2.2 LAG-3 (Lymphocyte Activation Gene-3)
LAG-3 (Lymphocyte Activation Gene-3) is a ∼55 kDa type I trans-membrane glycoprotein consisting of four extracellular immunoglobulin (Ig)-like domains (D1–D4) , a inter-connecting peptide, and an intracellular region that transmits inhibitory signals to the T cell upon binding to MHC class II and other ligands [47, 48]. It is expressed on activated T cells, some activated B cells, Tregs, natural killer (NK) cells, plasmacytoid dendritic cells and neurons, and subjected to epigenetic regulation [48, 49, 50]. Research on LAG-3 knockout mice and LAG-3 antibodies has shown that LAG-3 primarily plays a role in negatively regulating the activation, proliferation, effector function, and homeostasis of T cells [51, 52, 53]. LAG-3 has structural similarities to the CD4 co-receptor, including a similar domain architecture and approximately 25% amino acid sequence identity. It binds to MHC class II but has distinct functional properties [53, 54]. .
The primary/ canonical  ligand for LAG-3 is MHC class II molecules, which are expressed on antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B cells [55, 56]. Other LAG-3 ligands include galectin-3 (Gal-3), fibrinogen-like protein 1, α-synuclein and Liver Sinusoidal Endothelial Cell Lectin (LSECtin) [57]. LAG-3 binds to MHC with a higher affinity than CD4, thereby disrupting CD4–MHC-II interactions [58]. When LAG-3 binds to MHC class II molecules, it transmits inhibitory signals to the T cells, leading to reduced T cell proliferation, cytokine production, and overall activity. This interaction helps maintain immune homeostasis and prevents excessive immune responses that could lead to tissue damage or autoimmunity [57, 59].
In the context of cancer, the inhibitory signals delivered by LAG-3 is seen to contribute to the immune evasion mechanisms of tumors [60, 61]. Tumor cells and the TME can exploit LAG-3 to suppress anti-tumor immune responses, allowing tumors to grow and spread unchecked. Therefore, targeting LAG-3 has emerged as a promising strategy in cancer immunotherapy [62]. The co-expression of LAG-3 and PD-1 in T cells serves as a biomarker for significant T-cell dysfunction in cancer and is linked to resistance against anti-PD-1/anti-PD-L1 immunotherapies [58, 63]. In a study involving three distinct transplantable tumors, Woo et al. demonstrated that the immune inhibitory molecules LAG-3 and PD-1 work together to regulate T-cell function, thereby facilitating tumor immune escape [64]. Wang et al documented that, high expression of LAG-3 in residual tissues, especially in combination with PD-L1, was associated with poor prognosis in 148 pre- and 114 post-neoadjuvant chemotherapy (NACT) specimens of human Triple-Negative Breast Cancer (TNBC) tissue [65].
Preclinical studies and early-phase clinical trials had shown potential of LAG-3 inhibitors, particularly in combination with other checkpoint inhibitors such as PD-1/PD-L1 inhibitors [66, 67]. LAG-3 targeting molecules include: anti-LAG-3 monoclonal antibodies, bispecific molecules, LAG-3 fusion protein and CAR-T cells [68]. Wierz et al. documented that, dual blockade of PD-1 and LAG-3 immune checkpoints restricts tumor development in a murine model of chronic lymphocytic leukemia [67].  In mouse models, Thudium et al., documented that simultaneous blockade of LAG-3 and PD-1 using surrogate antibodies led to enhanced antitumor activity that surpassed the effects observed with blockade of either receptor alone [69]. In a study by Matsuzaki  et al., dual blockade of LAG-3 and PD-1 during T-cell priming significantly enhanced the proliferation and cytokine production of NY-ESO-1 (”cancer-testis” antigen) -specific CD8+ T cells in epithelial ovarian cancer [63]. In a phase 1 clinical trial involving patients with stage-IV renal cell carcinoma, the administration of IMP321—a recombinant soluble LAG-3 Ig fusion protein that activates dendritic cells via MHC class II, resulted in the induction of effector CD8+ T cells in all patients. Additionally, high doses of IMP321 led to reduced tumor growth and improved progression-free survival [NCT00351949] [66]. RELATIVITY-047 (NCT03470922), a phase II/III trial demonstrated that the combination of relatlimab (LAG-3 inhibitor) and nivolumab ( PD-1 blocking antibody ) is a well-tolerated regimen that provides superior progression-free survival compared to nivolumab monotherapy in patients with unresectable or metastatic melanoma [70]. In March 2022, the U.S. FDA approved the fixed-dose combination of relatlimab and nivolumab for treating unresectable or metastatic melanoma in adult patients and pediatric patients aged 12 years and older, weighing at least 40 kg [71]. Relatlimab, the first LAG-3 inhibitor to be approved, marks the third immune checkpoint inhibitor to enter clinical practice following PD-1 and CTLA-4 inhibitors [71].