Lymphocyte Activation Gene-3 Research Articles

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.

Tumor microenvironment signaling and therapeutics in cancer progression.

Tumor development and metastasis are facilitated by the complex interactions between cancer cells and their microenvironment, which comprises stromal cells and extracellular matrix (ECM) components, among other factors. Stromal cells can adopt new phenotypes to promote tumor cell invasion. A deep understanding of the signaling pathways involved in cell-to-cell and cell-to-ECM interactions is needed to design effective intervention strategies that might interrupt these interactions. In this review, we describe the tumor microenvironment (TME) components and associated therapeutics. We discuss the clinical advances in the prevalent and newly discovered signaling pathways in the TME, the immune checkpoints and immunosuppressive chemokines, and currently used inhibitors targeting these pathways. These include both intrinsic and non-autonomous tumor cell signaling pathways in the TME: protein kinase C (PKC) signaling, Notch, and transforming growth factor (TGF-β) signaling, Endoplasmic Reticulum (ER) stress response, lactate signaling, Metabolic reprogramming, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and Siglec signaling pathways. We also discuss the recent advances in Programmed Cell Death Protein 1 (PD-1), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4), T-cell immunoglobulin mucin-3 (TIM-3) and Lymphocyte Activating Gene 3 (LAG3) immune checkpoint inhibitors along with the C-C chemokine receptor 4 (CCR4)- C-C class chemokines 22 (CCL22)/ and 17 (CCL17), C-C chemokine receptor type 2 (CCR2)- chemokine (C-C motif) ligand 2 (CCL2), C-C chemokine receptor type 5 (CCR5)- chemokine (C-C motif) ligand 3 (CCL3) chemokine signaling axis in the TME. In addition, this review provides a holistic understanding of the TME as we discuss the three-dimensional and microfluidic models of the TME, which are believed to recapitulate the original characteristics of the patient tumor and hence may be used as a platform to study new mechanisms and screen for various anti-cancer therapies. We further discuss the systemic influences of gut microbiota in TME reprogramming and treatment response. Overall, this review provides a comprehensive analysis of the diverse and most critical signaling pathways in the TME, highlighting the associated newest and critical preclinical and clinical studies along with their underlying biology. We highlight the importance of the most recent technologies of microfluidics and lab-on-chip models for TME research and also present an overview of extrinsic factors, such as the inhabitant human microbiome, which have the potential to modulate TME biology and drug responses.

LAG3+ erythroid progenitor cells inhibit HBsAg seroclearance during finite pegylated interferon treatment through LAG3 and TGF-β

HBsAg seroclearance, the ideal aim of anti-hepatitis B virus (HBV) treatment, cannot be achieved easily. Anemia is another common issue for chronic hepatitis B (CHB) patients, which leads to elevation of erythroid progenitor cells (EPCs) and immune suppression in cancer. This study investigated the role of EPCs in HBsAg seroclearance following pegylated interferon-α (PEG-IFN) treatment. CD45+EPC accumulation in CHB patients and an AAV/HBV mice model was found in the circulation and liver by flow cytometry and immunofluorescence tests. Wright-Giemsa staining showed that these pathological CD45+EPCs presented elevated erythroid cells with relative immature morphologies and atypical cells compared with the control cells. CD45+EPCs were associated with immune tolerance and decreased HBsAg seroclearance during finite PEG-IFN treatment. CD45+EPCs suppressed antigen non-specific T cell activation and HBV-specific CD8+T cells, partially through transforming growth factor β (TGF-β). RNA-seq revealed that CD45+EPCs in patients with CHB presented a distinct gene expression profile compared with CD45−EPCs and CD45+EPCs from cord blood. Notably, CD45+EPCs from patients with CHB expressed high level of Lymphocyte-activation gene 3 (LAG3), an immune checkpoint molecule, and were then defined as LAG3+EPCs. LAG3+EPCs diminished the function of antigen presenting cells through LAG3, which was another mechanism by which LAG3+EPCs’ suppressed HBV-specific CD8+T cells. Anti-LAG3 and anti-TGF-β combination treatment decreased serum HBeAg, HBV DNA levels and HBsAg level, as well as HBsAg-expression in hepatocytes during PEG-IFN treatment in the AAV/HBV mice model. Conclusions: LAG3+EPCs inhibited the efficacy of PEG-IFN treatment on HBsAg seroclearance induced by LAG3 and TGF-β. Anti-LAG3, anti-TGF-β and PEG-IFN combination treatment might facilitate HBV clearance.

