LAG3 Monoclonal Antibody

What is LAG-3 and how does it function in the immune system?

LAG-3 (CD223) is a cell surface inhibitory receptor belonging to the immunoglobulin superfamily that regulates immune homeostasis through multiple biological activities related to T-cell functions. Structurally similar to CD4, LAG-3 binds to MHC-II with considerably higher affinity, transmitting inhibitory signals through its cytoplasmic domain to down-regulate CD4+ T lymphocytes. This interaction negatively regulates T cell antigen-stimulated activation, proliferation, cytokine production, and cytotoxicity .

LAG-3 also interacts with the TCR complex on activated CD4 and CD8 T cells, downregulating TCR signaling. Beyond MHC-II, LAG-3 interacts with additional ligands including LSECtin, Galectin-3 (Gal-3), and Fibrinogen-like protein 1 (FGL1). The FGL1/LAG-3 interaction occurs via the LAG-3 D1 and D2 domains and the FGL1 fibrinogen-like domain in an MHC-II-independent manner .

Which cell types express LAG-3 and what regulates its expression?

LAG-3 is expressed on multiple immune cell types, primarily:

• CD4+ T cells (especially Th1 cells, with weak or no expression on Th0 and Th2 clones)

• CD8+ T cells

• Regulatory T cells (Tregs)

• Natural Killer (NK) cells

• B cells

• Dendritic cells

• Microglia and neurons in the central nervous system

Cytokines regulate LAG-3 expression, with IL-2, IL-7, and IL-12 upregulating its expression. IL-12 provides the strongest stimulus for LAG-3 expression, and LAG-3 expression on activated CD4+ subsets correlates with higher intracellular interferon-gamma production .

How does LAG-3 expression impact cancer progression and patient prognosis?

LAG-3 expression varies across tumor types and can significantly impact patient prognosis:

These variable outcomes across cancer types highlight the context-dependent role of LAG-3 and the importance of considering tumor-specific microenvironments when designing therapeutic approaches.

What are the different types of LAG-3-targeting therapeutic agents?

LAG-3-targeting therapeutic agents can be classified into three main categories:

• Anti-LAG-3 monoclonal antibodies: Single-specificity antibodies that directly target LAG-3, such as TSR-033, which is a high-affinity human IgG4 monoclonal antibody

• Anti-LAG-3 bispecific antibodies: Dual-targeting antibodies that simultaneously engage LAG-3 and another immune checkpoint, such as MGD013 (tebotelimab), which is a humanized, hinge-stabilized, IgG4-κ tetravalent bispecific antibody targeting both PD-1 and LAG-3 with high affinity

• Soluble LAG-3-Ig fusion proteins: Recombinant proteins consisting of the extracellular domain of LAG-3 fused to an immunoglobulin Fc region

The mechanism of action typically involves disrupting the interaction between LAG-3 and its ligands (primarily MHC-II, but also FGL1 and others), thereby preventing inhibitory signaling and enhancing T cell activation and function.

What is the mechanism of action for anti-LAG-3 monoclonal antibodies?

Anti-LAG-3 monoclonal antibodies function by:

• Binding with high affinity to LAG-3 expressed on immune cells, particularly activated T cells

• Blocking the interaction between LAG-3 and its ligands (MHC-II, FGL1, Galectin-3, LSECtin)

• Preventing the transmission of inhibitory signals through LAG-3's cytoplasmic domain

• Relieving LAG-3-mediated suppression of T cell activation, proliferation, and effector functions

• Enhancing antitumor immune responses through increased T cell activity

By targeting LAG-3, these antibodies can restore T cell function in the tumor microenvironment, particularly in cases where T cells are dysfunctional or exhausted due to prolonged antigen exposure and inhibitory checkpoint expression.

How do bispecific antibodies targeting LAG-3 differ from conventional monoclonal antibodies?

