessed efti plus pembrolizumab in second-line anti–PD-(L)1-refractory metastatic patients with NSCLC. MethodsAfter confirmed progression on anti-PD-(L)1-based first-line therapy, patients received efti (30 mg subcutaneously every 2 weeks for eight 3-week cycles and then every 3 weeks for up to 54 weeks) plus pembrolizumab (200 mg intravenously every 3 weeks for up to 105 weeks). The primary endpoint was the objective response rate by modified Response Evaluation Criteria in Solid Tumors version 1.1 for immune-based therapies. Secondary endpoints included disease control rate, progression-free survival, overall survival (OS), and tolerability. Exploratory endpoints included tumor growth kinetics and predefined subgroup analyses. Programmed cell death-ligand 1 tumor proportion score was assessed centrally. ResultsThirty-six patients were enrolled from April 2019 to August 2021 using Simon’s two-stage design. Most patients (81.8%) had low or negative (<50%) PD-(L)1 tumor proportion score. First-line therapy was anti–PD-(L)1-based for all patients, combined with chemotherapy for 66.7%. The confirmed objective response and disease control rates were 8.3% and 33.3%. The median progression-free survival was 2.1 months and the median OS was 9.9 months. Patients exhibiting high PD-(L)1 expression or acquired resistance to PD-(L)1 inhibitors revealed superior response and survival outcomes, and OS was closely correlated with disease control. No treatment-emergent adverse event led to permanent discontinuation of study treatment. ConclusionsEfti plus pembrolizumab was well-tolerated and revealed signs of antitumor activity in patients with NSCLC resistant to PD-(L)1 inhibitors, warranting further investigation. Trial registration number: NCT03625323.

In vitro vascularized immunocompetent patient-derived model to test cancer therapies

This work describes a patient-derived tumoroid model (PDTs) to support precision medicine in lung oncology. The use of human adipose tissue-derived microvasculature and patient-derived peripheral blood mononuclear cells (PBMCs) permits to achieve a physiologically relevant tumor microenvironment. This study involved ten patients at various stages of tumor progression. The vascularized, immune-infiltrated PDT model could be obtained within two weeks, matching the requirements of the therapeutic decision. Histological and transcriptomic analyses confirmed that the main features from the original tumor were reproduced. The 3D tumor model could be used to determine the dynamics of response to antiangiogenic therapy and platinum-based chemotherapy. Antiangiogenic therapy showed a significant decrease in vascular endothelial growth factor (VEGF)-A expression, reflecting its therapeutic effect in the model. In an immune-infiltrated PDT model, chemotherapy showed the ability to decrease the levels of lymphocyte activation gene-3 protein (LAG-3), B and T lymphocyte attenuator (BTLA), and inhibitory receptors of Tcells functions.

A soluble LAG-3 protein (eftilagimod alpha) and an anti-PD-L1 antibody (avelumab) tested in a phase I trial: a new combination in immuno-oncology

Eftilagimod alpha (efti) is a major histocompatibility complex class II agonist activating antigen-presenting cells which leads to greater systemic type 1 T helper response and more cytotoxic CD8+ T-cell activation. This phase I trial evaluated the administration of efti, a soluble lymphocyte activation gene-3 (LAG-3) protein, combined with the anti-programmed death-ligand 1 (PD-L1) antibody avelumab in advanced solid tumors. Patients with heavily pretreated metastatic solid tumors received intravenous avelumab (800 mg) combined with subcutaneously administered efti (6 or 30 mg) for up to 12 cycles, followed by avelumab monotherapy. The primary endpoint was the assessment of the recommended phase II dose (RP2D) of efti in combination with avelumab. Twelve patients with different tumor entities were enrolled (six patients in each cohort). During treatment, no dose-limiting toxicities occurred, and the severity of most adverse events was grade 1 or 2. In total, nine serious adverse events were documented, resulting in a fatal outcome in two cases, but none of them were assessed to be treatment related. Five patients (42%) achieved partial response. The median progression-free survival was 1.96 months and the median overall survival was not reached, with a 12-month survival rate of 75%. Subcutaneously administered efti plus avelumab was well tolerated, and efti of 30 mg was determined to be RP2D. The activity is promising and warrants further investigation in future phase II trials.

