Molecular Pathways and Mechanisms of LAG-3 in Cancer Therapy

Abstract

Immunotherapy targeting co-inhibitory receptors has been highly successful in treating a wide variety of malignancies, however only a subset of patients exhibits durable responses. The first FDA-approved immunotherapeutics targeting co-inhibitory receptors PD1 and CTLA4, alone or in combination, significantly improved survival but were also accompanied by substantial toxicity in combination. The third FDA-approved immune checkpoint inhibitor targets LAG3, a co-inhibitory receptor expressed on activated CD4⁺ and CD8⁺ T cells, especially in settings of long-term antigenic stimulation, such as chronic viral infection or cancer. Mechanistically, LAG3 expression limits both the expansion of activated T cells and the size of the memory pool, suggesting that LAG3 may be a promising target for immunotherapy. Importantly, the mechanism(s) by which LAG3 contributes to CD8⁺ T cell exhaustion may be distinct from those governed by PD1, indicating that the combination of anti-LAG3 and anti-PD1 may synergistically enhance antitumor immunity. Clinical studies evaluating the role of anti-LAG3 in combination with anti-PD1 are underway, and recent phase III trial results in metastatic melanoma demonstrate both the efficacy and safety of this combination. Further ongoing clinical trials are evaluating this combination across multiple tumor types and the adjuvant setting, with accompanying translational and biomarker-focused studies designed to elucidate the molecular pathways that lead to improved antitumor T cell responses following dual blockade of PD1 and LAG3. Overall, LAG3 plays an important role in limiting T cell activation and has now become part of the repertoire of combinatorial immunotherapeutics available for the treatment of metastatic melanoma.

Introduction/Background

Co-inhibitory receptors (IRs), such as programed death-1 (PD1) and cytotoxic T lymphocyte antigen-4 (CTLA4), are upregulated on T cells in settings of chronic antigen stimulation or inflammation and serve to limit tissue damage and autoimmune-mediated host tissue pathology (1, 2). Importantly, such IRs play an essential role in suppressing autoimmunity, but conversely this immunoregulation can limit viral clearance and suppress antitumor immunity. Expression of these IRs on the surface of CD4⁺ and CD8⁺ T cells reflects a distinct transcriptional and epigenetic T cell lineage known as exhausted T cells (3). Cancer immunotherapeutics have sought to antagonize the interactions between IRs and their cognate ligands to reverse this exhausted state and to reinvigorate the antitumor immune response. Blockade of PD1 with monoclonal antibodies (mAbs) is an FDA-approved strategy that has been effective across a wide range of malignancies including melanoma, renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and multiple microsatellite instable (MSI) cancers, among others (4). Despite this success, not all patients benefit, and some tumor types are largely refractory to current immunotherapy. As multiple IRs are expressed on exhausted T cells, immunotherapies targeting more than one IR in combination could more effectively restore T cell function, leading to improved clinical efficacy.

The combination of nivolumab (anti-PD1) and ipilimumab (anti-CTLA4) has been shown to induce a higher response rate and progression-free survival (PFS), recently reaching superiority in 5-year survival versus monotherapy with nivolumab (5). However, this came at the cost of considerably increased toxicity. On March 18 2022, LAG3 became the third FDA-approved target of an immune checkpoint inhibitor with relatlimab (anti-LAG3) plus nivolumab for treatment of patients with unresectable or metastatic melanoma. Nearly a dozen more agents that target LAG3, alone or in combination with anti-PD1, are currently being evaluated clinically. In this review, we discuss the current understanding of LAG3 biology, including its expression pattern, structure, function and signaling pathways. We also summarize the current state of clinical investigation of LAG3-targeted monotherapies, combinations and bispecifics, as well as the rationale for synergistic enhancement of antitumor immunity with LAG3-targeted combinations. A timeline for the discovery and clinical development of LAG3 through FDA-approval is provided in Figure 1.

Figure 1. Timeline of scientific and clinical development of LAG3.

Discovery, development and clinical evaluation of LAG3 as an immunotherapeutic has taken place over the course of 30 years, beginning with the discovery of LAG3 in 1990 and appreciation of its inhibitory role on T cells in 2002. A subsequent major milestone was the discovery that LAG3 and PD1 synergistically inhibit T cell function in murine models of cancer, leading to subsequent assessment of LAG3 monoclonal and bispecific antibodies in the clinic, and culminating with FDA-approval of relatlimab plus nivolumab in treatment of patients with metastatic or unresectable melanoma by demonstration of efficacy over nivolumab monotherapy in the RELATIVITY-047 clinical trial.