Bispecific antibodies targeting LAG-3, such as MGD013 (tebotelimab), offer several advantages over conventional monoclonal antibodies:

• Dual targeting capability: They simultaneously target LAG-3 and another immune checkpoint (typically PD-1), allowing for concurrent blockade of two inhibitory pathways with a single molecule

• Enhanced specificity for dysfunctional T cells: They can specifically target PD-1+ LAG-3+ highly dysfunctional T cells, which represent a particularly suppressed subset of immune cells in the tumor microenvironment

• Increased efficacy: PD-1/LAG-3 co-blockade has demonstrated increased cytokine secretion and enhanced T-cell responses compared to single blockade of either PD-1 or LAG-3 alone

• Potential to overcome resistance: Bispecific antibodies may help address resistance to single-agent checkpoint inhibitors by targeting multiple inhibitory pathways simultaneously

MGD013, for example, has demonstrated favorable biophysical and manufacturability properties with a prolonged half-life, and preliminary clinical data show encouraging responses and acceptable pharmacokinetics .

What are optimal protocols for assessing LAG-3 expression in different tissue samples?

For researchers studying LAG-3 expression in tissue samples, consider these methodological approaches:

• Immunohistochemistry (IHC):

  • Use validated anti-LAG-3 antibodies such as clone 3DS223H

  • Include positive controls (activated T cells or known LAG-3+ tumors)

  • Quantify expression using digital pathology when possible

  • Always correlate with other immune markers (CD3, CD4, CD8) for context

• Flow cytometry:

  • Fresh tissue samples should be processed rapidly to maintain cell viability

  • Use multiple fluorescent markers to identify LAG-3 expression on specific cell subsets

  • Include FMO (Fluorescence Minus One) controls to accurately determine positive populations

  • Consider analysis of both surface and intracellular LAG-3 where relevant

• Multiplex immunofluorescence:

  • Enables simultaneous detection of LAG-3 with other immune checkpoint molecules

  • Provides spatial context for LAG-3 expression within the tumor microenvironment

  • Useful for analyzing co-expression patterns (LAG-3/PD-1/TIM-3/CTLA-4)

• RNA analysis:

  • qRT-PCR and RNA sequencing provide mRNA expression levels

  • Important to validate transcriptional findings at protein level due to post-transcriptional regulation

When evaluating LAG-3 expression in clinical samples, it's critical to standardize processing times and preservation methods, as these can significantly affect detection sensitivity .

How can researchers effectively evaluate the functional activity of anti-LAG-3 antibodies?

To evaluate the functional activity of anti-LAG-3 antibodies, researchers should employ multiple complementary assays:

• Binding assays:

  • ELISA to determine binding affinity (KD) to recombinant LAG-3

  • Flow cytometry to confirm binding to native LAG-3 on cells

  • Surface plasmon resonance for detailed binding kinetics

• Functional T cell assays:

  • Measure IL-2 and IFN-γ production in presence of anti-LAG-3 antibodies

  • Assess T cell proliferation using CFSE dilution or Ki-67 staining

  • Evaluate cytotoxic activity using chromium release or flow-based killing assays

  • Mixed lymphocyte reactions to assess impact on T cell activation

• Competitive binding assays:

  • Determine if the antibody blocks LAG-3 interaction with MHC-II, FGL1, or other ligands

  • Compare activity to benchmark antibodies with known mechanisms

• In vivo models:

  • Syngeneic tumor models to assess antitumor efficacy

  • Humanized mouse models for human-specific antibodies

  • Monitor changes in tumor-infiltrating lymphocyte (TIL) number and phenotype

Remember that antibody isotype significantly impacts functional outcomes, with IgG4 antibodies typically chosen for therapeutic applications due to reduced Fc-mediated effector functions .

What are the key considerations for designing effective LAG-3 blockade experiments?