Abstract 6371: Identification of a lymphocyte activation gene-3-binding peptides using phage displayed-peptide libraries for cancer immunotherapy

Abstract Treatment with immune checkpoint blockades against CTLA-4, PD-1, and PD-L1 is currently giving improved results to many cancer patients. Nevertheless, a significant proportion of cancer patients treated with immune checkpoint blockades are still largely unsatisfied with the benefits of these drugs. One of the reasons is that cancer cells also use other immune checkpoints such as lymphocyte activation gene-3 (LAG-3), T cell immunoreceptor with Ig and ITIM domains, and T-cell immunoglobulin and mucin-domain containing-3. In this study, we screened phage displayed-peptide libraries for peptides that selectively bind to LAG-3. After five rounds of screening, we selected a candidate peptide and named it LAG3Pep. LAG3Pep preferentially bound to human LAG-3-transfected HEK 293T cells over mock-transfected cells and phorbol myristate acetate/ionomycin/chloroquine-stimulated Jurkat T cells over unstimulated cells. LAG3Pep also bound to splenocytes isolated from tumor-bearing mice, suggesting the reactivity across human and mouse LAG-3. Pull-down assay of human recombinant LAG-3 protein using biotin-labeled LAG3Pep yielded a protein band of LAG-3. LAG3Pep showed a high-affinity binding to the LAG-3 protein in SPR assays. Pre-treatment of a LAG-3-blocking antibody competed the binding of LAG3Pep to stimulated Jurkat T cells. LAG3Pep inhibited the binding of the LAG-3 protein to THP-1 cells expressing HLA-DR, a well-known LAG-3 ligand. In addition, treatment of T-cells with LAG3Pep in combination with a PD-L1-blocking antibody recovered the secretion of IL-2 by T-cells which was suppressed by FGL-1, another ligand of LAG-3, secreted from HepG2 tumor cells in co-culture. Intravenous administration of LAG3Pep in combination with a PD-L1-blocking peptide showed an anti-tumor growth activity in the MC38 syngeneic mouse colon tumor model. These results show that LAG3Pep acts as LAG-3 blockade and, in combination with a PD-L1-blockade, could be a promising tool for cancer immunotherapy. Citation Format: Seok-Min Lee, Byungheon Lee. Identification of a lymphocyte activation gene-3-binding peptides using phage displayed-peptide libraries for cancer immunotherapy. [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 6371.

Implications of LAG3 and CTLA4 immune checkpoints beyond PD-1/PD-L1 as a potential target in determining the prognosis of uveal melanoma patients

BackgroundResponse rate of PD-1/PD-L1 immunotherapeutic blockade agents in uveal melanoma (UM) is poor. Lymphocyte activation gene 3 (LAG3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) are the two promising immune checkpoint...

Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors

BackgroundNext-generation cancer immunotherapies are designed to broaden the therapeutic repertoire by targeting new immune checkpoints including lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Yet, the...

Prognostic values of stromal tumor-infiltrating lymphocytes and lymphocyte-activation gene-3 in advanced gastric adenocarcinoma