Mechanics

LAG3 Expression Pattern and Clinical Significance

LAG3 is not expressed on naïve T cells, but induced upon stimulation of CD4⁺ and CD8⁺ T cells to limit activation and prevent autoimmunity (6). However, as a result of persistent antigenic stimulation within the tumor, sustained expression of LAG3 along with other IRs including PD1, TIM3, and TIGIT, results in a state of T cell dysfunction, associated with a lack of proliferation, cytokine secretion and cytolysis (7). Co-expression of PD1 and LAG3 has been demonstrated on CD4⁺ and CD8⁺ tumor infiltrating lymphocytes in numerous murine transplantable tumor models (B16-F10 melanoma, MC38 colon adenocarcinoma and SA1N fibrosarcoma) (8). Immunohistochemistry studies identified high LAG3 in a range of tumor types including ovarian cancer, melanoma, NSCLC and HNSCC, as correlating with poor prognosis and disease-free survival (9). Importantly, extensive immune profiling of melanoma patients treated with immune checkpoint blockade demonstrated that LAG3 expression on CD8⁺ T cells in peripheral blood resulted in poorer outcomes after immunotherapy compared to patients with no LAG3 expression. Patients with melanoma who possessed a LAG3⁺ CD8⁺ immunotype had poorer outcomes after immune checkpoint blockade with a median survival of 22.2 months compared to 75.8 months for those with a LAG3-negative immunotype (10). This demonstrates that expression of LAG3 may act as a mechanism of resistance to PD1 blockade, and thus can be used as a biomarker to identify patients who may or may not respond to treatment. Further, this also suggests that LAG3 could potentially be targeted to overcome such resistance.

LAG3 is also expressed on CD4⁺ Foxp3⁺ regulatory T (T_reg) cells and while it has been shown to be required for control of homeostatic T cell expansion and suppressive activity of T_reg cells themselves, it has been demonstrated that LAG3 acts as a brake on T_reg cells to limit proliferation and function in a model of diabetes (11–13). However, LAG3 expression on T_reg cells in melanoma and colorectal cancer patients’ peripheral blood, tumor-associated lymph nodes and the tumor itself has been correlated with production of immunosuppressive cytokines (IL-10, TGF-β) compared with LAG3-negative cells (14). Likewise, LAG3 co-expression with CD49b defines an IL-10 producing CD4⁺ type 1 T regulatory (Tr1) cell population, in which LAG3 also contributes to its suppressive properties (15). This population has also been associated with disease progression in colorectal cancer.

While persistent antigen exposure that drives the exhausted T cell program within tumors and chronic viral infection, IL-2, IL-7 and IL-12 have also been reported to drive LAG3 expression on activated T cells (16). Other mechanisms reported to drive expression of LAG3 include glycogen synthase kinase-3 (GSK-3), as inhibition was shown to down-regulate LAG3 expression in T cells (17). GSK-3 inactivation was shown to potentiate the anti-tumor effects of anti-LAG3 to eliminate tumor cells and may promote a more efficacious anti-tumor immune response.

In addition to CD4⁺ and CD8⁺ T cells, LAG3 can be found on other immune populations such as γδ T cells, NKT cells, plasmacytoid dendritic cells (pDCs) and activated B cells, although it is unclear if LAG3 expression on these populations plays a role with regard to antitumor immunity (18–20). LAG3 has also been found to be highly expressed by malignant cells in diffuse large B cell lymphoma (DLBCL) (21).

LAG3 Structure and Ligands

The Lag3 gene is comprised of eight exons and its chromosomal location is adjacent to the gene for CD4 on the distal part of chromosome 12, with a similar intron-exon organization that is likely to have evolved due to a gene duplication event (22). The Lag3 gene encodes a 70 kDa type I transmembrane protein, made up of 498 amino acids with structural homology to CD4, containing four extracellular immunoglobulin-like superfamily (IgSF) regions (D1-D4) with one variable (V type) and three constant (C type) Ig-like domains. The membrane distal D1 domain has an additional 30 amino acid proline-rich loop that is conserved across multiple species, but not present in CD4 (23). Biochemical analysis of LAG3 has suggested that it can be expressed as monomers, dimers and higher order oligomers on the cell surface, independent of ligand engagement (24).

As a result of the structural homology to CD4, the canonical ligands for LAG3 are major histocompatibility complex (MHC) class II molecules although additional ligands have been described (Figure 2). The presence of a proline-rich D1 loop in LAG3 allows it to bind to MHC class II with a much higher affinity than CD4, leading to the original hypothesis that LAG3 primarily functions by competing with CD4 for binding to MHC class II (18, 25, 26). However, mutagenesis studies as well as experiments performed with C9B7W (a rat mAb that binds to the D2 domain of LAG3), which blocks the inhibitory effects of LAG3 but does not disrupt the MHC class II:LAG3 interaction, implied that LAG3 does not primarily function by disrupting CD4:MHC class II interactions (18). While further studies have shown that LAG3 inhibits T cell responses by binding to stable complexes of peptide and MHC class II, inhibitory signals are transmitted via the intracellular region (27). Moreover, MHC class II expression by human melanoma is associated with poor prognosis, and ligation with LAG3 has been shown to be an escape mechanism to protect against apoptosis as LAG3 mutants unable to bind to MHC class II have reduced inhibitory function (28, 29). Despite this, these studies raise the possibility that LAG3 binding to other ligands may mediate its function, particularly with respect to non-MHC class II restricted CD8⁺ T cells expressing LAG3 within the tumor.

