Unraveling Relatlimab-Specific Biology Using Biomarker Analyses in Patients with Advanced Melanoma in RELATIVITY-047

TPS636 Background: The current standard of care for 1L treatment of patients with advanced/metastatic HCC is atezolizumab + BEV, which demonstrated significantly prolonged progression-free survival (PFS) and overall survival (OS) compared to sorafenib in treatment-naïve patients. However, only 29.8% of patients show objective responses and additional therapy options are needed in the 1L setting. Programmed death-1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) are distinct inhibitory immune checkpoint pathways that synergistically reduce T-cell function. RELA is a first-in-class human immunoglobulin G4 LAG-3-blocking antibody that binds to LAG-3 and restores the effector function of T cells. Dual checkpoint inhibition of the PD-1 and LAG-3 pathways with NIVO + RELA has the potential to boost immune surveillance in HCC. Preclinical data presume that BEV, a human vascular endothelial growth factor inhibitor, reverses abnormal vascularization to allow NIVO + RELA to inhibit hypoxia-induced programmed cell death ligand 1 and LAG-3 expression and enhance depth of response and OS in HCC. Here we describe the RELATIVITY-106 study investigating the triplet therapy of NIVO + RELA + BEV in the 1L treatment of advanced/metastatic HCC. Methods: RELATIVITY-106 (NCT05337137) is a phase 1/2, randomized, double-blind, placebo-controlled trial to assess the safety and efficacy of NIVO + RELA + BEV compared with NIVO + BEV in treatment-naïve patients with advanced/metastatic HCC. Key inclusion criteria include age ≥ 18 years; histologic confirmation of advanced/metastatic HCC in patients naïve to systemic therapy for advanced/metastatic HCC (prior neoadjuvant or adjuvant immunotherapy permitted if recurrence occurs ≥ 6 months after treatment completion); Child-Pugh A; and ECOG performance status 0 or 1. Key exclusion criteria include known fibrolamellar HCC, sarcomatoid HCC, or mixed hepatocellular cholangiocarcinoma; prior allogenic stem cell or solid organ transplantation; untreated symptomatic central nervous system metastases; clinically significant ascites; increased risk of bleeding; significant vascular disease or inadequately controlled hypertension; and major surgical procedure within 4 weeks prior to study treatment. Primary endpoints include incidence of dose-limiting toxicities assessed for up to 6 weeks and PFS by blinded independent central review (BICR) per RECIST v1.1 in all randomized patients in phase 1 and phase 2, respectively. Secondary endpoints include overall response rate (ORR) by BICR and OS in all randomized patients; ORR and PFS by BICR and OS in all randomized LAG-3-positive patients (≥ 1% by immunohistochemistry); and safety. Key exploratory endpoints include pharmacokinetics and immunogenicity assessed by antidrug antibody positivity. The study, initiated in May 2022, is currently enrolling globally. Clinical trial information: NCT05337137 .