Objective: To analyze density of stromal tumor-infiltrating lymphocytes (sTIL) and expression of lymphocyte-activation gene-3 (LAG-3) protein in advanced gastric adenocarcinomas, and to investigate the correlation of sTIL and LAG-3 with the prognosis in patients with advanced gastric adenocarcinoma. Methods: The clinicopathological characteristics and follow-up data of 260 patients with advanced gastric adenocarcinoma were collected at Fujian Cancer Hospital, from January 2011 to December 2014. The percentage of sTILs was reported semi-quantitatively using histological section evaluation, the LAG-3 protein was detected using immunohistochemistry, and the expression was correlated with the clinicopathological features and patient outcomes. Results: Among the 260 cases, high density of sTIL was detected in 173 cases (66.5%) while LAG-3 high expression was observed in 160 cases (61.5%). These cases were divided into four groups. Group Ⅰ: 48 cases (18.5%) were sTIL low/LAG-3 low; group Ⅱ: 52 cases (20.0%) were sTIL high/LAG-3 low; group Ⅲ: 39 cases (15.0%) were sTIL low/LAG-3 high; group Ⅳ: 121 cases (46.5%) were sTIL high/LAG-3 high. Kaplan-Meier survival analyses showed that patient prognoses were related to age, tumor size, tumor location, Lauren classification, perineural invasion, vascular invasion, TNM staging, postoperative adjuvant chemotherapy and molecular classification (P<0.05). Meanwhile, higher densities of sTIL and higher expression of LAG-3 were associated with better prognosis. Multivariate survival analysis showed age, tumor size, Lauren classification and postoperative adjuvant chemotherapy were independent prognostic factors for patient survival. The results showed a poor prognosis in low-sTIL/low-LAG-3 patients. Conclusions: Compared with low density of sTIL and low expression of LAG-3, high density of sTIL and high expression of LAG-3 are associated with better outcomes in patients with advanced gastric adenocarcinoma, respectively. Combined detecton of sTIL and LAG-3 may be more useful in gastric cancer than using either alone. Age, tumor size, Lauren classification and postoperative adjuvant chemotherapy are independent prognostic factors for patients with advanced gastric adenocarcinoma.

LAG-3 as a Potent Target for Novel Anticancer Therapies of a Wide Range of Tumors.

LAG-3 (Lymphocyte activation gene 3) protein is a checkpoint receptor that interacts with LSEC-tin, Galectin-3 and FGL1. This interaction leads to reduced production of IL-2 and IFN-γ. LAG-3 is widely expressed in different tumor types and modulates the tumor microenvironment through immunosuppressive effects. Differential expression in various tumor types influences patient prognosis, which is often associated with coexpression with immune checkpoint inhibitors, such as TIM-3, PD-1 and CTLA-4. Here, we discuss expression profiles in different tumor types. To date, many clinical trials have been conducted using LAG-3 inhibitors, which can be divided into anti-LAG-3 monoclonal antibodies, anti-LAG-3 bispecifics and soluble LAG-3-Ig fusion proteins. LAG-3 inhibitors supress T-cell proliferation and activation by disallowing for the interaction between LAG-3 to MHC-II. The process enhances anti-tumor immune response. In this paper, we will review the current state of knowledge on the structure, function and expression of LAG-3 in various types of cancer, as well as its correlation with overall prognosis, involvement in cell-based therapies and experimental medicine. We will consider the role of compounds targeting LAG-3 in clinical trials both as monotherapy and in combination, which will provide data relating to the efficacy and safety of proposed drug candidates.

Analysis of the immune checkpoint lymphocyte activation gene-3 (LAG-3) in endometrial cancer: An emerging target for immunotherapy