MP56-05 LAG-3/FGL1 AXIS PREDICTS RESPONSE TO IMMUNE CHECKPOINT INHIBITORS IN ADVANCED UROTHELIAL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023MP56-05 LAG-3/FGL1 AXIS PREDICTS RESPONSE TO IMMUNE CHECKPOINT INHIBITORS IN ADVANCED UROTHELIAL CARCINOMA Takashi Yoshida, Takahiro Nakamoto, Chisato Ohe, Junichi Ikeda, Yoshiki Yasukochi, Naho Atsumi, Ryoichi Saito, Koji Tsuta, and Hidefumi Kinoshita Takashi YoshidaTakashi Yoshida More articles by this author , Takahiro NakamotoTakahiro Nakamoto More articles by this author , Chisato OheChisato Ohe More articles by this author , Junichi IkedaJunichi Ikeda More articles by this author , Yoshiki YasukochiYoshiki Yasukochi More articles by this author , Naho AtsumiNaho Atsumi More articles by this author , Ryoichi SaitoRyoichi Saito More articles by this author , Koji TsutaKoji Tsuta More articles by this author , and Hidefumi KinoshitaHidefumi Kinoshita More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003309.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: PD-1/PDL-1 blockade is standard therapy in advanced urothelial carcinoma (UC), although few cases respond to this therapy. Lymphocyte activation gene-3 (LAG-3), T-cell immunoglobulin and mucin domain-containing-3 (i.e., TIM-3), and T cell immunoreceptor with Ig and ITIM domains (i.e., TIGIT) are major, co-expressed immune checkpoint receptors associated with tumor immune escape. However, the correlation of these receptors and their ligands with immunotherapeutic sensitivity remains unclear in UC. Therefore, we conducted this study to clarify how these checkpoint molecules are associated with response to immunotherapy. METHODS: We retrospectively analyzed a total of 851 patients from the public database and Kansai Medical University database. Clinical and molecular analyses and multiplex immunohistochemistry were performed to characterize immune cell infiltration in UC. RESULTS: Among the three checkpoint receptors, only LAG-3 was associated with the responsiveness of PD-1/PDL-1 blockade. In tumors with high LAG-3 levels, the increased expression of fibrinogen-like protein-1 (FGL-1), not for other LAG-3 ligands, such as major histocompatibility complex II and galectin-3, showed a significantly negative impact on response to anti-PD1/PDL-1 therapy and overall survival. Moreover, although the tumor infiltration of CD8+ T-cells was remarkable regardless of FGL-1 expression, high FGL-1 mRNA levels were linked to the upregulation of CD39 and neuropilin-1, as well as an increase in CD4+ regulatory T-cells and M2-like macrophage gene signatures, which may indicate CD8+ T-cell exhaustion. Further, enrichment analyses suggested that high LAG-3-FGL-1 co-expression was significantly correlated with the activation of epithelial-mesenchymal transition, transforming growth factor-beta, and angiogenesis signaling pathways. CONCLUSIONS: This study indicates that not only high LAG-3 expression but also co-expression of FGL-1 are crucial factors for predicting adverse oncological outcomes, associated with the presence of immunosuppressive contextures in tumors in cases of PD-1/PDL-1 blockade. Emerging combination therapy of anti-LAG3 and PD-1 blockade could be promising management for such UC patients. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e775 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Yoshida More articles by this author Takahiro Nakamoto More articles by this author Chisato Ohe More articles by this author Junichi Ikeda More articles by this author Yoshiki Yasukochi More articles by this author Naho Atsumi More articles by this author Ryoichi Saito More articles by this author Koji Tsuta More articles by this author Hidefumi Kinoshita More articles by this author Expand All Advertisement PDF downloadLoading ...

Clinical Significance and Prognostic Value of the Expression of LAG-3 and FGL1 in Esophageal Squamous Cell Carcinoma.

In this retrospective study, we analyzed the expression of lymphocyte activating gene 3 (LAG-3) and fibrinogen-like protein 1 (FGP1) mRNA and the corresponding proteins in 78 patients with esophageal squamous cell carcinoma (ESCC) to evaluate the clinical significance and prognostic value. mRNA and protein expression were analyzed by reverse transcription PCR and Western blotting, respectively. The expression of LAG-3 and FGL1 mRNA and the corresponding proteins in tumor tissues were significantly increased in comparison with the normal esophageal mucosa. The overexpression of LAG-3 significantly correlated with the content of tumor-infiltrating lymphocytes (TILs), tumor differentiation, and TNM stage. The overexpression of FGL1 also significantly correlated with TILs, TNM stage, and lymph node metastasis. Kaplan-Meier survival analysis showed that tumor diameter, TNM stage, lymph node metastasis, LAG-3 and FGL1 protein expression were related to the progression-free survival (p<0.05). Multivariate Cox regression showed that the level of FGL1 and TNM stage were independent prognostic factors of progression-free survival. We speculated that the tumor microenvironment of ESCC induces immunosuppression due to up-regulated expression of LAG-3 and FGL1 in the tumor tissues, which promotes tumor growth.