8527 Background: The addition of RELA HD, a lymphocyte activation gene-3 (LAG-3) inhibitor, to NIVO + PDCT has improved clinical benefits vs NIVO + PDCT for pts with PD-L1 expression ≥1% and NSQ histology in RELATIVITY-104 study. We report exploratory biomarker analyses from this study to elucidate mechanisms underlying NIVO + RELA HD + PDCT activity. Methods: Baseline and on-treatment blood samples were analyzed by flow cytometry for pharmacodynamic (PD) changes in immune cell populations including proliferating LAG-3-expressing CD4+ and CD8+ effector memory (EM) and central memory (CM) T cells. Baseline tumor samples were analyzed by monoplex immunohistochemistry (IHC) for tumor cell PD-L1, LAG-3, and CD8 expression. Associations between biomarkers and overall response rate (ORR) and progression free survival (PFS) were assessed. Results: NIVO + RELA HD + PDCT significantly modulated levels of proliferating LAG-3 expressing EM and CM T cells in the periphery on-treatment; no such PD change was observed with NIVO + PDCT. Among pts with NSQ histology, baseline tumor LAG-3 expression ≥1% showed improved ORR and median PFS in both treatment arms compared with LAG-3 <1%. Further, the benefit of RELA HD addition to NIVO + PDCT was also seen in patients with LAG-3 <1%, suggesting that baseline LAG-3 expression at 1%, unlike PD-L1 expression, would not help identify patients who can benefit from LAG-3 inhibition (Table). In contrast to NSQ, the same association trend of PD-L1 and LAG-3 expression with efficacy was not observed in pts with SQ histology, which could be partly attributable to the limited sample size in some SQ subgroups. Interestingly, PD-L1 ≥1% is more strongly correlated with CD8 T cells in NSQ as compared to SQ. Conclusions: These data represent the first in-depth biomarker analyses from a randomized phase 2 study to reveal that RELA can expand proliferating LAG-3 expressing T cells in NSCLC. NIVO + RELA HD + PDCT activity might be particularly robust in pts with NSQ histology and PD-L1 expression ≥1%, where CD8 T cells are enriched. The ongoing phase 3 RELATIVITY-1093 study is evaluating 1L NIVO + RELA HD + PDCT vs standard-of-care pembrolizumab + PDCT in mNSCLC. Clinical trial information: NCT04623775 . Efficacy of NIVO + RELA HD + PDCT vs NIVO + PDCT in pts with NSCLC by baseline histology, PD-L1 and LAG-3 expression. NIVO + RELA HD + PDCTvsNIVO + PDCT NSQ, PD-L1 ≥1%(n = 50 vs 48) NSQ, PD-L1 <1%(n = 48 vs 46) NSQ,LAG-3 ≥1%(n = 56 vs 42) NSQ,LAG-3 <1%(n = 38 vs 42) SQ,PD-L1 ≥1%(n = 29 vs 23) SQ,PD-L1 <1%(n = 22 vs 21) SQ,LAG-3 ≥1%(n = 38 vs 34) SQ,LAG-3 <1%(n = 13 vs 10) PFS HR(90% CI) 0.55(0.36–0.85) 1.24(0.84, 1.83) 0.81(0.54, 1.22) 0.79(0.51, 1.23) 0.78(0.46, 1.34) 1.25(0.7, 2.23) 0.97(0.61, 1.52) 0.98(0.45, 2.15) ORR, % 58% vs 39.6% 35.4% vs 34.8% 57.1% vs 45.2% 34.2% vs 26.2% 44.8% vs 43.5% 81.8% vs 66.7% 52.6% vs 55.9% 84.6% vs 50%

BackgroundDual inhibition of the lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) receptors has significantly improved clinical outcomes in patients with metastatic melanoma compared to treatment with...

Lymphocyte-activation gene 3 in non-small-cell lung carcinomas: correlations with clinicopathologic features and prognostic significance