BackgroundLymphocyte activation gene-3 (LAG-3) is a novel molecule that participates in the immune escape of tumor cells and is a target for immunotherapy. However, the expression of LAG-3 in patients with endometrial cancer (EC) has not been comprehensively characterized. ObjectivesWe elucidated the expression of LAG-3 and investigated its correlation with clinicopathological parameters, ProMisE subtypes, CD8+ T-cell infiltration and relapse-free survival (RFS) in a retrospective cohort of 421 patients with endometrial cancer. MethodsNext-generation sequencing of the polymerase epsilon (POLE) and immunohistochemistry of mismatch repair (MMR)-related protein (MLH1, PMS2, MSH2, and MSH6), p53, CD8 and LAG-3 protein in whole sections were performed. ResultsPositive LAG-3 was detected in tumor cells (TCs) and immune cells (ICs) in 31.6% (133/421) and 24.0% (101/421) of the patients, respectively. LAG-3 positivity in ICs was more common in high-grade, high-intermediate risk, high-risk, and advanced/metastatic subgroups and was relevant to lymphovascular space invasion, while that in TCs was more common in older individuals (≥54 years). LAG-3 expression was more prevalent in POLE ultramutated (POLEmut) and MMR-deficient (MMRd) EC than in p53-abnormal (p53abn) and p53-wild (p53wt) EC in TCs (34.4 % and 66.3% in POLEmut and MMRd versus 28.6% and 19.5% in p53abn and p53wt, P < 0.001) and ICs (78.1 % and 65.1% in POLEmut and MMRd versus 2.9% and 5.2% in p53abn and p53wt, P < 0.001). Positive expression of LAG-3 in TCs and ICs was associated with high levels of tumor-associated CD8+ T-cell immune infiltration. Additionally, LAG-3 positivity in TCs was related to improved RFS. ConclusionsThis study suggests that immunotherapy targeting LAG-3 may play a role in EC patients with POLEmut or MMRd molecular markers. Positive LAG-3 expression in TCs may be a predictor of improved RFS.

Abstract 599: Identification of lymphocyte activation gene 3 binding peptides using phage displayed peptide libraries for cancer immunotherapy

Abstract Treatment with immune checkpoint blockades against cytotoxic T lymphocyte associated protein-4, programmed cell death-1, and programmed death-ligand 1 is currently giving improved results to many cancer patients. Nevertheless, a significant proportion of cancer patients treated with immune checkpoint blockades are still largely unsatisfied with the benefits of these drugs. One of the reasons is that cancer cells also use other immune checkpoints such as lymphocyte activation gene-3 (LAG-3), T cell immunoreceptor with Ig and ITIM domains, and T-cell immunoglobulin and mucin-domain containing-3. LAG-3 negatively regulates activated T cells. In this study, we screened phage displayed-peptide libraries for peptides that selectively bind to LAG-3. After five rounds of screening, we selected a candidate peptide and named it LAG3Pep. LAG3Pep preferentially bound to human LAG-3-transfected HEK 293T cells over mock-transfected cells and phorbol myristate acetate/ionomycin/chloroquine-stimulated Jurkat T cells over unstimulated cells. LAG3Pep also bound to splenocytes isolated from tumor-bearing mice, suggesting the reactivity across human and mouse LAG-3. Pull-down of Jurkat T cell lysates using biotin-labeled LAG3Pep yielded a protein band of LAG-3. A competition by the pre-treatment of Jurkat T cells with a LAG-3-blocking antibody inhibited the cell binding of LAG3Pep. In addition, LAG3Pep inhibited the binding of LAG-3 protein to THP-1 cells expressing HLA-DR, a well-known LAG-3 ligand. Moreover, LAG3Pep recovered IL2 production by stimulated Jurkat T cells, which were suppressed by fibrinogen-like protein 1, another ligand of LAG-3, in the culture medium of HepG2 tumor cells. These results show that LAG3Pep selectively binds to LAG-3 and has a potential as a LAG-3 blocker for cancer immunotherapy. Citation Format: Seok-Min Lee, Byungheon Lee. Identification of lymphocyte activation gene 3 binding peptides using phage displayed peptide libraries for cancer immunotherapy [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 599.

Lymphocyte activation gene-3-associated protein networks are associated with HDL-cholesterol and mortality in the Trans-omics for Precision Medicine program

