LAIR2 Human

What is LAIR2 and how does it differ from LAIR1?

LAIR2 is a soluble homolog of the membrane-bound inhibitory receptor LAIR1, sharing 83% identity with the LAIR1 extracellular region. Unlike LAIR1, which contains inhibitory ITIM motifs in its cytoplasmic tail, LAIR2 functions as a secreted protein that can act as a natural decoy receptor. LAIR2 binds to collagens with higher affinity than LAIR1 and serves as a natural antagonist, blocking LAIR1-mediated inhibitory signaling. This competitive interaction represents a novel mechanism of immune regulation, where the soluble LAIR2 can modulate the inhibitory potential of membrane-bound LAIR1 by competing for collagen binding .

What is the expression pattern of LAIR2 in human tissues?

LAIR2 gene expression broadly mirrors that of LAIR1 in human peripheral blood mononuclear cells (PBMCs). In vitro studies have identified CD4+ T cells as the main producers of LAIR2, though other cell types may produce it in vivo. The protein is expressed as a soluble receptor and can be detected in biological fluids. Notably, LAIR2 is found in high amounts in the urine of pregnant women and at increased levels in the synovial fluid of patients with rheumatoid arthritis compared to osteoarthritis patients. LAIR2 expression is also detected in human placental villus and decidua during the first trimester of pregnancy .

Is LAIR2 conserved across species?

LAIR2 is a human-specific gene that is not expressed in mice or other non-primate species. Comparative genomic analysis identified LAIR2 among 2,272 human-specific genes, with 807 of these being expressed in the placenta. This species-specificity suggests that LAIR2 may have evolved to serve specific functions in human biology, particularly in immune regulation and potentially in placental development. While LAIR2 protein is not present in rodents, it can bind to rat and mouse collagens due to the highly conserved nature of collagens across species .

What methods are available for measuring LAIR2 protein in biological samples?

LAIR2 protein can be detected and quantified using various immunological techniques. The most common approach is enzyme-linked immunosorbent assay (ELISA), with commercial DuoSet ELISA kits available specifically for human LAIR2 detection. These kits typically include optimized capture and detection antibody pairs along with recombinant standards. For research requiring custom approaches, researchers can develop sandwich ELISAs using specific antibodies against LAIR2. Flow cytometry can also be employed for functional assays, particularly when examining LAIR2's ability to block LAIR1-collagen interactions .

How can researchers generate recombinant LAIR2 for functional studies?

Recombinant LAIR2 proteins can be generated through several approaches:

• Gene synthesis and expression in mammalian cell lines: The human LAIR2 gene can be synthesized and genetically fused with protein tags (such as Fc domains) to create fusion proteins with desired properties.

• Stable cell line development: Stable CHO cell lines expressing recombinant human LAIR2 fusion proteins can be developed using systems such as the Lonza GS system. This involves transfecting cells by electroporation with linearized plasmid DNA containing the LAIR2 sequence.

• Protein purification: LAIR2 fusion proteins can be purified from cell culture supernatants using affinity chromatography, with protein purity determined by HPLC and SDS-PAGE.

For enhanced functionality, researchers have developed dimeric LAIR2 constructs, such as NC410, which consists of LAIR2 fused to a human IgG1 Fc domain. Such dimeric structures provide higher avidity for collagen binding compared to monomeric endogenous LAIR2 .

What functional assays can be used to study LAIR2 activity?

Several functional assays have been developed to evaluate LAIR2 activity:

• Collagen binding assays: These assess the ability of LAIR2 to bind various collagen types, utilizing biotinylated LAIR2 proteins and measuring binding through techniques like ELISA or surface plasmon resonance.

• LAIR1-collagen blocking assays: These determine LAIR2's capacity to prevent LAIR1 binding to collagen, often using LAIR1-Fc fusion proteins as competitors.

• Reporter cell assays: Systems employing cells expressing the human LAIR1 extracellular domain fused to CD3ζ, coupled with NFAT-GFP reporters, can visualize LAIR1-mediated signal induction and its inhibition by LAIR2.

• T cell functional assays: These measure how LAIR2 affects T cell expansion and effector function, using techniques like flow cytometry to assess proliferation and cytokine production.

• In vivo tumor models: Humanized mouse models with human PBMC engraftment can be used to evaluate how LAIR2 affects tumor growth and immune response in a more complex biological system .

What is the potential role of LAIR2 in cancer immunotherapy?

Researchers have developed NC410, a dimeric LAIR2 human IgG1 Fc fusion protein, which demonstrated efficacy in preclinical studies:

• NC410 increased human T cell expansion and effector function in vivo in mouse xenogeneic-graft versus-host disease models

• In humanized mouse tumor models, NC410 reduced tumor growth in a T cell-dependent manner

• Immunohistochemical analysis showed NC410 binding to collagen-rich areas where LAIR1+ immune cells localize

The therapeutic potential may extend beyond monotherapy, as combining LAIR2-based therapeutics with established checkpoint inhibitors like anti-PD-1 antibodies may enhance efficacy. Recent studies have shown that abrogating LAIR1 immunosuppression through LAIR2 overexpression sensitizes resistant lung tumors to anti-PD-1 therapy .

How does LAIR2 expression correlate with pregnancy and pregnancy complications?

LAIR2 exhibits increased expression during normal pregnancy, with high amounts detectable in the urine of pregnant women. This elevated expression appears physiologically relevant, as reduced LAIR2 expression has been associated with pregnancy complications. LAIR2 is expressed in human placental villus and decidua during the first trimester, suggesting a potential role in placental development and function.