To investigate the impact of glucocorticoids (GCs) and anti-rheumatic drugs on lymphocyte activation gene-3 (LAG-3) and on programmed cell death-1 (PD-1) expression by synovial and peripheral cells ex vivo. Synovial fluid mononuclear cells (SFMCs) from psoriatic arthritis (PsA, n = 26) and rheumatoid arthritis (RA, n = 13) patients, synovial fluid cells (SFCs) from osteoarthritis (OA, n = 5) patients and peripheral blood mononuclear cells (PBMCs) of healthy donors (n = 14) were co-cultured with GCs, glucocorticoid receptor antagonist RU486, methotrexate (MTX) and biologics. LAG-3 and PD-1 expression on immune subsets were analysed by flow cytometry. GCs in PsA inhibited SFMC growth vs medium [2.3 (0.4) × 105vs 5.3 (0.7) × 105, respectively, P < 0.01] and markedly up-regulated CD14+LAG-3+ cells [11.7 (2.4)% vs 0.8 (0.3)%, P < 0.0001, respectively], but not CD3+LAG-3+ and CD14+PD-1+ cells. MTX had no effect on CD14+LAG-3+ cells [0.7 (0.3)%]. The TNF inhibitors infliximab (IFX) and etanercept, but not IL-12/23 inhibitor, up-regulated CD14+LAG-3+ cells vs medium [2.0 (0.6)% and 1.6 (0.4)% vs 0.5 (0.1)%, P < 0.03, respectively]. SFMC growth inhibition by GC in both PsA and RA correlated with CD14+LAG-3+ cell up-regulation (r = 0.53, P = 0.03). RU486 inhibited GC-induced CD14+LAG-3+ cells up-regulation in a dose-dependent manner compared with GC alone [5 µM 5.3 (1.2)% and 50 µM 1.3 (0.5)% vs 7.0 (1.4)%, P < 0.003], but had no significant effect on CD14+LAG-3+ cells co-cultured with IFX. GCs in healthy donors' PBMCs up-regulated the immune subsets CD3+LAG-3+, CD14+LAG-3+ and CD14+PD-1+ cells. This study proposes a novel regulatory mechanism of GCs and of TNF inhibitors mediated by LAG-3 up-regulation in synovial cells and PBMCs. LAG-3 modulation may be a promising target for development of novel therapies for inflammatory arthritis.

The immune checkpoint receptor lymphocyte activation gene 3 (LAG-3) inhibits proliferation and cytokine production by activated CD4 and CD8 T-cells upon binding to major histocompatibility complex class II (MHC II). Another inhibitory receptor, programmed death 1 (PD-1), and its ligands PD-L1 and PD-L2, are critical for the establishment and maintenance of peripheral T-cell tolerance. Because PD-1 and LAG-3 signaling in the tumor microenvironment enables tumors to escape immune surveillance, combined targeting of PD-1 and LAG-3 has emerged as a promising therapeutic strategy. REGN3767 is a human monoclonal antibody that binds LAG-3 with high affinity and rescues T-cell function by blocking its interaction with MHC II. Similarly, the human monoclonal antibody REGN2810 blocks PD-1 interaction with PD-L1 and PD-L2. To test the in vivo activity of REGN3767 alone or in combination with REGN2810, we generated humanized PD-1/LAG-3 knock-in mice, in which the extracellular domains of mouse Pdcd1 and Lag3 genes were replaced with the corresponding regions of human PD-1 and human LAG-3, respectively. In these mice, combined REGN2810 and REGN3767 therapy reduced MC38.Ova tumor growth and prolonged survival, compared to REGN2810 and REGN3767 monotherapies. To explore the underlying molecular mechanisms, we conducted RNA sequencing and RT PCR analyses of MC38.Ova tumors in PD-1/LAG-3 humanized mice following administration of one or two doses of REGN2810, REGN3767, or the combination. Both REGN3767 and REGN2810 promoted robust transcriptional changes in tumors, and combined PD-1 and LAG-3 blockade resulted in enhanced immune activation signatures. Analysis of differentially expressed genes revealed T-cell expansion and activation induced by either antibody monotherapy. While tumor immune responses to REGN3767 were delayed compared to REGN2810, both agents induced similar gene changes via shared pathways. In addition to T-cell activation, REGN2810 or REGN3767 treatments engaged other tumor-infiltrating leukocytes, including neutrophils, myeloid and NK cells. Stronger tumor growth inhibition in response to REGN2810 or REGN3767 therapy was associated with more robust immune activation signatures. Combination of REGN2810 and REGN3767 enhanced antitumor efficacy, resulting in gene expression changes not seen with either monotherapy. The combination therapy also enhanced immune responses promoted by either antibody alone, including genes associated with T-cell activation and effector function. REGN2810 and REGN3767 combination also increased the expression of other checkpoint and costimulatory molecules, including CTLA-4, CD40 and VISTA. The robust intratumoral immune activation associated with antitumor efficacy in preclinical setting supports the clinical development of REGN2810 and REGN3767 as a combination cancer immunotherapy. Citation Format: Elena Burova, Gabor Halls, Omaira Allbritton, Wen Zhang, William Olson, Markus Mohrs, Gavin Thurston, Ella Ioffe. The anti-LAG-3 antibody REGN3767 promotes immune activation in the tumor microenvironment and enhances antitumor activity of anti-PD-1 antibody REGN2810 in PD-1/LAG-3 humanized mice [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A174.