MP22-14 LAG3 EXPRESSION ASSOCIATED WITH SURVIVAL IN PATIENTS WITH BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP22-14 LAG3 EXPRESSION ASSOCIATED WITH SURVIVAL IN PATIENTS WITH BLADDER CANCER Tamir Sholklapper, Laura Bukavina, Andres Correa, Alexander Kutikov, and Philip Abbosh Tamir SholklapperTamir Sholklapper More articles by this author , Laura BukavinaLaura Bukavina More articles by this author , Andres CorreaAndres Correa More articles by this author , Alexander KutikovAlexander Kutikov More articles by this author , and Philip AbboshPhilip Abbosh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003247.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Expression of lymphocyte activation gene-3 (LAG3), an immune cell transmembrane protein with a regulatory impact on tumor microenvironments, has been identified as a promising checkpoint for emerging immunotherapies. We sought to evaluate the impact of LAG3 expression on bladder cancer survival and the immune regulatory environment. (Figure 1A) METHODS: RNA seq data for bladder cancer was obtained in TCGA from TCGA Data Portal (https://tcga-data.nci.nih.gov/docs/publications/tcga/). Wilcoxon rank-sum test was used for all pairwise comparisons. Spearman Rho was used for all correlations, and P-values were calculated using either the exact permutation distributions (for small sample sizes) or large-sample approximations. All P-values are from two-sided tests, and P < .05 was used as the threshold for statistical significance. The Kaplan-Meier method and log-rank test were used to evaluate overall survival and compare inter-group survival, respectively. Sub-analyses were performed to compare gene enrichment values (GEV) among various immunologic cell types and immune markers with known associations with oncologic outcomes. RESULTS: We identified 424 patients with LAG3 expression and survival data in the TCGA BLCA cohort. Patients with low LAG3 expression had a statistically significant higher OS compared to patients with MIBC and lower expression (p=0.0006). When stratified by gender and age, receipt of chemotherapy, high LAG3 expression remainder significant prognostic factors for OS (p=0.023) (Figure 8B). As LAG-3 expression across immune cells, we compared LAG-3 expression across immune cell subtypes. High LAG-3 expression in muscle invasive bladder cancer (MIBC) decreased infiltration of CD8+/M2b macrophages/Th17/Tregs and B cells (p<0.001). Likewise, comparison of high and low LAG-3 expression demonstrated increased Treg expression, increased CD8+ T cell with enhanced PD1 expression, reflecting marked immunoevasive environment with CD8+ T cell dysfunction. CONCLUSIONS: Our study reported LAG-3+ cells abundance was an independent predictor for poor OS in MIBC patients within TCGA cohort. Blockade with LAG-3 inhibitors in bladder cancer is critical to explore as potential novel adjunctive therapy. Source of Funding: NA © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e302 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tamir Sholklapper More articles by this author Laura Bukavina More articles by this author Andres Correa More articles by this author Alexander Kutikov More articles by this author Philip Abbosh More articles by this author Expand All Advertisement PDF downloadLoading ...

Soluble immune checkpoints as correlates for HIV persistence and T cell function in people with HIV on antiretroviral therapy.

In people with HIV (PWH) both off and on antiretroviral therapy (ART), the expression of immune checkpoint (IC) proteins is elevated on the surface of total and HIV-specific T-cells, indicating T-cell exhaustion. Soluble IC proteins and their ligands can also be detected in plasma, but have not been systematically examined in PWH. Since T-cell exhaustion is associated with HIV persistence on ART, we aimed to determine if soluble IC proteins and their ligands also correlated with the size of the HIV reservoir and HIV-specific T-cell function. We used multiplex bead-based immunoassay to quantify soluble programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain and mucin domain 3 (TIM-3), PD-1 Ligand 1 (PD-L1) and PD-1 Ligand 2 (PD-L2) in plasma from PWH off ART (n=20), on suppressive ART (n=75) and uninfected controls (n=20). We also quantified expression of membrane-bound IC and frequencies of functional T-cells to Gag and Nef peptide stimulation on CD4+ and CD8+ T-cells using flow cytometry. The HIV reservoir was quantified in circulating CD4+ T-cells using qPCR for total and integrated HIV DNA, cell-associated unspliced HIV RNA and 2LTR circles. Soluble (s) PD-L2 level was higher in PWH off and on ART compared to uninfected controls. Higher levels of sPD-L2 correlated with lower levels of HIV total DNA and higher frequencies of gag-specific CD8+ T-cells expressing CD107a, IFNγ or TNFα. In contrast, the concentration of sLAG-3 was similar in uninfected individuals and PWH on ART, but was significantly elevated in PWH off ART. Higher levels of sLAG-3 correlated with higher levels of HIV total and integrated DNA, and lower frequency of gag-specific CD4+ T cells expressing CD107a. Similar to sLAG-3, levels of sPD-1 were elevated in PWH off ART and normalized in PWH on ART. sPD-1 was positively correlated with the frequency of gag-specific CD4+ T cells expressing TNF-a and the expression of membrane-bound PD-1 on total CD8+ T-cells in PWH on ART. Plasma soluble IC proteins and their ligands correlate with markers of the HIV reservoir and HIV-specific T-cell function and should be investigated further in in large population-based studies of the HIV reservoir or cure interventions in PWH on ART.