Deficiency of the immune checkpoint lymphocyte activation gene-3 (LAG3) protein is significantly associated with both elevated HDL-cholesterol (HDL-C) and myocardial infarction risk. We determined the association of genetic variants within ±500 kb of LAG3 with plasma LAG3 and defined LAG3-associated plasma proteins with HDL-C and clinical outcomes. Whole genome sequencing and plasma proteomics were obtained from the Multi-Ethnic Study of Atherosclerosis (MESA) and the Framingham Heart Study (FHS) cohorts as part of the Trans-Omics for Precision Medicine program. In situ Hi-C chromatin capture was performed in EBV-transformed cell lines isolated from four MESA participants. Genetic association analyses were performed in MESA using multivariate regression models, with validation in FHS. A LAG3-associated protein network was tested for association with HDL-C, coronary heart disease, and all-cause mortality. We identify an association between the LAG3 rs3782735 variant and plasma LAG3 protein. Proteomics analysis reveals 183 proteins significantly associated with LAG3 with four proteins associated with HDL-C. Four proteins discovered for association with all-cause mortality in FHS shows nominal associations in MESA. Chromatin capture analysis reveals significant cis interactions between LAG3 and C1S, LRIG3, TNFRSF1A, and trans interactions between LAG3 and B2M. A LAG3-associated protein network has significant associations with HDL-C and mortality.

Fewer LAG-3+ T Cells in Relapsing-Remitting Multiple Sclerosis and Type 1 Diabetes.

The coinhibitory receptor lymphocyte activation gene 3 (LAG-3) is an immune checkpoint molecule that negatively regulates T cell activation, proliferation, and homeostasis. Blockade or deletion of LAG-3 in autoimmune-prone backgrounds or induced-disease models has been shown to exacerbate disease. We observed significantly fewer LAG-3+ CD4 and CD8 T cells from subjects with relapsing-remitting multiple sclerosis (RRMS) and type 1 diabetes. Low LAG-3 protein expression was linked to alterations in mRNA expression and not cell surface cleavage. Functional studies inhibiting LAG-3 suggest that in subjects with RRMS, LAG-3 retains its ability to suppress T cell proliferation. However, LAG-3 expression was associated with the expression of markers of apoptosis, indicating a role for low LAG-3 in T cell resistance to cell death. In T cells from subjects with RRMS, we observed a global dysregulation of LAG-3 expression stemming from decreased transcription and persisting after T cell stimulation. These findings further support the potential clinical benefits of a LAG-3 agonist in the treatment of human autoimmunity.

Eftilagimod alpha, a soluble lymphocyte activation gene-3 (LAG-3) protein plus pembrolizumab in patients with metastatic melanoma

BackgroundTo evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of eftilagimod alpha (efti), a soluble lymphocyte activation gene-3 protein, in combination with the programmed cell death-1 (PD-1) antagonist pembrolizumab.MethodsThe study was...

499P - Identification of first-in-class, naturally occurring LAG3 checkpoint inhibitor

BackgroundDespite the impressive impact of CTLA4 and PD-1/L1 cancer immunotherapy, a large proportion of patients with many tumor types fail to respond. Lymphocyte activation gene 3 (LAG3) is the third checkpoint receptor that plays an important role in the pathogenesis of cancer. We have previously described a novel methodology in the identification of therapeutic antibodies (US Patent #9810694). Here we report the discovery of first-in-class, naturally occurring LAG3 checkpoint inhibitor in cancer patients. MethodsAnti-LAG3 antibody presence was evaluated in 35 individuals: 11 healthy donors (controls) and 24 cancer patients in 3 different laboratories blinded to clinical information. Surface plasmon resonance analysis was done using the optical biosensor Biacore T200 where LAG3 protein was covalently immobilized on the optical chip and biosensor signals from human serum samples were analyzed. Western Blotting used recombinant LAG3 loaded on a 10% SDS PAGE followed by blotting with plasma samples followed by biotinylated anti-human Ig and IrDye 800 streptavidin. Plasma anti-LAG3 IP utilized recombinant LAG3 crosslinked to MagResyn Carboxyl beads followed by incubation with plasma, followed by a biotinylated anti-human Ig and IrDye streptavidin. Affinity purification protocol with elution of antigen-specific antibody with pH gradient was developed. REmAb™ Protein Sequencing with WILD™ analysis (Rapid Novor) is performed. ResultsNo anti-LAG3 antibodies were detected in the control group. Among three assays there was a complete correlation for presence of anti-LAG3 antibody in 5 patients. Three of these patients had sufficient plasma quantity and anti-LAG3 concentration (21 – 33 µg/ml) to allow for the antibody purification. Immunoglobulin isotypes were IgG and IgM. The ELISA results of purified antibody samples showed a high quantity of LAG3-specific antibody (0.22mg, 0.52mg, 0.3mg in each sample, respectively). The results of the characterization of novel LAG3 checkpoint inhibitor will be presented at the congress. ConclusionsTo our knowledge, this is the first report of checkpoint antibody presence in humans. Further study with cloning and evaluation of therapeutic properties of novel anti-LAG3 antibody in xenograft models will be performed. Legal entity responsible for the studyThe authors. FundingILGEN Inc. DisclosureI. Tsimafeyeu: Research grant / Funding (self), Shareholder / Stockholder / Stock options, Full / Part-time employment: ILGEN Inc. G. Bratslavsky: Shareholder / Stockholder / Stock options, Officer / Board of Directors: ILGEN Inc.