When designing LAG-3 blockade experiments, researchers should consider:

• Model selection:

  • Choose models with documented LAG-3 expression in the tumor microenvironment

  • Consider models resistant to PD-1 monotherapy to evaluate combination potential

  • For immunocompetent models, ensure species cross-reactivity of the antibody

• Experimental controls:

  • Include isotype-matched control antibodies

  • Consider using LAG-3 knockout models as controls

  • Include PD-1 blockade arms for comparison and combination studies

• Dosing and scheduling:

  • Determine optimal antibody concentration through dose-response studies

  • Consider different dosing schedules (concurrent vs. sequential for combinations)

  • Ensure adequate serum levels throughout the experimental period

• Comprehensive immune monitoring:

  • Monitor multiple cell populations (CD4+ T cells, CD8+ T cells, Tregs, NK cells)

  • Assess changes in both peripheral blood and tumor-infiltrating immune cells

  • Evaluate systemic and local cytokine production

• Potential confounding factors:

  • Consider the impact of tumor size at treatment initiation

  • Account for microbiome variations in animal studies

  • Control for stress and other environmental factors that may affect immune response

How do LAG-3 monoclonal antibodies perform in combination with other checkpoint inhibitors?

Combination therapies with LAG-3 monoclonal antibodies and other checkpoint inhibitors have shown promising results:

• LAG-3 and PD-1 co-blockade:

  • Dual blockade of LAG-3 and PD-1 has demonstrated enhanced efficacy compared to single-agent therapy

  • In murine models, combination therapy showed strong anti-tumor effects in mice resistant to single antibody treatment without obvious evidence of autoimmunity

  • Clinical trials confirm these findings, with long-term disease control observed in patients with prior acquired resistance to PD-(L)1 therapy

• LAG-3, PD-1, and CTLA-4 combinations:

  • Triple blockade against PD-1/LAG-3/CTLA-4 resulted in tumor-free survival in 20% of treated mice

  • Dual blockade of LAG-3 and CTLA-4 pathways in PD-1 knockout mice led to tumor-free survival in 40% of treated mice

• Bispecific antibodies:

  • MGD013 (tebotelimab), a PD-1/LAG-3 bispecific antibody, has shown antitumor activity in multiple tumor types

  • One complete response was observed after single MGD013 administration in a patient who had received chimeric antigen receptor (CAR)-T-cell therapy

  • Combination of margetuximab (anti-HER2) with PD-1xLAG-3 DART® enhanced lytic activity of immune cells, with an objective response rate of 42.9% in relapsed or refractory HER2+ solid tumors

High baseline LAG-3/PD-1 expression and IFN-γ high gene signatures (CXCL9, CXCL10, CXC11, STAT1) were associated with objective clinical responses to combination therapy .

What is the current landscape of clinical trials for LAG-3-targeting therapies?

As of early 2025, the clinical trial landscape for LAG-3-targeting therapies is extensive:

• Clinical trial volume and distribution:

  • 97 clinical trials evaluating at least 16 LAG-3-targeting molecules

  • Two trials have reached phase III (sponsored by BMS and Merck)

  • Phase III studies in melanoma and colorectal cancer are demonstrating encouraging results

  • Most trials are testing LAG-3-antagonistic molecules, including combinations with other immune checkpoint inhibitors

• Types of investigational agents:

  • Conventional anti-LAG-3 monoclonal antibodies

  • Next-generation bispecifics with dual targeting capabilities

  • Various combination strategies with other immunotherapeutic agents

• Clinical outcomes:

  • LAG-3-targeting cancer immunotherapies have demonstrated good safety profiles and tolerability

  • Adequate pharmacokinetics and pharmacodynamics have been observed

  • Promising antitumor efficacy has been reported, particularly in combination settings

  • Simultaneous co-expression of LAG-3 with other immune checkpoint molecules characterizes highly dysfunctional T cells in cancer patients, making them prime targets for bispecific approaches

The co-blockade of LAG-3 with PD-1 shows particular promise for counteracting resistance to current immunotherapies, though long-term clinical efficacy remains to be fully established.

What biomarkers predict response to LAG-3-targeting therapies?