Health-related quality of life with nivolumab plus relatlimab versus nivolumab monotherapy in patients with previously untreated unresectable or metastatic melanoma: RELATIVITY-047 trial

BackgroundIn the phase II/III RELATIVITY-047 trial, a novel fixed-dose combination (FDC) of nivolumab plus relatlimab (NIVO + RELA; a programmed death-1 and a lymphocyte-activation gene 3 inhibitor, respectively) significantly improved progression-free survival (PFS) versus NIVO in patients with previously untreated unresectable or metastatic melanoma (median follow-up, 13.2 months) with stable health-related quality of life (HRQoL), although grade three or four treatment-related adverse events (TRAEs) were more frequent with the combination. Updated HRQoL results (median follow-up, 19.3 months) are presented. MethodsPatients were randomised to receive intravenous NIVO + RELA (480 mg and 160 mg, respectively) or NIVO (480 mg) every 4 weeks. HRQoL was assessed using the Functional Assessment of Cancer Treatment-Melanoma (FACT-M) and EQ-5D-3L questionnaires at baseline, before dosing at each treatment cycle, and at follow-up (posttreatment) visits. ResultsConsistent with the initial analysis, HRQoL remained stable with NIVO + RELA on treatment and was similar to that with NIVO. Mean changes from baseline did not exceed clinically meaningful thresholds. HRQoL results were consistent across instruments and scales/subscales. Despite an increased rate of grade three or four TRAEs with NIVO + RELA versus NIVO, the proportion of patients reporting that they were bothered 'quite a bit' or 'very much' by TRAEs was low and comparable between treatments. ConclusionResults from the RELATIVITY-047 trial show that the PFS benefit with NIVO + RELA FDC over NIVO was obtained with stable patient-reported HRQoL, supporting NIVO + RELA as a first-line treatment option for patients with advanced melanoma.

The involvement of Lymphocyte activation gene 3 and the inhibitor in non-small cell lung cancer

Non-small cell lung cancer, or NSCLC, is the most typical malignant tumor in lung carcinoma. NSCLC causes a relatively high fatality and prevalence rate compared to other cancers worldwide. A combination of surgery, chemotherapy, and chemoradiotherapy interventions are commonly used as standard or front-line therapy for patients with NSCLC. However, a relatively novel treatment, immunotherapy, has become a popular target for many cancer-related clinical researches in recent years, including melanoma, urothelial cancer, and NSCLC. The general purpose of immunotherapy is to target the host’s immune system and increase the immune response opposing the tumor. Within immunotherapy, immune checkpoint inhibitor (ICI) was gaining increasing attention as a therapeutic method for many cancers through recent studies. Immune checkpoints (ICs) regulate the immune response and suppress the activity of immune cells when expressed. This mechanism of action prevents immune cells from developing autoimmunity, which, if it happens, immune cells will start attacking healthy cells in the host. However, one disadvantage of IC is that they indirectly enhance tumor survival by deactivating immune cells. Thus, many ICIs are developed to directly inhibit the activity of the targeted IC. Among all types of immune checkpoints, lymphocyte activation gene 3 (LAG-3) acts as an essential IC in the host’s cell. The primary objective of this article is not only to elucidate the association between LAG-3 and NSCLC but also to explain the effect of anti-LAG-3 drugs in patients with NSCLC, its synergistic effect with other checkpoint inhibitors, and the limitations of immune checkpoint inhibitors for future investigation.

Lymphocyte-activating gene 3 expression in tumor cells predicts immune checkpoint inhibitor response in triple negative breast cancer.

Immune checkpoint inhibitor (ICI) is one of the standard treatment strategies in triple negative breast cancer (TNBC). However, the benefit of ICI with chemotherapy is limited in metastatic TNBC. In this study, we evaluated the effect of PD-L1 and LAG-3 expression on tissue microenvironment of mTNBC treated with ICI. We reviewed representative formalin-fixed paraffin embedded specimens from metastatic or archival tumor tissues of TNBCs who treated with PD-1/PD-L1 inhibitors in metastatic setting. We used the Opal multiplex Detection kit with six antibodies (anti-PD-L1, anti-LAG-3, anti-CD68, anti-panCK, anti-CD8, anti-CD107a/LAMP antibody). We evaluated the association between LAG-3+cells and survival outcome regarding CK expression. Stromal LAG-3+/CK+ and LAG-3+/CK- cells were not associated with ICI-progression free survival(PFS) (P=0.16). However, LAG-3+ cell distributions in the tumor area impacted on ICI-PFS. A high density of LAG-3+CK+ cells was associated with shorter ICI-PFS compared with low densities of both LAG-3+CK+ and LAG-3+CK- cells (1.9 vs. 3.5 months). In addition, a high density of LAG-3+CK- cells had a relatively longer ICI-PFS compared with other groups (P=0.01). In terms of total area, the pattern of densities of LAG-3+CK+ cells and LAG-3+CK- cells were similar to those in the tumor area In addition, ICI-PFS of LAG-3+CK- and LAG-3+CK+ cell densities in the total area was equal to that in the tumor area. In conclusion, our findings revealed tumor-intrinsic LAG-3 expression was the resistance mechanism toward PD-1/PD-L1 inhibitors in mTNBCs. Multivariate analysis also suggested that LAG-3 expression in tumor cells was an independent predictive biomarker.