Figure 2. LAG3 ligand/receptor interactions and their antagonism with therapeutic agents alone or in combination with anti-PD1 therapies.

LAG3 interacts with several known ligands that can lead to inhibition of T cell function including MHC class II, CD4, TCR, Gal3, LSECtin and FGL1. Surface expression of LAG3 is regulated by ADAM10/17, which cleaves LAG3 from the surface releasing soluble LAG3. In contrast to LAG3 mediated regulation of T cell function, a LAG3-Ig fusion protein agonist has been generated to activate APCs via interaction with MHC class II. On T cells expressing LAG3, T cell activation, function and memory are inhibited following LAG3 ligation via intracellular signaling via an intracellular EP motif domain on LAG3. Blockade of LAG3 with relatlimab in conjunction with disruption of the PD1 axis with nivolumab, results in synergistic reactivation of T cell function. Bispecific antibodies such as FS118 or tebotelimab simultaneously block PD1/PDL1 and LAG3.

Other ligands have been reported to interact with glycans on LAG3 including galectin-3 (Gal-3) and liver sinusoidal endothelial cell lectin (LSECtin) that are both lectins with carbohydrate-recognition domains. Gal-3 is a soluble galactose-binding lectin secreted from tumor cells and tumor stromal cells (30). LAG3:Gal-3 engagement was suggested to reduce the frequency of IFNγ-producing CD8⁺ T cells upon TCR stimulation (31). LSECtin is expressed in the liver, but is also found on the surface of melanoma cells where engagement with LAG3 may inhibit IFNγ production by antigen-specific effector T cells (32, 33).

Fibrinogen-like protein 1 (FGL1), which is secreted by the hepatocytes in the liver and is highly upregulated in tumors, has also been suggested as a ligand for LAG3 with immunosuppressive activities (34). FGL1 binds to the D1 and D2 domains of LAG3, independent of MHC class II interaction, and blockade of this interaction increases IFNγ levels. FGL1 may be a biomarker of poor prognosis itself as high FGL1 levels in plasma of NSCLC and melanoma patients were associated with resistance to anti-PD1 therapy (34).

Recently, it has been postulated that LAG3 may interact in cis with the T cell receptor (TCR):CD3 complex in the presence or absence of MHC class II ligation in both CD4⁺ and CD8⁺ T cells. In support of this hypothesis, prior to and following TCR stimulation, multiple LAG3 molecules associate and track with the (TCR):CD3 complex to the immunological synapse and via its cytoplasmic tail allows LAG3 to promote an inhibitory effect on TCR signaling (35).

LAG3 Signaling and Inhibitory Function

Many immune IRs (checkpoints), such as CTLA4, PD1 and TIM3, inhibit T cell activation through cytoplasmic inhibitory domains such as an immunoreceptor tyrosine-based inhibitory motif (ITIM) or immunoreceptor tyrosine-based switch motif (ITSM), but these are not present in the LAG3 intracellular domain. Despite structural homology to CD4, LAG3 does not have the C-X-X-C binding site for the tyrosine kinase p56^Lck that facilitates coreceptor downstream signal transduction, although curiously LAG3 function was shown some time ago to be co-receptor dependent (23, 26). Instead, LAG3 possesses three conserved regions in the cytoplasmic tail: [i] a ‘KIEELE’ motif, [ii] an acidic glutamic acid-proline (EP) repetitive sequence, and [iii] a conserved putative serine phosphorylation site (Figure 2) (6). The ‘KIEELE’ motif has been shown to be required for LAG3 inhibitory activity in T cell hybridomas (26). Another group has suggested that LAG3 transduces inhibitory signals through an FXXL motif in the membrane-proximal region (36).

Recent studies with LAG3 mutants have shown that the EP motif in the cytoplasmic tail, and in particular, the highly conserved and repetitive glutamic acid residues are responsible for the dissociation of p56^lck from CD4 or CD8 co-receptors, consistent with previous studies highlighting coreceptor dependency of LAG3 (26), thereby abrogating coreceptor mediated signaling (35). Although LAG3 lacks the di-cysteine binding motif for p56^lck, a large number of LAG3 molecules moves into the immunological synapse via its association with the (TCR):CD3 complex and the resulting number of glutamic acid residues binds to Zn²⁺ and leads to a reduction in the local pH of the immunological synapse. This reduction in pH causes p56^lck to disassociate from CD4 or CD8 resulting in a reduction in phosphorylation of ZAP70 and the consequent downstream signaling events. Therefore, LAG3 through dissociation of p56^lck signaling via this unique mechanism can limit TCR signaling and T cell activation (35).

