Lag3

LAG3 was discovered in 1990 (Triebel F et al. 1990) as a transmembrane molecule expressed on CD4+ and CD8+ T cells, natural killer T cells (NKT), natural killer cells (NK), plasmacytoid dendritic cells (pDCs), and regulatory T cells (Tregs ).

LAG3 is the acronym for "Lymphocyte-activation gene 3". LAG3, also known as CD223, is a transmembrane protein with structural similarities to CD4. In humans, the protein is encoded by LAG gene3, which is located on chromosome 12 (12p13.32). The LAG3 locus is adjacent to the gene encoding the CD4 co-receptor and has a similar intron/exon organization. Like CD4, LAG3 also binds to MHC class II, but with a much higher affinity.

LAG3 belongs to the family of anti-inflammatory immune checkpoints (ICs). These ICs inhibit the immune reactivity of T lymphocytes, in contrast to proinflammatory ICs, which increase the immune reactivity of T lymphocytes. The immune checkpoints are activated by precisely fitting cytokines (ligands) that are presented and released by other cells.

There is ample evidence that LAG-3 is an inhibitory coreceptor and plays a central role in autoimmunity, tumor immunity and anti-infection immunity (Triebel F et al. 1990). Thus, LAG-3 suppresses the activation of T cells and the secretion of cytokines, thereby providing immune homeostasis. The protein exerts different inhibitory effects on different types of lymphocytes and showed synergy with PD-1 in inhibiting immune responses.

LAG3 structure, function and ligands: In most cell types, LAG3 expression is regulated by activation, with the exception of pDCs and Tregs, where expression appears to be constitutive (Zhang Q et al. 2017).

Structure of LAG: Both CD4 and LAG3 are composed of four, extracellular immunoglobulin superfamily-like, domains (D1-D4), although the structure of LAG3 has not been definitively elucidated. However, LAG3 appears to possess two unique structural features.

1. unlike CD4, the interaction between LAG3 and MHC class II is mediated by a unique, proline-rich, thirty amino acid loop within D1 (Li N et al. 2004).
2. has a longer connecting peptide between the fourth Ig domain and the transmembrane region, making LAG3 more susceptible to shedding from the cell surface by a disintegrin and metalloproteinase domain-containing protein (ADAM) (Li N et al. 2004).

The cytoplasmic tail of LAG3 consists of three conserved motifs. However, the function and downstream signaling events remain unknown. The first motif contains a putative serine phosphorylation site, which in humans contains two serine residues. To date, no function has been attributed to this motif. The second motif is a unique, conserved six-amino acid sequence (KIEELE) that has been shown to be required for LAG3 to downregulate T cell function (Workman CJ et al 2003). The third motif is a glutamic acid and proline dipeptide (EP) repeat. It has been suggested that this motif binds LAG3-associated protein (LAP), allowing co-localization of LAG3 with CD3, CD4 and/or CD8 molecules within lipid rafts (Workman CJ et al 2003).

Ligands: The primary, canonical ligand for LAG3 is MHC class II, which binds to a conserved extended loop in the LAG3 D1 domain. Once bound to MHC class II, LAG3 transmits inhibitory signals through its cytoplasmic domain. The mechanism of signal transduction remains unclear (Workman CJ et al 2004). Note: Human melanoma cancer cells exhibit increased expression of MHC class II, which has been associated with poor patient prognosis. Binding of LAG3 to MHC class II contributes to tumor escape from apoptosis and recruitment of tumor-specific CD4+ T cells, which in turn leads to a reduction in CD8+ T cell response (Hemon P et al 2011; see also under immune evasion).

Other ligands:

• However, other potential ligands are now known, such as Galectin-3 (Gal-3). This is a galactose-binding lectin that regulates T-cell activation. Gal-3 can be expressed on different cell types and thus exerts its regulatory function on CD8+ T cells through multiple mechanisms ((Kouo T et al. 2015).
• LSECtin, a C-type lectin receptor: This binds to the four glycosylated sites on LAG3. LSECtin is expressed in liver and melanoma tumor cells, suggesting a mechanism by which LAG3 may regulate CD8+ T cell and NK cell function in these settings (Xu F et al (2014).
• Furthermore, fibrinogen-like protein 1 (FGL1) has been described as a ligand for LAG3 (Wang J et al. 2019). FGL1 is a member of the fibrinogen family that has a similar structure to fibrinogen beta and gamma, with no known effects on platelets or in clot formation. FGL1 is normally secreted by hepatocytes in the liver, but tumor cells can also express large amounts of FGL1, which correlates with poor patient prognosis and resistance to immunotherapy (Wang J et al 2019). Strikingly, blocking the interaction between LAG3 and FGL1, increases intratumoral T cell responses, leading to a reduction in melanoma tumor size in mouse models (novel mechanism for targeted immunotherapy?).

