Do you really understand the molecular structure of LAG-3?

preface 

Over the past decade, the discovery of T cell immune checkpoints (ICPs) and the development of CTLA-4 and PD-1/PD-L1 monoclonal antibody inhibitors have revolutionized the field of
immuno-oncology.
New other immune checkpoints related to the tumor microenvironment have also become hot spots
in the development of oncology drugs.
In these new generations of ICPs, lymphocyte-activating gene-3 (LAG-3) has become one of
the most promising and promising targets for cancer treatment.

LAG-3 was first discovered in 1990, but so far there is still little
information about the molecule's fine structure.
Recently, Nature Immunology published an important article "Overcoming the LAG3 phase problem", using the yeast display library method to select LAG-3 variants with higher expression levels and higher affinity for ligands than wild-type LAG-3, by forming a new crystal structure of LAG-3, which opens the door to structural and biophysical research of this molecule, and also provides new insights for future basic and applied research
。

Why study the LAG-3 so deeply structurally?

A major function of LAG-3 is negative regulation of major histocompatibility complex class II (MHCII)-mediated T cell activation
.
LAG-3 has 20% sequence homology to CD4, and similar to CD4, LAG-3 binds to αβT cell receptors (TCRs
) on the cell surface.
However, in contrast to CD4, which enhances TCR signaling, when TCR binds to MHCII molecules on antigen-presenting cells, LAG-3 also binds to MHCII molecules and inhibits TCR signaling
by driving co-receptor-Lck dissociation.
Therefore, the distribution and aggregation of LAG-3 on the cell surface affects its activity
.

In addition, the biology of LAG-3 is complex because, in addition to MHCII, multiple other ligands of LAG-3 have been discovered, namely fibrin-like protein 1 (FGL1), LSECtin, galectin 3, and α-synuclein
.
Although LAG3 is expressed in T cells in a TCR signal-dependent manner, its presence and activity on immune cells lacking TCR expression (such as natural killer cells, B cells, and plasmacytoid dendritic cells) suggests that LAG-3 may have multiple functions that may result from the interaction
of its multiple ligands.

Therefore, in-depth research on the molecular structure, epitope, and function of LAG-3 will enhance our understanding of the LAG-3 signaling axis, thereby clarifying how LAG-3 regulates changes in T cell activity to guide the development of the most effective immunotherapies
targeting LAG-3.

What does the new structure of LAG-3 tell us?

To identify the structure of the LAG-3 extracellular domain, the authors cocrystallized the LAG-3 D1–D4 domain with a single-stranded variable fragment (scFv) of the F7 antagonist.

In addition, the structure of the mouse LAG-3 D1D2 domain, which is more stable and biochemically better, as well as the structure of
human LAG-3 D3D4, were characterized.

Although the crystallization resolution of the intact LAG-3 extracellular domain is low, it is enough to indicate that LAG-3 is a homodimer formed by the D2 domain, and the remaining domains form an elongated and curved arrangement
.
The dimer interface in D2 is at an angle, so the D1 domain deviates from the central axis of the dimer and forms a V-shaped pore size
.

While the dimer interface in the human and mouse LAG-3 structure shares a broadly conserved set of residues, the D1D2 dimer forms at very different angles (23.
9° vs 69.
6°), which may result in different relative positions
of MHCII and FGL1 binding sites.
In the human LAG-3 structure, the potential MHCII-binding loop 1 (called the "extra ring") of the D1 domain is rotated approximately 90° away from the central axis, while in mice it is towards the wells
in the D1D2 structure.
This conformational difference between human and mouse LAG-3 may reflect two different functional states of LAG-3 and have considerable plasticity to switch functional
states through dimer arrangement.
In fact, this interdomain flexibility and conformational variability of the LAG3 structure suggests that the dynamics of the LAG-3 structure play an important role
in its biology.

In addition to analyzing the structural information of the LAG-3 extracellular domain, the yeast display technique was used to analyze the structure of FGL1 and draw the interface
between LAG-3 and FGL1.
This structure identifies key interface residues in LAG-3 D1 loop 2 and demonstrates that LAG-3 binds MHCII and FGL1
through different molecular surfaces.
Combined with the FGL1 structure and interface information, it is concluded that the LAG-3 dimer cannot form a reasonable 2:2 complex with the FGL1 dimer
.
This conclusion is interesting because it gives us a glimpse of how FGL1 changes TCR signal conditioning via LAG-3
.
By aggregating LAG-3 on the cell surface, FGL1 can reversibly modulate LAG-3 activity without directly interfering with MHCII binding
.
The structural elucidation of LAG-3 and FGL1 provides some key information for deciphering the mechanism of action of LAG-3, however many questions remain to be answered, particularly about the structural basis of LAG3's interaction with its various ligands, and why different MHCII isoforms exhibit different affinities for LAG-3.

Clinical development of LAG-3

Currently, many biopharmaceutical companies are investing in and developing LAG-3 projects, and there are currently at least a hundred LAG-3-related clinical trials in clinical trials
.
To date, at least 20 drugs
have been developed against LAG-3.
Includes anti-LAG-3 blocking antibodies (relatlimab (BMS-986016), Sym022, TSR-033, REGN3767, LAG525, INCAGN2385-101, MK-4280, and BI754111) and antagonistic bispecific antibodies ( MGD013 (anti-PD-1/LAG-3), FS118 (Anti-LAG-3/PD-L1), XmAb22841 (anti-CTLA-4/LAG-3)), etc
.

Other LAG-3-targeted drugs are also being used in cancer treatment
.
IMP321 is a soluble recombinant fusion protein consisting of the extracellular region of LAG-3 and the Fc region of IgG, which activates antigen-presenting cells through MHCII-mediated reverse signaling, resulting in an increase in IL-12 and TNF and upregulation
of CD80 and CD86.
In the clinical trials currently conducted, IMP321 has been mediocre
in efficacy as monotherapy or in combination with other therapies.
The details of the reverse signal through MHCII are still unknown and need to be carefully studied
.

Anti-LAG-3 depleting antibody (GSK2831781) and agonist antibody (IMP761) have also been reported
as potential therapeutic agents for the treatment of autoimmune diseases.
Although these antibodies are designed to clear or inhibit pathogenic T cells, they may also deplete or inhibit Treg cells
.
Further research to elucidate the function of this antibody and the biological properties of LAG-3 are expected to contribute to its development
.

brief summary

Structural studies of LAG-3 not only provide insight into the biology of LAG-3, but also provide insight into the evaluation of agonist and antagonist antibodies against LAG-3 and the rational development of antibodies against LAG3- Small molecule inhibitors of ligand interaction lay the groundwork
.
Although LAG-3 structure and function studies may lag behind other checkpoint inhibitors, a series of recent important studies will take a critical step
in our understanding of LAG-3's structure and function.

References:

1.
LAG-3: from molecular functions to clinical applications.
J Immunother Cancer.
2020 Sep; 8(2):e001014.

2.
 The Next-Generation Immune Checkpoint LAG-3 and Its Therapeutic Potentialin Oncology: Third Time’s a Charm.
Int J Mol Sci.
 2021 Jan; 22(1): 75.

3.
 Overcoming the LAG-3 phase problem.
Nat Immunol.
2022; 23(7): 993–995.

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