While many ligands could be involved in triggering LAG3 signaling, the consequences of this signaling in T cells lead to a reduction in T cell proliferation and function. Early studies demonstrated that LAG3 knockout mice experienced uncontrolled T cell proliferation following administration of a superantigen and ovalbumin-specific T cells lacking LAG3 similarly experienced uncontrolled proliferation following immunization (37, 38). Additionally, LAG3 was found to limit the size of the memory pool in mice following infection with Sendai virus (39). In chronic viral infection, LAG3 is co-expressed with other IRs (39) that together result in a progressive reduction in effector function due to lower expression of cytolytic effectors such as granzyme B and perforin, loss of expression of effector cytokines such as IFNγ and TNFα in CD8⁺ T cells (39) and IL-2, IL-4 and IFNγ in conventional CD4⁺ T cells (40). LAG3 has also been shown to regulate the metabolic profile of naïve CD4⁺ T cell metabolism to control homeostatic expansion (41). LAG3 has also been shown to regulate both the STAT5 and AKT pathways. The enhanced STAT5 signaling in LAG3-deficient CD4⁺ T cells resulted in increased aerobic glycolysis and effector function following activation. STAT5 signaling is thus required for LAG3 blockade to affect homeostatic proliferation in vivo and thus it is inferred that LAG3 may modulate the STAT5/IL-2 signaling axis during homeostatic proliferation (41). Overall, the binding of ligands to LAG3 expressed on the cell surface of T cells leads to inhibition of proliferation and loss of T cell effector functions.

Regulation of LAG3 Expression and Function

Expression of LAG3 is controlled at multiple levels including genetic, epigenetic, cleavage at the cell surface and via intracellular trafficking. Early studies suggested that LAG3 may be subjected to additional levels of genetic control as three splice variants have been proposed to exist, which include a version lacking the last three exons producing a soluble truncated version of the full length protein (42, 43). There is also evidence for epigenetic regulation of LAG3 expression, via DNA methylation of the Lag3 promoter, in some tumor types including melanoma and clear cell RCC (44, 45). This epigenetically-mediated regulation of Lag3 by tumor cells has been associated with increased immune cell infiltration and improved overall survival.

LAG3 levels on the cell surface are also regulated by intracellular trafficking and cell surface cleavage. Rapid translocation of LAG3 from intracellular stores in lysosomal compartments to the cell surface occurs upon TCR stimulation (46, 47). Interestingly, unlike healthy individuals, a proportion of cancer patients demonstrate robust intracellular, but not surface, LAG3 expression in peripheral CD8⁺ T cells (48). The divergence between intracellular and surface LAG3 is the result of rapid shedding of LAG3 from the cell surface by a disintegrin and metalloprotease domain-containing protein-10 and −17 (ADAM10/17)-mediated cell surface shedding following trafficking to the surface. ADAM10/17 cleave LAG3 at the connecting peptide region between the membrane proximal D4 domain and the transmembrane domain releasing a monomeric soluble form of LAG3 (sLAG3) (49). While ADAM10 constitutively cleaves LAG3 under resting conditions, shedding activity is enhanced upon TCR activation and ADAM17 shedding only occurs upon PKC activation (49). It has been demonstrated in vitro that the shedding of LAG3 is essential for T cell function and proliferation by generation of non-cleavable LAG3 (LAG3^NC) mutant (49). Furthermore, generation of a conditional knock-in mouse in which LAG3^NC is only expressed in T cells was shown to be resistant to anti-tumor immune effects of PD1 blockade – including impact on tumor growth and functionality of LAG3^NC-expressing CD4⁺ T cells within the tumor (50). In addition, the translational relevance of LAG3 shedding was demonstrated by assessment of LAG3 and reciprocal ADAM10 expression, which were correlated to disease stage and prognosis of patients with HNSCC, and the LAG3:ADAM10 ratio in fact predicted response to nivolumab in a cohort of melanoma patients (50). These data demonstrate that regulation of LAG3 expression and function by ADAM-mediated cell surface shedding has an important effect on patient responsiveness to immunotherapy. Overall, LAG3 levels on T cells are tightly regulated by several mechanisms.