Regulation of LAG3 expression: Cell surface expression of LAG3 is regulated by two separate mechanisms, highlighting the importance of tight control of LAG3 expression and function for optimal immune homeostasis:

• Storage: 1. LAG3 is stored in lysosomal compartments to allow rapid translocation to the cell surface after TCR stimulation to control T cell responses. In resting T cells, LAG3 is grown in the lysosomal compartments. This degradation is the limiting step for surface expression of LAG3, and inhibition of lysosomal activity increases its surface expression. Moreover, its translocation to the cell surface is mediated by protein kinase C signaling via the cytoplasmic domain (Bae J et al. 2014).
• Cleavage: 2. LAG3 cell surface expression is regulated by proteolytic cleavage, resulting in the release of a soluble form of LAG3 (sLAG3). Cleavage of LAG3 is mediated by a disintegrin and metalloproteinase domain-containing protein 10 and 17(ADAM10 and ADAM17) (Moss ML et al. 2017).

LAG3 is an IR expressed on activated and dysfunctional T cells. When activated, it negatively regulates T cell function. Therefore, LAG3 is a novel target for modulating T cell responses in diseases.

Autoimmune and inflammatory diseases:

• IRs play a central role in the regulation of autoimmune and inflammatory diseases. Indeed, symptoms of autoimmunity are often a side effect of checkpoint blockade in solid tumors due to loss of self-tolerance (Stamatouli AM et al. 2018).
• Targeting LAG3 has also generated interest in clinical applications for autoimmune diseases in humans. An anti-LAG3 mAb (GSK2831781; GlaxoSmithKline) has been developed, a humanized monoclonal "afucosylated" antibody with enhanced antibody-dependent cell cytotoxicity (ADCC) that decimates LAG3-expressing immune cells in patients with autoimmune diseases. This compound has completed a Phase 1, first-in-human study in patients with plaque psoriasis and will soon move into a Phase 2 study.
• Another agonistic monoclonal antibody (mAb) targeting LAG3 (IMP761; Immutep) is currently in preclinical development for the treatment of additional autoimmune diseases.

Chronic viral and parasitic infections:

• IRs are highly expressed on dysfunctional and depleted T cells in chronic viral and parasitic infections. The role of LAG3 in acute and chronic infections in vivo is well known (Blackburn SD et al (2009). Blockade of LAG3 alone appears to only slightly reduce viral load in best viral infections. However, if co-blockade of LAG3 and PDL1 synergistically enhances CD8+ T cell responses, viral load is significantly reduced (Blackburn SD et al (2009). Animal experiments showed that LAG3 is also strongly expressed on CD4+ T cells and NK cells in the lungs and especially in the granulomatous lesions of macaques during Mycobacterium tuberculosis (MTB) infections. Similarly, LAG3 is expressed by depleted parasite-specific CD4+ T cells during malaria (Plasmodium falciparum) infection in mice (Butler NS et al. (2011). Correlative studies in humans have shown that Plasmodium falciparum infection resulted in increased expression of LAG3 and PD1, which was associated with T cell dysfunction (Butler NS et al (2011).

Tumor entities: There are currently 7 mAbs in development: Relatilimab (BMS-986016, Bristol-Myers Squibb; fully human IgG4 mAb), LAG525 (Novartis; humanized IgG4), MK-4280 (Merck; humanized IgG4), REGN3767 (Regeneron; human IgG4), TSR-033 (Tesaro; humanized IgG4), Sym022 (Symphogen; Fc-inert human mAb), and INCAGN02385 (InCyte; Fc-engineered IgG1κ). There is significant interest in developing mAbs targeting LAG3 in human cancers, particularly in combination with anti-PD1.

Relatlimab (BMS-986016) is a fully human LAG3-specific antibody isolated after immunization of transgenic mice expressing human immunoglobulin (Ig) genes. It is the first humanized anti-LAG3 mAb to be developed, and of all anti-LAG3 mAbs, it is the most advanced in clinical development. The first Phase I/IIa trial, initiated in 2013, was designed to evaluate the safety and efficacy of LAG3 blockade as monotherapy or in combination with nivolumab in patients with advanced solid malignancies (cervical, ovarian, bladder, colorectal, HPV-positive HNSCC, gastric cancer, hepatocellular carcinoma, RCC) who had not previously received immunotherapy . It is currently being studied in 18 Phase I and II/III trials in a variety of solid and hematologic malignancies.