P-61 Relatlimab + nivolumab in patients with advanced hepatocellular carcinoma who are naive to immuno-oncology therapy but progressed on tyrosine kinase inhibitors, a phase 2, randomized, open-label study: RELATIVITY-073

e16517 Background: LAG-3 (lymphocyte activation gene 3) is of interest as a target molecule for next-generation immune checkpoint inhibitors in several solid tumors, including renal cell carcinoma (RCC). The aim of this study is uncovering the tumor immune microenvironment (TIME) of the LAG-3-dominant RCC. Methods: Firstly, we performed an automated single-cell count for immunolabelled LAG-3, TIM-3 and TIGIT and TIME evaluation panel molecules (CD3, CD8, CD39, PD1, PDL1, Ctla4, CD68, CD163, CD47, Ki67, CD73, IDO1, GLUT1, CD34 and D2-40) to 105 primary ccRCC tumor samples and created the new classification model. The TIME and clinical value of LAG-3-dominant RCC were evaluated. Secondly, The TIME features of LAG-3 dominant RCC in metastatic sites was evaluated in 47 metastatic samples. Lastly, mRNA seq data from the public cohort and clinical trial data (RCC, n = 1144, other solid cancers, n = 3691) was analyzed with the digital cytometry and evaluated whether the TIME features of LAG-3 dominant RCC are RCC-specific or applicable to other solid tumors. Results: In our cohort and our validated cohort (n = 96), the LAG-3 group had significantly shorter OS (p = 0.037 and p = 0.003). The LAG-3 high expression RCC also showed shorter OS in the TCGA RCC cohort (p = 0.016). As the result of TIME evaluation, LAG-3 dominant RCCs had statistically high level of CD8, high CD39+CD8+, and high M2 macrophages. In metastasis samples, LAG-3 dominant RCCs preserved high level of CD8, high CD39+CD8+. In public mRNA seq analysis, LAG-3 dominant RCCs showed high level of CD8, high CD8+Tregs and high M2 macrophages. High Tregs in LAG-3 dominant tumor were seen in melanoma, HCC and prostate cancer. High M2 macrophage TIME in LAG-3 dominant tumor was unique to RCC, not seen in other solid cancer. Conclusions: LAG-3 dominant RCC had poor prognosis and could exhibit a unique, immunosuppressive TIME.

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.

We aimed to determine whether lymphocyte activation gene 3 (LAG-3), also known as CD223, is associated with microvessel density (MVD) in primary hepatocellular carcinoma (HCC), as well as their clinical significance in predicting survival. One hundred and twenty-seven patients were enrolled in the study. Samples were obtained on resection at the Department of Hepatobiliary Surgery of the Qingdao Municipal Hospital from June 2014 to June 2016. Immunohistochemistry was used to determine vessel density and LAG-3 abundance. Statistical analyses were performed to test for correlation of LAG-3 density and other clinicopathological variables with overall survival (OS). High LAG-3 abundance was significantly correlated with increased MVD in primary HCC (P < 0.05). The χ2 test revealed a significant association of LAG-3 with preoperative AFP level, tumor diameter, N stage, and the presence of HBV infection (P < 0.05). Patients with high LAG-3 expression had shorter OS compared to those with low LAG-3 expression (P < 0.05). The Cox proportional hazards model showed that both higher LAG-3 and MVD density, age, the number of tumors, preoperative AFP level, tissue differentiation, Child-Pugh grade, and lymph node metastasis correlated with survival. High expression of LAG-3 is associated with angiogenesis and poor prognosis in HCC patients. With the deepening of research, LAG-3 is likely to become a novel biomarker for clinical diagnosis and prognosis and can even be a therapeutic target of HCC.