Soluble LAG3 as a Biomarker

While both in vitro and in vivo studies have not uncovered a function of soluble LAG3 (sLAG3) compared to the transmembrane counterpart, sLAG3 in patient sera is prognostic of outcome in several malignancies. Serum levels of sLAG3 were significantly elevated in hepatocellular carcinoma (HCC) patients compared to healthy donor controls (51) and in hormone receptor (HR)-positive breast cancer patients (52). High sLAG3 expression was found in relation to better prognosis with gastric cancer (53). Patients with advanced (Stage III-IV) non-small cell lung cancer (NSCLC) also had significantly lower sLAG3 expression than patients with Stage I-II NSCLC (54). However, in HNSCC, high levels of sLAG3 were associated with poor prognosis (55). Although LAG3 is shed from the surface of T cells, pDCs are the major contributor to sLAG3 levels in vivo, complicating the interpretation of the biological significance of sLAG3 (19). Further work is needed to better understand the significance of the role of LAG3 on pDCs and the consequent release of sLAG3, as little is known in tumors other than a role in regulation of pDC homeostasis (19). Regardless of the origins of sLAG3 in vivo, promoting LAG3 shedding activity by enhancing ADAM10/17 activity may lead to enhanced T cell function by abrogating LAG3-mediated intracellular signaling.

Clinical Implications

LAG3-Ig agonist as an APC activator

In 2006, a dimeric, recombinant, soluble form of LAG3 called eftilagimod alpha was the first-in-class agent to enter the clinic. Consisting of four LAG3 extracellular domains fused to the Fc portion of human IgG1, this agent was clinically developed as an immune adjuvant to activate antigen-presenting cells (APCs) and is not to be confused with the monomeric sLAG3 released as a byproduct of metalloprotease-mediated shedding. The rationale for eftilagimod alpha-based therapy lies in the observation that a soluble LAG3-Ig fusion protein directly stimulates DCs to upregulate co-stimulatory molecules and produce IL-12 and TNFα (56). Recent data suggests a survival benefit with the combination of eftilagimod alpha and paclitaxel as a first-line chemo-immunotherapy assessed in a Phase IIb study of patients with HER2-negative, HR-positive metastatic breast cancer (MBC), but only in a pre-specified subgroup of patients aged under 65 years and not the overall population. This combination was well-tolerated with manageable adverse events. Clinical trials involving combination of eftilagimod alpha with pembrolizumab (anti-PD1) have been initiated in a phase IIb study in PDL1-positive HNSCC and NSCLC patients (57), as well as in melanoma with encouraging antitumor activity (58).

Rationale for anti-PD1 and anti-LAG3 blockade

The inhibitory effects of LAG3 on CD8⁺ T cells appear to be distinct from those of PD1 or CTLA4, providing a rationale for blockade of multiple IRs. Biochemical reconstruction of the PD1 signaling pathway has indicated that the co-stimulatory receptor CD28 is more highly dephosphorylated following PD1 ligation with PDL1 than the TCR (59). Thus, PD1 blockade may serve to reinvigorate T cells via rescue of co-signaling pathways as opposed to directly regulating signals that drive T cell proliferation through the TCR. Similarly, CTLA4 serves to reduce T cell activation by depriving T cells of signaling through the CD28 co-receptor, although CTLA4 does this through competitive binding with the CD80 and CD86 co-stimulatory ligands for CD28 rather than dephosphorylating CD28 following signaling through PD-L1 (60). CTLA4 may also remove CD80/CD86 directly from APCs as an alternative mechanism of limiting co-stimulation. Thus, although blockade of PD1 and CTLA4 may lead to expansion of different subpopulations of T cells (61, 62), they may do so through a similar mechanism of action. Additionally, previous studies have suggested that LAG3 may also mediate bidirectional inhibitory signaling into APCs. Conversely, as outlined above, the mechanism of action of LAG3 may limit T cell activation by reducing signaling through the TCR. This unique mechanism of action of LAG3 suggests the potential for synergistic enhancement of antitumor immunity with less toxicity in combination with PD1 due to their distinct mechanisms.

LAG3 Blockade with Antagonistic mAbs

Following the success of targeting PD1/PDL1, clinical trials began to move beyond monotherapy to combinations of immunomodulatory agents. The rationale for this is that, in many instances, blockade of a single immunomodulatory IR may be insufficient to rescue CD8⁺ T cell function, and that targeting multiple co-inhibitory pathways will lead to a more robust reversal of immune exhaustion in the tumor microenvironment. This has been demonstrated with combination of ipilimumab with nivolumab in advanced melanoma resulting in a 52% 5 year survival compared to 44% and 26% in the nivolumab and ipilimumab groups, respectively, associated with severe toxicity (63).

Preclinical investigation of LAG3 has suggested a strong rationale for combination therapy with anti-PD1. Compared to CTLA4 blockade alone, LAG3 blockade as a monotherapy leads to a small reduction in tumor growth in several murine tumor models including MC38 (8). However, combination of anti-LAG3 with PD1 blockade dramatically limits tumor growth (~80% of mice tumor-free), compared to administration of anti-PD1 alone (~40% of mice tumor-free). Increased tumor-free survival was associated with augmented CD8⁺ T cell infiltration and enhanced cytokine production (8). Synergistic effects with combinatorial blockade have also been demonstrated in the ID8 ovarian cancer model, EG7 lymphoma model and the 5T33 multiple myeloma model (64, 65). This evidence suggests that PD1 and LAG3 blockade in combination leads to potent reinvigoration of antitumor immune responses. Combination of anti-LAG3 with an agonist mAb targeting the immunostimulatory 4–1BB molecule was also shown to synergistically control B16 tumor outgrowth (66). The observation that LAG3 alone does not have a substantive antitumor effect also suggests that the efficacy of LAG3 in combination is reliant on perturbing distinct pathways, which may suggest the potential for fewer adverse events. In addition in an adoptive chimeric antigen receptor (CAR)-T cell model, LAG3 blockade along with anti-PD1 and TIM3 was shown to overcome dysfunction in a synergistic manner to boost effector function (67).