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...

Fibrinogen-like protein 1 promotes liver-resident memory T-cell exhaustion in hepatocellular carcinoma.

The key role of tissue-resident memory T (TRM) cells in the immune regulation of hepatocellular carcinoma (HCC) has been investigated and reported, but the regulatory mechanism of tumor microenvironment on TRM cells is still unclear. Lymphocyte activating gene 3 (LAG-3) is a promising next-generation immune checkpoint that is continuously expressed due to persistent antigen exposure in the tumor microenvironment. Fibrinogen-like protein 1 (FGL1) is a classical ligand of LAG-3 and can promote T cell exhaustion in tumors. Here, we excavated the effect of FGL1-LAG3 regulatory axis on TRM cells in HCC. The function and phenotype of intrahepatic CD8+ TRM cells in 35 HCC patients were analyzed using multicolor flow cytometry. Using a tissue microarray of 80 HCC patients, we performed the prognosis analysis. Moreover, we investigated the suppressive effect of FGL1 on CD8+ TRM cells both in in vitro induction model and in vivo orthotopic HCC mouse model. There was an increase in LAG3 expression in CD8+ TRM cells in end-stage HCC; moreover, FGL1 levels were negatively correlated with CD103 expression and related to poor outcomes in HCC. Patients with high CD8+ TRM cell proportions have better outcomes, and FGL1-LAG3 binding could lead to the exhaustion of CD8+ TRM cells in tumors, indicating its potential as a target for immune checkpoint therapy of HCC. Increased FGL1 expression in HCC may result in CD8+ TRM cell exhaustion, causing tumor immune escape. We identified CD8+TRM cells as a potential immunotherapeutic target and reported the effect of FGL1-LAG3 binding on CD8+ TRM cell function in HCC.

Phase 1 study of fianlimab, a human lymphocyte activation gene-3 monoclonal antibody, in combination with cemiplimab in advanced melanoma: Expansion cohort analysis

Phase 1 study of fianlimab, a human lymphocyte activation gene-3 monoclonal antibody, in combination with cemiplimab in advanced melanoma: Expansion cohort analysis

89Zr-immuno-PET using the anti-LAG-3 tracer [89Zr]Zr-BI 754111: demonstrating target specific binding in NSCLC and HNSCC

PurposeAlthough lymphocyte activation gene-3 (LAG-3) directed therapies demonstrate promising clinical anti-cancer activity, only a subset of patients seems to benefit and predictive biomarkers are lacking. Here, we explored the potential use of the anti-LAG-3 antibody tracer [89Zr]Zr-BI 754111 as a predictive imaging biomarker and investigated its target specific uptake as well as the correlation of its tumor uptake and the tumor immune infiltration.MethodsPatients with head and neck (N = 2) or lung cancer (N = 4) were included in an imaging substudy of a phase 1 trial with BI 754091 (anti-PD-1) and BI 754111 (anti-LAG-3). After baseline tumor biopsy and [18F]FDG-PET, patients were given 240 mg of BI 754091, followed 8 days later by administration of [89Zr]Zr-BI 754111 (37 MBq, 4 mg). PET scans were performed 2 h, 96 h, and 144 h post-injection. To investigate target specificity, a second tracer administration was given two weeks later, this time with pre-administration of 40 (N = 3) or 600 mg (N = 3) unlabeled BI 754111, followed by PET scans at 96 h and 144 h post-injection. Tumor immune cell infiltration was assessed by immunohistochemistry and RNA sequencing.ResultsTracer uptake in tumors was clearly visible at the 4-mg mass dose (tumor-to-plasma ratio 1.63 [IQR 0.37-2.89]) and could be saturated by increasing mass doses (44 mg: 0.67 [IQR 0.50–0.85]; 604 mg: 0.56 [IQR 0.42–0.75]), demonstrating target specificity. Tumor uptake correlated to immune cell-derived RNA signatures.Conclusions[89Zr]Zr-BI-754111 PET imaging shows favorable technical and biological characteristics for developing a potential predictive imaging biomarker for LAG-3-directed therapies.Trial registrationClinicalTrials.gov, NCT03780725. Registered 19 December 2018