TPS10085 Background: Novel checkpoint inhibitors are a promising treatment for advanced melanoma, as only a fraction of patients have durable responses to current FDA-approved immunotherapy. Lymphocyte activation gene 3 (LAG3) is an inhibitory checkpoint receptor on CD4+ and CD8+ T cells, where engagement results in suppression of T cell activation and proliferation. LAG3 and PD1 are co-expressed on T cells during T cell receptor signaling and are down-regulated after antigen clearance. Persistent stimulation leads to prolonged LAG3 and PD1 expression and to T cell exhaustion, a possible mechanism of resistance to immunotherapy. Both LAG3 and PD1 are expressed on tumor-infiltrating T cells in melanoma. Murine tumors treated with both anti-LAG3 and anti-PD1 have demonstrated increased tumor regression than tumors in mice treated with either single agent. Further, a phase I trial has demonstrated safety of combined anti-LAG3 monoclonal antibody, relatlimab and anti-PD1 monoclonal antibody, nivolumab. Methods: This phase II, single-center clinical trial is designed to enroll treatment naive patients with unresectable or metastatic melanoma to ultimately receive combined relatlimab and nivolumab after a lead-in arm where patients are randomized to receive relatlimab, nivolumab, or the combination for the first 4 week cycle. For the lead-in phase, patients will have baseline and post-treatment blood and tumor sampling. Disease assessment by imaging will occur after the lead-in phase at 4 weeks. After completion of the lead-in phase, all patients proceed to combination therapy with disease assessment at 12-week intervals. The primary endpoint for the lead-in phase is to evaluate changes in immune cell populations in peripheral blood and tumor with the single agents and combination treatment. The primary endpoint for the combination phase is best overall anti-tumor response. Secondary clinical endpoints include progression-free survival, overall survival, duration of response and toxicity. Exploratory endpoints are to determine the mechanistic effects of anti-LAG3 and anti-PD1 on the blood and tumor microenvironment, cytokine signatures, and correlation of these with clinical response. The study is currently accruing with enrollment of 9 out of 42 patients. Clinical trial information: NCT03743766.

High Expression of LAG3, LPL and ZAP-70 Genes in B-CLL Strongly Correlates with Unmutated IgVH and Early Therapy Requirement.

Combined inhibition of lymphocyte-activation gene 3 (LAG-3) and PD-1 improves outcomes in patients with melanoma. Increased LAG-3 expression in colorectal cancer correlates with reduced survival. Higher mucin and PD-L1 expression in the mismatch repair-proficient (pMMR) colorectal cancer tumor microenvironment was associated with increased LAG-3 and retrospectively with prolonged progression-free survival upon PD-1 blockade. This led to the hypothesis that LAG-3/PD-1 inhibition would improve clinical outcomes in this pMMR colorectal cancer subset. NCT03642067 was a phase II study evaluating the combination of relatlimab (LAG-3 inhibitor) and nivolumab (PD-1 inhibitor) in patients with previously treated metastatic pMMR colorectal cancer. Patients were enrolled into one of three cohorts: A, mucin/PD-L1-high; B, mucin/PD-L1-low; or C, mucin/PD-L1 unselected. The primary endpoint for each cohort was the objective response rate. We enrolled 59 evaluable patients; best treatment responses were partial response (3), stable disease (6), and progressive disease (50). Response rates did not differ significantly between cohorts. Subgroup analyses demonstrated that two of five patients with lung-only metastases had a partial response. Comparison of liver and lung metastases identified higher baseline dendritic cell densities in lung lesions. Nivolumab/relatlimab resulted in increased intratumoral cytotoxic T cells. Lower baseline intratumoral regulatory T cells and ADAM10+ cancer cells correlated with clinical response. This investigation did not reach its primary endpoint for any of the three treatment cohorts but does provide critical insight into the effects of combining nivolumab/relatlimab on the colorectal cancer tumor microenvironment and identifies subgroups that may derive greater benefit from this combination.