Given the strong rationale for antagonistic blockade of LAG3 to enhance CD8⁺ T cell function (8), numerous mAbs have been developed and are progressing through clinical trials targeting a variety of solid and hematological tumor types (Table 1). All these clinical trials have an anti-PD1 arm to capitalize on the synergistic effects expected with dual blockade. The clinical success of an anti-LAG3/PD1 combination therapy was recently demonstrated by RELATIVITY-047, a global, randomized, double-blinded Phase 2/3 study of patients with metastatic or unresectable melanoma in the first-line setting, which resulted in the FDA approval of relatlimab with nivolumab. Combination of relatlimab and nivolumab versus nivolumab alone, showed superior PFS (10.2 months) comparing the combination therapy to nivolumab alone (4.6 months) (68). Patients with LAG3 expression greater than 1% did show improved PFS, however in both stratified groups there was a benefit with the addition of relatlimab to nivolumab (69). Further, PFS at 12 months was 48% in the combination arm and 36% with nivolumab alone. While there was also a clinically meaningful improvement in overall survival with the combination, this was not statistically significant. Grade 3 or 4 adverse events in the combination arm (21.1%), were manageable and this safety profile was substantially more favorable than observed with anti-PD1/CTLA4 combination therapy (where Grade 3–4 toxicity was encountered in 59% of patients) (70). Myocarditis occurred in 1.7% of patients in the relatlimab-nivolumab group, compared with 0.6% of patients in the nivolumab alone group, although all myocarditis events resolved completely (69). Recent work presented at the Annual Society of Clinical Oncology (ASCO) Annual Meeting revealed that addition of relatlimab to nivolumab as a second-line treatment benefited patients who have progressed previously on anti-PD1/PDL1 therapy (71). Other work at ASCO revealed that pathologic complete response correlated with improved survival in the setting of neoadjuvant nivolumab and relatlimab, supporting synergistic activity when targeting these two IRs (72). Initial clinical activity, with a manageable safety profile, has also been demonstrated with fianlimab (anti-LAG3) given in combination with cemiplimab (anti-PD1) in patients with advanced melanoma at ASCO (73). Additionally, work presented at ASCO showed favezelimab (anti-LAG3) plus pembrolizumab recently demonstrated safety in a Phase I trial in microsatellite stable colorectal cancer (MSS-CRC) (74). Overall, these early results suggest similar efficacy with substantively reduced toxicity for anti-LAG3/PD1 therapy versus anti-CTLA4/PD1 blockade.

Table 1.