The combination of relatlimab and nivolumab (anti-PD1) showed remarkable efficacy in melanoma patients who were refractory to prior immunotherapy (PA M.I. Ascierto, Bhatia S, et al.(2017). The results demonstrate that the combination of nivolumab and relatlimab was well tolerated, with grade 3 or 4 treatment-related immunologic adverse events occurring in 9% of patients, which is similar to the frequency with nivolumab alone. This suggests that a nivolumab/relatlimab combination may be safer than a nivolumab/ipilimumab (anti-CTLA4) combination.

Melanoma: Immune checkpoint inhibitors alone or in combination have transformed treatment and improved survival rates for patients with metastatic or unresectable melanoma. However, there remain a substantial number of patients who could benefit from a novel combination therapy that potentially utilizes complementary signaling pathways, thereby improving anti-tumor activity. The results of versch. studies suggest that targeting the LAG-3 pathway in combination with PD-1 inhibition may be a key strategy to enhance the immune response and improve outcomes for these patients."

Furthermore, sustained antigen stimulation, such as in cancers and chronic viral infections, leads to increased chronic LAG3 expression, which in turn results in T-cell exhaustion and subsequent impairment of T-cell function (Wherry EJ 2011). Numerous immunotherapies (autoimmune diseases, chronic infections, tumor diseases) targeting LAG3 are in clinical trials in combination with antibodies against other IRs, such as PD1/PDL1, for the treatment of cancer (see references below).

1. Bae J et al (2014) Trafficking of LAG-3 to the surface on activated T cells via its cytoplasmic domain and protein kinase C signaling. J Immunol 193: 3101-3112.
2. Blackburn SD et al (2009) Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol 10: 29-37.
3. Butler NS et al (2011) Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection. Nat Immunol 13: 188-195.
4. Goldberg MV et al (2011) LAG-3 in cancer immunotherapy, Curr Top Microbiol Immunol 344: 269-78.
5. Hemon P et al (2011) MHC class II engagement by its ligand LAG-3 (CD223) contributes to melanoma resistance to apoptosis, J Immunol 186: 5173-5183.
6. Kouo T et al (2015) Galectin-3 Shapes Antitumor Immune Responses by Suppressing CD8+ T Cells via LAG-3 and Inhibiting Expansion of Plasmacytoid Dendritic Cells. Cancer Immunol Res 3: 412-423.
7. Li N et al (2004) Biochemical analysis of the regulatory T cell protein lymphocyte activation gene-3 (LAG-3; CD223). J Immunol 173: 6806-6812.
8. Moss ML et al. (2017) Recent Advances in ADAM17 Research: A Promising Target for Cancer and Inflammation, Mediators Inflamm: 9673537.
9. PA M.I. Ascierto, Bhatia S, et al.(2017) Initial efficacy of anti-lymphocyte activation gene-3 (anti-LAG-3; BMS-986016) in combination with nivolumab in patients with melanoma previously treated with ant-PD-1/PD-L1 therapy. J Clin Oncol 35 suppl; abstr 9520.
10. Stamatouli AM et al (2018) Collateral damage: insulin-dependent diabetes induced with checkpoint inhibitors. Diabetes 67: 1471-1480.
11. Triebel F et al (1990) LAG-3, a novel lymphocyte activation gene closely related to CD4. J Exp Med 171: 1393-405.
12. Wang J et al (2019) Fibrinogen-like protein 1 Is a Major Immune Inhibitory Ligand of LAG-3. Cell 176: 334-347.
13. Wherry EJ (2011) T cell exhaustion, Nat Immunol 12: 492-499.
14. Zhang Q et al (2017) LAG3 limits regulatory T cell proliferation and function in autoimmune diabetes. Sci Immunol 2:eaah4569
15. Workman CJ et al (2004) Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo. J Immunol 172: 5450-5455.
16. Workman CJ et al (2003) The CD4-related molecule, LAG-3 (CD223), regulates the expansion of activated T cells, Eur J Immunol 33: 970-979.
17. Xu F et al (2014) LSECtin expressed on melanoma cells promotes tumor progression by inhibiting antitumor T-cell responses. Cancer Res 74: 3418-3428.