Abstract GS5-03: Evaluation of anti-PD-1 Cemiplimab plus anti-LAG-3 REGN3767 in early-stage, high-risk HER2-negative breast cancer: Results from the neoadjuvant I-SPY 2 TRIAL

Abstract Background: I-SPY2 is a multicenter, phase 2 trial using response-adaptive randomization within biomarker subtypes defined by hormone-receptor (HR), HER2, and MammaPrint (MP) status to evaluate novel agents as neoadjuvant therapy for high-risk breast cancer. The primary endpoint is pathologic complete response (pCR). Cemiplimab is an anti-PD-1 inhibitor approved for the treatment of NSCLC and cutaneous basal and squamous cell CA. Lymphocyte activation gene 3 (LAG-3) binds MHC class II leading to inhibition of T-cell proliferation and activation and is often co-expressed with PD-1. REGN3767 is a fully humanized mAb that binds to LAG-3 and blocks inhibitory T-cell signaling. Concurrent blockade of LAG-3 with an anti-PD-1 may enhance efficacy of an anti-PD-1. Methods: Women with tumors ≥ 2.5cm were eligible for screening. Only HER2 negative (HER2-) patients were eligible for this treatment; HR positive (HR+) patients had to be MP high risk. Treatment included Paclitaxel 80 mg/m2 IV weekly x 12 and Cemiplimab 350 mg and REGN3767 1600 mg both given q3weeks x 4, followed by doxorubicin/cyclophosphamide (AC) every 2 weeks x 4. The control arm was weekly paclitaxel x 12 followed by AC every 2-3 weeks x 4. Cemiplimab/REGN3767 was eligible to graduate in 3 of 10 pre-defined signatures: HER2-, HR-HER2-, and HR+HER2-. The statistical methods for evaluating I-SPY 2 agents has been previously described. To adapt to changing standard of care, we constructed “dynamic controls” comprising ‘best’ alternative therapies using I-SPY 2 and external data and estimated the probability of Cemiplimab/REGN3767 being superior to the dynamic control. Response predictive subtypes (Immune+ vs Immune-) were assessed using pre-treatment gene expression data and the ImPrint signature. Results: 73 HER2- patients (40 HR+ and 33 HR-) received Cemiplimab/REGN3767 treatment. The control group included [357 patients with HER2- tumors (201 HR+ and 156 HR-) enrolled since March 2010. Cemiplimab/REGN3767 graduated in both HR-/HER2- and HR+/HER2- groups; estimated pCR rates (as of June 2022) are summarized in the table. Safety events of note for Cemiplimab/REGN3767 include hypothyroidism 30.8%, adrenal insufficiency (AI) 19.2%, hyperthyroidism 14.1%, pneumonitis 1.3%, and hepatitis 3.8%. All were G1/2 except for 6 (7.7%) G3 AI and 3 (3.8%) G3 colitis. Rash occurred in 62.8%, 9% G3 and 2 pts (2.6%) had pulmonary embolism. X% of adrenal insufficiency cases required replacement therapy. 40 patients (11 HR+ and 29 HR-) in Cemiplimab/REGN3767 were predicted Immune+; 32 (29 HR+ and 3 HR-) were predicted Immune-. In the HR+ group pCR was achieved in 10/11 (91%) patients with Immune+ subtype compared with 8/29 (28%) with Immune- subtype. Additional biomarker analyses are ongoing and will be presented at the meeting. Conclusion: The I-SPY 2 study aims to assess the probability that investigational regimens will be successful in a phase 3 neoadjuvant trial. Dual immune blockade with a LAG-3 inhibitor and anti-PD1 therapy resulted in a high predicted pCR rate both in HR-/HER2- (60%) and HR+/HER2- (37%) disease. The novel Imprint signature identified a group of HR+ patients most likely to benefit from this active regimen. Table 1: Estimated pCR rates Citation Format: Claudine Isaacs, Rita Nanda, Jo Chien, Meghna S. Trivedi, Erica Stringer-Reasor, Christos Vaklavas, Judy C. Boughey, Amy Sanford, Anne Wallace, Amy S. Clark, Alexandra Thomas, Kathy S. Albain, Laura C. Kennedy, Tara B. Sanft, Kevin Kalinsky, Hyo S. Han, Nicole Williams, Mili Arora, Anthony Elias, Carla Falkson, Smita Asare, Ruixiao Lu, Maria Pitsouni, Amy Wilson, Jane Perlmutter, Hope Rugo, Richard Schwab, W. Fraser Symmans, Nola M. Hylton, Laura Van’t Veer, Douglas Yee, Angela DeMichele, Donald Berry, Laura J. Esserman, I-SPY Investigators. Evaluation of anti-PD-1 Cemiplimab plus anti-LAG-3 REGN3767 in early-stage, high-risk HER2-negative breast cancer: Results from the neoadjuvant I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-03.