A signal-decreased electrochemical immunosensor for the sensitive detection of LAG-3 protein based on a hollow nanobox-MOFs/AuPt alloy

In this work, hollow nanobox metal-organic framework (HNM) nanocomposites were synthesised and utilised for the first time in a signal decreased electrochemical immunosensor for the ultrasensitive quantitative determination of lymphocyte activation gene-3 (LAG-3) protein, which is a newly discovered biomarker. With the aid of signal materials, namely, SiO2-tagged anti-LAG-3 antibody (SiO2-Ab2) and the biotin-streptavidin system, the sensor can achieve signal amplification. Encapsulation of tin dioxide-functionalised reduced graphene oxide (rGO-SnO2) and gold and platinum alloys (AuPt alloys) onto the surface of hollow nanobox metal-organic frameworks (MOFs) was performed to prepare rGO-SnO2/hollow nanobox-MOFs/AuPt alloys (rGO-SnO2/HNMs/AuPt) as the matrix. SiO2-Ab2, which is used as the signal-decreased label, can be utilised to enhance the distinction of the electrochemical signal after the specific recognition between antibodies and antigens, owing to its large steric hindrance property. In this sensor, this proposed sandwich immunosensor can achieve a high sensitivity, especially in the presence of low concentrations of the LAG-3 protein. Under optimal conditions, this sandwich-designed immunosensor exhibited a sensitive detection of the LAG-3 protein from concentrations of 0.01 ng mL−1 to 1 μg mL−1, with a lower detection limit of 1.1 pg mL−1 (based on 3σ). We proposed that this ultrasensitive biosensor can be utilised for the detection of the LAG-3 protein in early clinical tumour diagnosis.

Research progress on the mechanism of co-inhibition of lymphocyte activation gene-3 in choronic infection and tumor immunity

Background Sepsis means a host create a Specific reaction to infections which leads to life-threatening organ dysfunctions. In case of chronic infection and cancer, T cells occur dysfunction as a result of prolonged antigen exposure, and have a reduced ability to clear pathogens or to eliminate cancer cells. Insights have recently been gained into the role of receptors that negatively regulate T cells which promote T cell dysfunction. Lymphocyte activation gene-3 protein (LAG3, also known as CD223), a inhibitory receptor, come into notice. Objective To evaluate the therapeutic effects of targeting the LAG3 in the situations such as chronic infection and cancer. Content The paper focuses on the Effects on LAG3 on the effector T cell, immune cells, and soluble LAG3, and summarize from clinical trails and discuss the potential mechanism of immune cells. Trend In the tumor environment, it is very useful in clinic application that LAG3 can negative regulate T cells. LAG3 may also have multiple roles in immune regulation. In the studies of the oncotherapy, how to make using of the immune functions of LAG3 can suppress the proliferation of tumor cells, and take the effect of LAG3 blockage in patients in the future. Key words: Lymphocyte activation gene-3; T cells exhaustion; Co-inhibitory factor

Lymphocyte activation gene 3 and coronary artery disease.