Several potential biomarkers have been identified for predicting response to LAG-3-targeting therapies:

• LAG-3 expression levels:

  • Baseline LAG-3 expression on tumor-infiltrating lymphocytes correlates with response

  • The LAG-3/CD8 ratio in tumor samples may provide prognostic information

• Co-expression patterns:

  • Co-expression of LAG-3 with PD-1 identifies highly exhausted T cells that may respond to dual blockade

  • High baseline LAG-3/PD-1 expression has been associated with objective clinical responses to combination therapy

• Gene expression signatures:

  • IFN-γ high gene signature (CXCL9, CXCL10, CXC11, STAT1) correlates with response

  • In lung adenocarcinoma, CTSF-associated LAG-3 expression patterns may predict outcomes

• Tumor mutational profile:

  • Tumors with KRAS mutation demonstrate higher numbers of LAG-3+ cells than those with EGFR mutation in lung adenocarcinoma

  • Microsatellite instability and EBV status in gastric cancer influence LAG-3 expression patterns and therapeutic outcomes

The complex interplay between these factors highlights the need for comprehensive biomarker assessment to guide patient selection for LAG-3-targeting therapies.

How does the tumor microenvironment modulate LAG-3 expression and function?

The tumor microenvironment (TME) significantly impacts LAG-3 expression and function through multiple mechanisms:

• Cytokine milieu:

  • IL-12 in the TME strongly upregulates LAG-3 expression on T cells

  • Chronic exposure to IL-2 and IL-7 within the tumor can enhance LAG-3 expression

  • TGF-β promotes regulatory T cell development and can indirectly affect LAG-3 expression patterns

• Hypoxia and metabolic factors:

  • Hypoxic conditions common in solid tumors may alter LAG-3 expression and function

  • Metabolic competition within the TME can drive T cell exhaustion and upregulate multiple checkpoint molecules including LAG-3

• Tumor-specific ligand expression:

  • Upregulation of FGL1 by tumor cells provides an MHC-II-independent mechanism for LAG-3-mediated immunosuppression

  • Galectin-3 expression on tumor cells can engage LAG-3 on T cells to suppress their function

  • LSECtin expressed by tumor cells interacts with LAG-3, inhibiting antitumor T-cell responses by reducing CDK2, CDK4, and CDK6 expression

• Cellular cross-talk:

  • LAG-3+ regulatory T cells within the TME exhibit enhanced suppressive function

  • LAG-3 expression on NK cells may result in inhibitory feedback signals from surrounding MHC-II+ cells

  • In gastric cancer, LAG-3+ cell infiltration can associate with immunoevasive contexture featuring decreased IFN-γ+ cells and increased regulatory T cells and M2-like macrophages

Understanding these complex interactions is critical for developing effective therapeutic strategies targeting LAG-3 in different tumor contexts.

What mechanisms contribute to resistance to LAG-3-targeted therapies?

Several mechanisms may contribute to resistance to LAG-3-targeted therapies:

• Compensatory checkpoint upregulation:

  • Blockade of LAG-3 may lead to upregulation of alternative immune checkpoints

  • A study by Ding et al. demonstrated LAG-3 upregulation in TILs from NSCLC patients with acquired resistance to checkpoint blockers, suggesting dynamic regulation of multiple checkpoints

• Alternative ligand interactions:

  • Even with MHC-II binding blocked, LAG-3 may still interact with alternative ligands like FGL1

  • Complete disruption of all LAG-3 ligand interactions may be necessary for optimal therapeutic effect

• T cell-intrinsic dysfunction:

  • Chronic antigen exposure may lead to epigenetic changes in T cells that persist despite checkpoint blockade

  • Terminal exhaustion states may be refractory to LAG-3 blockade alone

• TME immunosuppressive mechanisms:

  • Myeloid-derived suppressor cells and regulatory T cells may maintain immunosuppression through LAG-3-independent mechanisms

  • Metabolic competition and hypoxia can impair T cell function despite LAG-3 blockade

• Tumor-intrinsic resistance mechanisms:

  • Downregulation of tumor antigens or MHC molecules

  • Activation of oncogenic pathways that promote immune evasion

  • Secretion of immunosuppressive factors that operate independently of LAG-3

These resistance mechanisms underscore the importance of combination approaches targeting multiple aspects of tumor-immune interactions.