Abstract P2-20-10: Prognostic impact of lymphocyte-Activation Gene-3 (LAG-3) expression in triple negative breast cancer

Abstract Introduction Recent advances in breast cancer treatment strategies have improved survival outcomes in metastatic breast cancer (mBC). Targeting immune checkpoint, especially programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1), has made a breakthrough in treating advanced malignancies including breast cancer. Immune checkpoint inhibitors (ICIs), such as pembrolizumab and atezolizumab, in combination with chemotherapy have prolonged survival outcomes in mBC with triple negative subtype. However, the low response rate and resistance of ICIs with anti-PD 1/PDL-1 antibodies is a major limitation and a challenge. Lymphocyte Activation Gene-3 (LAG3; CD223) is a potential cancer immunotherapeutic target due to its negative regulatory role on T cells and cytokines, thereby ensuring immune homeostasis. Several studies suggested that combination immunotherapy of anti-LAG-3 and anti-PD-1 has shown promising efficacy in fighting PD-1 resistance. Therefore, we evaluated the prognostic role of LAG3 in metastatic TNBC (mTNBC) treated with ICIs to figure out resistance mechanism of ICIs. Methods mTNBC patients with available archival tumor tissues, who has received ICIs in Samsung Medical Center, were enrolled in this study. For the evaluation of LAG3 expression in tumor microenvironment, Vectra Polaris Multispectral Imaging and Whole Slide Scanning technique (PerkinElmer, Inc. Hopkinton, MA) was used. Results In total, 64 mTNBC patients were treated with ICI’s with or without cytotoxic chemotherapy between 2019.02- 2021.11. Of 70 mTNBC patients, 41 patients had archival tissues and finally 40 patients were included in this study. Median age was 43.0 years of age (range: 24.5 ~ 64.5). Recurrent mTNBC was 92.5% and only 7.5% was de novo mTNBC. Geremline BRCA1 pathogenic variants were detected in 4 (10.0%) patients. Among 37 recurrent mTNBC patients, 72.5% were treated with neoadjuvant chemotherapy with anthracycline (97.3%) and taxane(97.3%). Among ICI’s, 52.5% were treated with pembrolizumab and 47.5% of atezolizumab. LAG3 expression varies among mTNBC tissues (median cell density: 366, range: 48, 2861 cells/mm2). Among cells expressing LAG3, LAG3 was expressed more in CK+ cells compared with CK- cells (median:150 (20, 2503) cells/mm2 in CK+ cells, median: 88 (2, 806) cells/mm2 in CK- cells, p=0.005). Patients with high LAG3 expression in CK+ cells showed short progression free survival(PFS) compared to those with low LAG3 expression in CK+ cells (median PFS of high vs. low LAG3 expression [months]:1.9 vs. 4.2, p=0.01). On the contrary, patients with high LAG3 expression in CK- cells had 9.1 months of PFS compared to 3.1 months of PFS in patients with low LAG3 expression in CK- cells (p=0.10). In addition, patients with high LAG3 in CK- cells had longer overall survival(OS) compared to those with low LAG3 expression in CK- cells (median OS of high vs. low LAG3 expression [months]: not reached, 15.7, p=0.05). Conclusion LAG3 expression was associated with PFS in patients with mTNBC treated with ICI’s independent of PDL-1 expression. And the prognostic significance of LAG3 expression was different between CK+ cells and CK- cells. These findings need to be proved in large scale clinical trials. Citation Format: ji-Yeon Kim, Jeehyun Kim, Hae Hyun Jung, eun Yoon Cho, Yeon Hee Park, Jin Seok Ahn, Kyoung-Mee Kim, Young-Hyuck Im. Prognostic impact of lymphocyte-Activation Gene-3 (LAG-3) expression in triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-10.

Method validation of a bridging immunoassay in combination with acid-dissociation and bead treatment for detection of anti-drug antibody

Anti-drug antibody (ADA) positivity is correlated with disease relapse risk when treated with monoclonal antibody (mAb) therapeutics. ADA evaluation can assist with interpreting pharmacokinetic, pharmacological, and toxicology results. Here, we established an ADA assay based on two steps of acid dissociation combined with a bridging immunoassay to provide a comprehensive validation strategy. The three-tiered sample analysis process included screening, confirmation, and titration assays using therapeutic HLX26 (targeting lymphocyte activation gene-3 [LAG-3]) as an example. The cut points were determined by testing 50 individual normal human serum samples, including screening cut point (SCP) (SNR: 1.08), confirmatory cut point (CCP) (% inhibition: 12.65), and titration cut point (TCP) (sample-to-noise ratio [SNR]: 1.17). The assay sensitivity, low positive control (LPC), and high positive control (HPC) titer acceptable range were also set up as 33.0 ng/mL, 41.0 ng/mL, and 320–1280, respectively. After full validation, both the intra-assay and inter-assay precision testing passed with coefficient of variations (CVs) < 20%. The assay enabled excellent drug tolerance up to 768.0 μg/mL at the HPC level and 291.0 μg/mL at the LPC level, while the tolerance of target interference was up to 74.0 ng/mL of soluble LAG3. Moreover, no false-positive results were observed in the presence of 5% hemolyzed serum samples and 150 mg/dL of triglyceride in the serum samples, no hook effect was observed, and the stability performed normally under room temperature for 24 h, 2–8 °C for 7 d, and six freeze/thaw cycles. In summary, this ADA assay is feasible and could be used for evaluating the immunogenicity of HLX26 in clinical trials.