Clinical Studies of LAG3-targeted immunotherapeutics

Category	Agent	Manufacturer	Description	Study Phase / Tumor Types	Therapeutic Combination
Antagonistic Antibodies	Relatlimab	Bristol-Myers Squibb	mAb to human LAG3 (human IgG4)	Phase I: HCC, melanoma, gastric cancer, esophageal cancer, gastroesophageal cancer; Phase I/II: Hematologic Neoplasms, Metastatic Ovarian Cancer, Peritoneal Cancer, Gastroesophageal Cancer. Phase II: Metastatic Uveal Melanoma, Soft Tissue Sarcoma, Advanced Chordoma, Melanoma, HNSCC, Acute Myeloid Leukemia, MSI-high tumors, Advanced Colorectal Cancer, HCC, Liver Cell Carcinoma, Basal Cell Carcinoma, NSCLC, RCC, Gastric Cancer, Stomach Cancer, Esophagogastric Junction; Phase II/III: Advanced Melanoma; Phase III: Melanoma	Nivolumab (anti-PD1); Ipilimumab (anti-CTLA4); BMS-986205 (IDO1 Inhibitor)
	Leramilimab	Novartis	mAb to human LAG3 (humanized IgG4)	Phase I: TNBC; Phase I/II: Advanced Solid Tumors; Phase II: SCLC, Gastric Adenocarcinoma, Esophagael Adenocarcinoma, Castration Resistant Prostate Adenocarcinoma, Soft Tissue Sarcoma, Ovarian Adenocarcinoma, Advanced Well-differentiated Neuroendocrine Tumors, Diffuse Large B Cell Lymphoma, TNBC	Spartalizumab (anti-PD1); Canakinumab (anti-IL1B)
	Favezelimab	Merck	mAb to human LAG3 (humanized IgG4)	Phase I: Neoplasms; Phase I/II: Hodgkin Disease, Non-Hodgkin Lymphoma, B-cell Lymphoma, SCLC, RCC; Phase II: Advanced NSCLC; Phase III: Colorectal Cancer (Previously Treated Metastatic PDL1 Positive)	Pembrolizumab (anti-PD1)
	TSR-033	Tesaro	mAb to human LAG3 (humanized IgG4)	Phase I: Multiple Neoplasms	Dostarlimab (anti-PD1) and Cobolimab (anti-TIM3) plus Bevacizumab (anti-VEGFA) and FOLFIRI (folinic acid/leucovorin, 5-fluorouracil and irinotecan)
	Fianlimab	Regeneron/ Sanofi	mAb to human LAG3 (human hinge-stabilized IgG4)	Phase I: Advanced Malignancies	Cemipilimab (anti-PD1)
	Sym022	Symphogen	mAb to human LAG3 (recombinant human, Fc inert)	Phase I: Metastatic Cancer, Solid Tumor, Lymphoma	Sym021 (anti-PD1)
	INCAGN02385	Agenus (Incyte Corporation)	mAb to human LAG3 (Fc-engineered IgG1k)	Phase I: Cervical Cancer, MSI-High Endometrial Cancer, Gastric Cancer, Esophageal Cancer, Hepatocellular Cancer, Melanoma, MCC, Mesothelioma, MSI-High Colorectal Cancer, NSCLC, Ovarian Cancer, SCCHN, SCLC, RCC, TNBC, UC, DLBCL. Phase I/II: Melanoma	INCMGA00012 (anti-PD1); INCAGN02390 (anti-TIM3)
	BI754111	Boehringer Ingelheim	mAb to human LAG3 (humanized IgG4)	Phase I: Neoplasms, NSCLC; Phase II: Neoplasm Metastasis	Ezabenlimab (anti-PD1); BI 907828 (MDM2-p53 antagonist)
	LBL-007	Nanjing Leads Biolabs	mAb to human LAG3 (humanized IgG4)	Phase I/II: Advanced Solid Tumor	Toripalimab (anti-PD1)
	IBI110	Innovent Biologics	mAb to human LAG3 (human IgG)	Phase I: Advanced Malignancies, NSCLC, DLBCL; Phase II: SCLC	Sintilimab (anti-PD1) plus Etoposide and Platinum or Carboplatin
	HLX26	Shanghai Henlius Biotech	mAb to human LAG3 (humanized IgG)	Phase I: Solid Tumors, Lymphoma	N/A
Bispecific Antibodies	FS118	F-Star	Bispecific anti-LAG3-anti-PDL1 composed of anti-human LAG3-binding Fc (Fcab) structurally incorporated into the Fc-region of IgG1 mAb to human PDL1	Phase I/II: Advanced Cancer, Metastatic Cancer, HNSCC	N/A
	RO7247669	Hoffmann-La Roche	Bispecific anti-LAG3-anti-PDL1	Phase I: Solid Tumors, Metastatic Melanoma, NSCLC, Esophageal Squamous Cell Carcinoma; Phase I/II: Melanoma, Advanced Liver Cancers; Phase II: Advanced or Metastatic Esophageal Squamous Cell Carcinoma	N/A
	ABL501	ABL Bio	Bispecific anti-LAG3/PDL1	Phase I: Advanced Solid Tumor	N/A
	EMB-02	Shanghai EpimAb Biotherapeutics	FIT-Ig bispecific antibody against PD1 and LAG3	Phase I/II: Advanced Solid Tumor	N/A
	IBI323	Innovent Biologics	Bispecific anti-LAG3/PDL1 antibody	Phase I: Advanced Malignancies	N/A
	XmAb22841	Xencor	Bispecific LAG3/CTLA4 Antibody	Phase I: Melanoma, Cervical Carcinoma, Pancreatic Carcinoma, TNBC, HCC, Urothelial Carcinoma, HNSCC, Nasopharyngeal Carcinoma, RCC, NSCLC, SCLC, Gastric or Gastroesophageal Junction Adenocarcinoma, Advanced or Metastatic Solid Tumors, Prostate Carcinoma, Epithelial Ovarian Cancer, Fallopian Tube Cancer, Primary Peritoneal Carcinoma, Intrahepatic Cholangiocarcinoma, Squamous Cell Anal Cancer, Squamous Cell Penile Cancer, Squamous Cell Vulvar Cancer, Colorectal Carcinoma, Endometrial Carcinoma	N/A
DART	Tebotelimab	MacroGenics	DART anti-LAG3/PD1 with human IgG4 Fc	Phase I: Melanoma, Gastric Cancer, TNBC, Biliary Tract Carcinoma, Endometrial Cancer, Hematologic Neoplasms, Ovarian Cancer, NSCLC, SCLC, HNSCC, Cholangiocarcinoma, Cervical Cancer; Phase I/II: HCC; Phase II: HNSCC; Phase II/III: Gastric Cancer, Gastroesophageal Junction Cancer	Margetuximab (anti-HER2)
LAG3-Ig Agonist	Eftilagimod alpha	Immutep	Soluble LAG3-Ig Protein	Phase I: Melanoma, Solid Tumors, Metastatic Breast Cancer, RCC; Phase II: Adenocarcinoma Breast Cancer, NSCLC, HNSCC	Pembrolizumab (anti-PD1); Avelumab (anti-PDL1)