&lt;div&gt;AbstractPurpose:&lt;p&gt;Combined inhibition of lymphocyte-activation gene 3 (LAG-3) and PD-1 improves outcomes in patients with melanoma. Increased LAG-3 expression in colorectal cancer correlates with reduced survival. Higher mucin and PD-L1 expression in the mismatch repair–proficient (pMMR) colorectal cancer tumor microenvironment was associated with increased LAG-3 and retrospectively with prolonged progression-free survival upon PD-1 blockade. This led to the hypothesis that LAG-3/PD-1 inhibition would improve clinical outcomes in this pMMR colorectal cancer subset.&lt;/p&gt;Patients and Methods:&lt;p&gt;NCT03642067 was a phase II study evaluating the combination of relatlimab (LAG-3 inhibitor) and nivolumab (PD-1 inhibitor) in patients with previously treated metastatic pMMR colorectal cancer. Patients were enrolled into one of three cohorts: A, mucin/PD-L1–high; B, mucin/PD-L1–low; or C, mucin/PD-L1 unselected. The primary endpoint for each cohort was the objective response rate.&lt;/p&gt;Results:&lt;p&gt;We enrolled 59 evaluable patients; best treatment responses were partial response (3), stable disease (6), and progressive disease (50). Response rates did not differ significantly between cohorts. Subgroup analyses demonstrated that two of five patients with lung-only metastases had a partial response. Comparison of liver and lung metastases identified higher baseline dendritic cell densities in lung lesions. Nivolumab/relatlimab resulted in increased intratumoral cytotoxic T cells. Lower baseline intratumoral regulatory T cells and ADAM10&lt;sup&gt;+&lt;/sup&gt; cancer cells correlated with clinical response.&lt;/p&gt;Conclusions:&lt;p&gt;This investigation did not reach its primary endpoint for any of the three treatment cohorts but does provide critical insight into the effects of combining nivolumab/relatlimab on the colorectal cancer tumor microenvironment and identifies subgroups that may derive greater benefit from this combination.&lt;/p&gt;&lt;/div&gt;

BackgroundThe use of adoptive T cell therapy has proven to be effective in some advanced malignancies. This study aimed to investigate the effects of lymphocyte-activation gene 3 (LAG-3) immune checkpoint receptor in the enrichment of tumor antigen-specific CD8+ T lymphocytes derived from peripheral blood mononuclear cells (PBMCs) in patients with colorectal cancer.Material/MethodsPeripheral blood samples were obtained from 20 patients with colorectal cancer and apheresis was performed with enrichment and cell sorting to obtain CD8+LAG-3+ T cells, which were expanded using high-dose interleukin-2 (IL-2). T cell subsets were detected using fluorescence-activated cell sorting (FACS), and T cell receptor (TCR) sequencing was used to determine specific clone types. Interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) and cell counting kit-8 (CCK-8) assays were used to measure cell avidity and cytotoxicity.ResultsThe cultured cells increased in number over time and had the greatest proliferative activity at 15 days, at which time the percentage of CD3+, CD3+CD8+, and CD8+CD28+ reached maximal levels. High purity CD8+LAG-3+ T cells were isolated by FACS and at 15 days. TCR sequencing showed that CD8+LAG-3+ T cells were oligoclonal, ELISpot identified increased production of tumor-specific IFN-γ, and the CCK-8 assay showed increased cytotoxicity when compared with pre-cultured CD8+LAG-3− T cells.ConclusionsIn patients with colorectal cancer, CD8+LAG-3+ T cells showed more specific anti-tumor activity following cell culture in vitro, which supported the potential role for the LAG-3 immune checkpoint receptor in enriching tumor-specific T cells in patients with cancer.