PP.42 Phase 1 Study of Fianlimab, a Human Lymphocyte Activation Gene-3 (LAG-3) Monoclonal Antibody, in Combination With Cemiplimab in Advanced NSCLC

PP.42 Phase 1 Study of Fianlimab, a Human Lymphocyte Activation Gene-3 (LAG-3) Monoclonal Antibody, in Combination With Cemiplimab in Advanced NSCLC

Conformational Dynamics of an α-Synuclein Fibril upon Receptor Binding Revealed by Insensitive Nuclei Enhanced by Polarization Transfer-Based Solid-State Nuclear Magnetic Resonance and Cryo-Electron Microscopy.

Many amyloid fibrils associated with neurodegenerative diseases consist of an ordered fibril core (FC) and disordered terminal regions (TRs). The former represents a stable scaffold, while the latter is rather active in binding with various partners. Current structural studies mainly focus on the ordered FC since the high flexibility of TRs hinders structural characterization. Here, by combining insensitive nuclei enhanced by polarization transfer-based 1H-detected solid-state NMR and cryo-EM, we explored the intact structure of an α-syn fibril including both FC and TRs and further studied the conformational dynamics of the fibril upon binding to lymphocyte activation gene 3 (LAG3)─a cell surface receptor that is involved in α-syn fibril transmission in brains. We found that both the N- and C-TRs of α-syn are disordered in free fibrils featuring similar conformation ensembles as those in soluble monomers. While in the presence of the D1 domain of LAG3 (L3D1), the C-TR directly binds to L3D1, meanwhile the N-TR folds into a β-strand and further integrates with the FC, which leads to alteration of the overall fibril structure and surface property. Our work reveals synergistic conformational transition of the intrinsically disordered TRs of α-syn, which sheds light on mechanistic understanding of the essential role of TRs in regulating the structure and pathology of amyloid fibrils.

Nivolumab and Relatlimab in Patients With Advanced Melanoma That Had Progressed on Anti-Programmed Death-1/Programmed Death Ligand 1 Therapy: Results From the Phase I/IIa RELATIVITY-020 Trial.

Nivolumab and relatlimab activity in advanced melanoma with prior progression on anti-programmed death-1/programmed death ligand 1 (PD-(L)1)-containing regimens is under investigation. RELATIVITY-047 demonstrated significantly improved progression-free survival (PFS) for nivolumab and relatlimab over nivolumab in previously untreated advanced melanoma. The phase I/IIa, open-label RELATIVITY-020 trial part D assessed efficacy and safety of nivolumab and relatlimab in advanced melanoma with progression during, or within 3 months of, 1 (D1) or ≥ 1 (D2) anti-PD-(L)1-containing regimens. Safety was a primary end point. Objective response rate (coprimary end point) and PFS by blinded independent central review (BICR) were assessed. Five hundred eighteen patients (D1 = 354; D2 = 164) received nivolumab and relatlimab. Among evaluable patients, the objective response rate by BICR was 12.0% (95% CI, 8.8 to 15.8) in D1 (n = 351) and 9.2% (95% CI, 5.2 to 14.7) in D2 (n = 163). Responses appeared to be enriched among patients with tumors expressing programmed death ligand 1 or lymphocyte activation gene 3; however, responses were observed regardless of programmed death ligand 1 and lymphocyte activation gene 3 expression (1%). The median duration of response was not reached (95% CI, 12.9 to not reached) in D1 and 12.8 months (95% CI, 6.9 to 12.9) in D2. The median PFS by BICR was 2.1 months (95% CI, 1.9 to 3.5) in D1 and 3.2 months (95% CI, 1.9 to 3.6) in D2; the 6-month PFS rate was 29.1% (95% CI, 24.2 to 34.1) and 27.7% (95% CI, 20.5 to 35.4), respectively. The grade 3-4 treatment-related adverse event incidence was 15.0% in D1 and 12.8% in D2. One case of grade 3 myocarditis and no treatment-related deaths occurred across part D. Nivolumab and relatlimab had a manageable safety profile and demonstrated durable clinical activity in a proportion of patients with heavily pretreated advanced melanoma with prior progression on anti-PD-(L)1-containing regimens.