How do LAG-3 variants and polymorphisms affect antibody binding and function?

LAG-3 variants and polymorphisms can significantly impact antibody binding and function, presenting important considerations for research and therapeutic development:

• Structural variants:

  • Alternative splicing can generate LAG-3 variants with altered domain structure

  • Deletions in key binding domains (D1-D4) may affect antibody recognition and function

  • Soluble LAG-3 variants can potentially interfere with antibody binding to membrane-bound LAG-3

• Single nucleotide polymorphisms (SNPs):

  • SNPs in the LAG-3 gene can alter protein structure and expression

  • Variations in binding epitopes may affect antibody recognition

  • Polymorphisms in regulatory regions can influence LAG-3 expression levels in response to various stimuli

• Species-specific differences:

  • Significant differences exist between human and murine LAG-3

  • These differences may limit the translational value of some preclinical models

  • Species-specific antibodies must be used in appropriate model systems

• Post-translational modifications:

  • Glycosylation patterns can vary and affect antibody binding

  • Differential processing of LAG-3 in various cell types may generate functionally distinct forms

  • Proteolytic cleavage of LAG-3 generates soluble forms that may have different immunological properties

For therapeutic development, understanding these variations is crucial as they may contribute to heterogeneous responses observed in clinical trials. Antibody engineering approaches that target conserved epitopes or multiple epitopes simultaneously may help address these challenges .

What novel LAG-3 targeting strategies are under development?

Beyond conventional antibodies, several innovative approaches to LAG-3 targeting are being explored:

• Multifunctional fusion proteins:

  • LAG-3 blockade combined with targeted cytokine delivery

  • Tri-specific antibodies targeting LAG-3, PD-1, and tumor-associated antigens

• Cell-based therapies:

  • Genetic modification of CAR-T cells to be resistant to LAG-3 inhibition

  • Combination of adoptive cell therapy with LAG-3 blockade

  • Ex vivo expansion of T cells in the presence of LAG-3 blockers

• Small molecule inhibitors:

  • Development of small molecules targeting the LAG-3/MHC-II interaction

  • Inhibitors of intracellular LAG-3 signaling pathways

• LAG-3 ligand targeting:

  • Antibodies against FGL1 to block its interaction with LAG-3

  • Strategies targeting Galectin-3 and LSECtin as alternative approaches

• Combinations with emerging immunotherapies:

  • LAG-3 blockade combined with STING agonists or TLR modulators

  • Integration with cancer vaccines to enhance antigen-specific responses

  • Combination with microbiome-targeting approaches

These innovative strategies aim to overcome limitations of current approaches and address resistance mechanisms encountered with existing LAG-3-targeted therapies.

How might LAG-3 research impact immunotherapy beyond oncology?

LAG-3 research has potential applications beyond cancer immunotherapy:

• Autoimmune diseases:

  • LAG-3 agonists might suppress pathological immune responses

  • Targeting LAG-3+ pathogenic T cells could provide selective immunosuppression

  • Modulation of regulatory T cell function through LAG-3 could restore immune tolerance

• Infectious diseases:

  • LAG-3 blockade might reinvigorate exhausted T cells in chronic viral infections

  • Potential application in HIV, HBV, and HCV treatment

  • Role in enhancing vaccine efficacy against challenging pathogens

• Neurodegenerative disorders:

  • LAG-3 is expressed on microglia and neurons in the central nervous system

  • Modulating neuroinflammation through LAG-3 targeting could impact disease progression

  • Potential role in Alzheimer's disease and multiple sclerosis pathology

• Transplantation medicine:

  • LAG-3 modulation might help induce transplant tolerance

  • Selective targeting of alloreactive T cells through LAG-3

  • Combination approaches with other tolerance-inducing strategies

The expanding understanding of LAG-3 biology continues to reveal potential applications beyond oncology, highlighting its role as a central regulator of immune homeostasis.