Bispecific agents targeting LAG3

In addition to mAbs described here, several dual antagonist bispecific agents are in clinical development targeting LAG3 with either an anti-PD1 or PDL1 pairing. FS118 is a dual checkpoint inhibitor targeting LAG3 and PDL1 that was shown to clear MC38 in pre-clinical settings and has now entered Phase I clinical study with a cohort of patients that have previously shown resistance to anti-PD1 as a monotherapy. Interestingly, recent data has demonstrated that the bispecific activity of FS118 drives the shedding of LAG3, which is of importance for its therapeutic activity (75). In pre-clinical murine studies, FS118 resulted in reduced surface expression of LAG3 and increased sLAG3 in the serum of tumor-bearing mice. This increased level of sLAG3 was FS118 dose-dependent and inhibited with ADAM10 metalloproteases (GI254023X), suggesting that FS118 may promote LAG3 shedding. Likewise, in the patient cohort, FS118 increased sLAG3 in patients’ sera and decreased LAG3 TIL expression (75). While this is the first immunotherapeutic agent to suggest LAG3 shedding as part of its mechanism of action, it is unknown whether other LAG3-targeting agents can modulate cell-surface expression. An additional PD1-LAG3 bispecific agent, called tebotelimab, has been generated using a dual-affinity re-targeting (DART) platform that covalently links two polypeptide chains between the variable domains of two antibodies via a disulfide bridge and a short linker that promotes heterodimerization (76). Encouraging results with tebotelimab have demonstrated early evidence of anti-tumor activity with a tolerable safety profile and further clinical results are highly anticipated with these bispecific agents to determine if these are more efficacious than combinations of antagonistic mAbs.

Future Directions

Combination therapy targeting PD1 and CTLA4 achieves a superior response rate and PFS advantages for cancer patients compared with either therapy alone, but at the cost of increased toxicity. LAG3 functions by limiting T cell proliferation and plays a role in inhibiting T cell effector function via mechanisms distinct from PD1, making it a rational choice for combination therapy. While the FDA-approval of relatlimab and nivolumab for the treatment of metastatic melanoma is exciting, there remain a number of important unanswered questions that are being addressed in current studies:

1. Which pharmacological compounds will be the most efficacious? Currently there are several monoclonal and bispecific antibodies undergoing clinical assessment, with the latter category of agents possibly being more efficacious due to dual blockade of both LAG3 and PD1/PDL1.

2. When should this combination be used? Whether the optimal setting for this combination is front-line, neo-adjuvant, or adjuvant remains unknown. While the combination showed improved PFS in the front-line setting in advanced melanoma it remains yet to be evaluated in the adjuvant setting.

3. Will anti-LAG3/PD1 combinations prove to be a viable treatment option for poorly immunogenic tumors? It remains to be seen whether the combination of LAG3 and PD1 blockade will be beneficial for tumors with a low TIL infiltrate such as MSS-CRC and clinical trials have been initiated (74). LAG3 expression is also being evaluated as a biomarker in MSS tumors (77).

4. Is there room for LAG3 targeting agents in hematological malignancies? Lag3 mRNA has been demonstrated to be a prognostic marker in chronic lymphocyte leukemia (CLL) and DLBCL, associated with reduced tumor-free survival (21, 78). Numerous clinical trials with LAG3 targeting agents are ongoing for hematological malignancies, and a recent phase I study with tebotelimab achieved a clinical response in 4 of 11 patients with relapsed or refractory disease (79).

5. What is the mechanism of action for this combination? Understanding the cell types (i.e. conventional CD4⁺ T cells, CD4⁺ T_reg, and CD8⁺ T cells) that respond to this therapeutic combination and the activation of downstream pathways following anti-PD1/LAG3 blockade has important implications for T cell biology and cancer immunology.

6. Is LAG3 expression a prognostic or predictive biomarker for response? In RELATIVITY-047, there was a clear benefit for patients receiving combined relatlimab and nivolumab regardless of LAG3 tumor expression, however patients stratified as >1% LAG3 expression did have superior responses (69).

Overall, LAG3 as a target is an exciting new addition to the repertoire of cancer immunotherapies and addressing these unanswered questions will further our understanding of LAG3 biology and allow us to optimize its utility in the clinic.