BACKGROUND: The lipoprotein scavenger receptor BI (SCARB1) rs10846744 noncoding variant is significantly associated with atherosclerotic disease independently of traditional cardiovascular risk factors. We identified a potentially novel connection between rs10846744, the immune checkpoint inhibitor lymphocyte activation gene 3 (LAG3), and atherosclerosis. METHODS: In vitro approaches included flow cytometry, lipid raft isolation, phosphosignaling, cytokine measurements, and overexpressing and silencing LAG3 protein. Fasting plasma LAG3 protein was measured in hyperalphalipoproteinemic (HALP) and Multi-Ethnic Study of Atherosclerosis (MESA) participants. RESULTS: In comparison with rs10846744 reference (GG homozygous) cells, LAG3 protein levels by flow cytometry (P < 0.001), in lipid rafts stimulated and unstimulated (P = 0.03), and phosphosignaling downstream of B cell receptor engagement of CD79A (P = 0.04), CD19 (P = 0.04), and LYN (P = 0.001) were lower in rs10846744 risk (CC homozygous) cells. Overexpressing LAG3 protein in risk cells and silencing LAG3 in reference cells confirmed its importance in phosphosignaling. Secretion of TNF-α was higher (P = 0.04) and IL-10 was lower (P = 0.04) in risk cells. Plasma LAG3 levels were lower in HALP carriers of the CC allele (P < 0.0001) and by race (P = 0.004). In MESA, race (P = 0.0005), age (P = 0.003), lipid medications (P = 0.03), smoking history (P < 0.0001), and rs10846744 genotype (P = 0.002) were independent predictors of plasma LAG3. In multivariable regression models, plasma LAG3 was significantly associated with HDL-cholesterol (HDL-C) (P = 0.007), plasma IL-10 (P < 0.0001), and provided additional predictive value above the Framingham risk score (P = 0.04). In MESA, when stratified by high HDL-C, plasma LAG3 was associated with coronary heart disease (CHD) (odds ratio 1.45, P = 0.004). CONCLUSION: Plasma LAG3 is a potentially novel independent predictor of HDL-C levels and CHD risk. FUNDING: This work was supported by an NIH RO1 grant (HL075646), the endowed Linda and David Roth Chair for Cardiovascular Research, and the Harold S. Geneen Charitable Trust Coronary Heart Disease Research award to Annabelle Rodriguez. MESA is conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with MESA investigators. Support for MESA is provided by contracts HHSN268201500003I, N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169, UL1-TR-001079, UL1-TR-000040, and DK063491. Cardiometabochip genotyping data for the MESA samples was supported in part by grants and contracts R01HL98077, N02-HL-64278, HL071205, UL1TR000124, DK063491, RD831697, and P50 ES015915.

LAG3 and PD1 co-inhibitory molecules collaborate to limit CD8+ T cell signaling and dampen antitumor immunity in a murine ovarian cancer model.

The immune co-inhibitory receptors lymphocyte activation gene-3 (LAG3) and programmed cell death 1 (PD1) synergistically contribute to autoimmunity and tumor evasion. Here we demonstrate how they collaborate and interact to regulate T cell function. We first show that LAG3 and PD1 are co-expressed on both OVA-specific and non-specific T cells infiltrating murine ovarian tumors. Dual antibody blockade or genetic knockout of LAG3 and PD1 significantly enhanced T effector function and delayed tumor growth. LAG3 and PD1 co-localized in activated CD8+ T cells in vitro at the trans-Golgi vesicles, early/recycling endosomal compartments, lysosomes, and microtubule organizing center. Importantly, LAG3 and PD1 cluster with pLck at the immunological synapse. Reciprocal immunoprecipitation of T cell extracts revealed physical interaction between LAG3 and PD1. Mutational analyses indicate that the cytoplasmic domain of LAG3 is not absolutely required for its association with PD1, while the ITIM and ITSM of PD1 are necessary for its association with LAG3. Finally, LAG3 protein also associates with the Src-homology-2 domain-containing phosphatases (SHP1/2) which are known to be recruited by PD1 during T cell signaling. Our data indicate that the association of LAG3 with PD1 contributes to their rapid trafficking to the immunological synapse, leading to a synergistic inhibitory effect on T cell signaling.

Abstract 4075: LAG3 and PD1 co-inhibitory molecules directly interact to limit CD8+ T cell signaling

Abstract Increasing evidence indicate that dual blockade of Lymphocyte Activation Gene-3 (LAG3) and programmed cell death 1 (PD1) synergistically enhances autoimmunity and anti-tumor immunity. Despite the fact that these co-inhibitory receptors individually attenuate T cell receptor signaling, it is not clear whether they interact at the physical, biochemical and/or molecular levels, to regulate the functional fate of T cells. To answer these questions, we first simulated possible direct interaction of LAG3 and PD1 using in-silico molecular docking techniques. Using confocal microscopy and ImageStream analyses, we demonstrated that LAG3 and PD1 molecules co-localized in a subset of activated murine CD8+ T cells. The co-localization occurred at the trans-Golgi vesicles, early/recycling endosomal compartments, lysosomes, and microtubule organizing center (MTOC). Importantly, LAG3 and PD1 cluster with pLck at the immunological synapse of CD8+ T cell and dendritic cell conjugates. Reciprocal immunoprecipitation of the T cell extracts with specific antibodies for LAG3 and PD1 revealed a physical association between LAG3 and PD1 and this interaction was absent in T cells isolated from LAG3 and PD1 knockout mice. Finally, LAG3 protein was also associated with Src family tyrosine phosphatase-2 (SHP2) that is recruited by PD1 during T cell signaling. The recruitment of phosphatases is additively reduced in CD8+ T cells deficient in both LAG3 and PD1. Taken together, our data suggest that LAG3 interacts and collaborates with PD1, following T cell activation, for rapid translocation from early/recycling endosomal compartments to the immunological synapse, leading to a synergistic inhibitory effect on T cell signaling. Citation Format: Ruea-Yea Huang, Cheryl Eppolito, Ahmed Fadiel, Adel Hamza, Shashikant Lele, Junko Mastuzaki, Kunle Odunsi. LAG3 and PD1 co-inhibitory molecules directly interact to limit CD8+ T cell signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2014-4075

Molecular cloning of bovine lymphocyte activation gene-3 and its expression characteristics in bovine leukemia virus-infected cattle

Lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II binding CD4 homologue has recently been shown as one of the mechanisms for down-regulating immune responses during chronic disease progression. For the first time, we cloned LAG-3 from two breeds of cattle (Holstein and Japanese Black), and analyzed its expression levels in cattle infected with bovine leukemia virus (BLV), a chronic viral infection that leads to immuno-suppression. The cloned cDNA of bovine LAG-3 have an open reading frame of 1551 nucleotides, encoding a polypeptide of 515 amino acids in length. Similar to the swine LAG-3, the bovine LAG-3 protein sequence consisted of four extracellular domains, a transmembrane domain and an inhibitory motif, KTGELE. We found that the bovine LAG-3 mRNA transcripts were expressed predominantly on T-cells such as CD4 + and CD8 + cells, among peripheral blood mononuclear cells. In subsequent expression analysis, LAG-3 mRNA expression on CD4 + T-cells from BLV-infected cattle was upregulated compared to that in normal cattle. Comparable results were obtained with CD8 + T-cells from cattle infected with BLV. We further observed strong upregualtion of MHC class II molecule, the ligand for LAG-3 in BLV-infected cattle. These findings indicate an important role for inhibitory receptor molecules such as LAG-3 in chronic bovine infections and future studies will elucidate the specific role of LAG-3 in bovine diseases.