Interestingly, human-specific LAIR2 may contribute to the aggressive invasive properties of human placental extravillous trophoblast cells (EVTs). In vitro studies demonstrated that knockdown of LAIR2 markedly improved cell viability but inhibited the invasive ability of the human trophoblast cell line HTR8/SVneo. This suggests LAIR2 may have evolved to enhance the invasiveness of human placenta to meet the physiological demands of human fetal development .

What is the relationship between LAIR2 and inflammatory conditions?

LAIR2 levels are elevated in inflammatory conditions, particularly in autoimmune diseases. Studies have detected increased LAIR2 protein in the synovial fluid of patients with rheumatoid arthritis compared to osteoarthritis patients, suggesting a potential role in the inflammatory process. LAIR2 is also found in the circulation of patients with autoimmune thyroid disease.

The correlation between LAIR2 and inflammation makes biological sense, as CD4+ T cells, which are key players in many inflammatory and autoimmune conditions, are major producers of LAIR2. The increased levels may represent an attempt to counteract excessive collagen-mediated immune suppression in inflammatory environments, as LAIR2 can block the inhibitory interaction between LAIR1 and collagen.

This relationship with inflammation may have therapeutic implications beyond cancer, potentially extending to autoimmune and inflammatory diseases where modulation of immune inhibitory signals could be beneficial .

How does the dimeric structure of engineered LAIR2 therapeutics impact their function?

The dimeric structure of engineered LAIR2 therapeutics, such as NC410, significantly enhances their functional properties compared to monomeric endogenous LAIR2. This structural modification creates important differences:

• Higher avidity binding: The dimeric structure provides higher avidity for collagen binding, potentially improving the ability to compete with LAIR1 for collagen binding sites in the tumor microenvironment.

• Extended half-life: Fusion with an IgG1 Fc domain increases the molecular weight and likely extends the in vivo half-life compared to native LAIR2.

• Enhanced tissue penetration: NC410 demonstrated the ability to penetrate collagen-rich areas in human tumors where LAIR1+ immune cells localize.

• Differential dose-response: Studies observed peak activity of NC410 at different doses in different tumor models (1 mg/kg in P815 vs. 10 mg/kg in HT-29), suggesting model-specific or mechanism-specific dose requirements that may relate to the dimeric structure.

These structural considerations have critical implications for therapeutic development, as the dimeric design may provide advantages over simply delivering additional monomeric LAIR2 protein or inducing endogenous LAIR2 expression .

What are the challenges in developing accurate animal models for studying LAIR2 function?

Developing animal models for studying LAIR2 function presents several significant challenges:

• Species-specificity: LAIR2 is human-specific and not present in rodents, making conventional mouse models inadequate for studying native LAIR2 biology.

• Humanized mouse limitations: While humanized mice with human PBMC engraftment provide a platform for investigating LAIR2's effects on human immune cells, these models lack human myeloid cells, potentially missing important aspects of LAIR2 function.

• Tumor microenvironment complexity: Subcutaneous tumor models used in current studies may not accurately recapitulate the organ-specific extracellular matrix (ECM), architecture, and immune composition found in human tumors.

• Cross-reactivity considerations: Though LAIR2 is human-specific, it can bind mouse collagens due to the conserved nature of collagens across species. This creates a complex situation where the human protein can interact with mouse ligands but may not engage with all relevant mouse signaling pathways.

To address these challenges, researchers are considering more sophisticated approaches, including orthotopic tumor models that better mimic organ-specific ECM and developing more complete humanized immune system models that include myeloid lineages .

What mechanisms beyond LAIR1 antagonism might contribute to LAIR2 biological effects?

While LAIR2's primary known function is antagonizing LAIR1-collagen interactions, several additional mechanisms may contribute to its biological effects:

• Complement pathway modulation: LAIR2 Fc can efficiently inhibit complement activation via classical and lectin pathways, suggesting LAIR2 may influence immune response through complement regulation.

• Impact on myeloid cells: Though most studies focus on T cells, LAIR2 may affect LAIR1+ myeloid cell function, including monocytes, macrophages, and dendritic cells, which express high levels of LAIR1.

• ECM remodeling effects: By binding to collagens, LAIR2 might influence ECM structure and properties beyond simply blocking LAIR1 access.

• Interaction with other receptors: LAIR2 may block other receptors or have additional functions beyond competing with LAIR1, potentially affecting various immune cell subsets.

• C1q interactions: LAIR1 can bind C1q, which is increased in tumors and may provide a local inhibitory effect. LAIR2 could potentially neutralize this interaction as well.

These potential mechanisms require further investigation and may be explored as part of ongoing clinical trials and additional preclinical models to fully understand the multifaceted nature of LAIR2 biology .

How might differential binding of LAIR2 to various collagen types impact therapeutic efficacy?

The differential binding of LAIR2 to various collagen types may significantly impact therapeutic efficacy in several ways:

• Tissue-specific effects: Different tissues and tumors express distinct collagen profiles. LAIR2's binding affinity for various collagen types (particularly collagen I and III) may determine its tissue-specific activity.

• Tumoral versus healthy collagen targeting: The hypothesis that LAIR2 may bind with higher avidity to tumoral collagen compared to healthy collagen could explain therapeutic selectivity, though this requires additional study.

• Collagen modification effects: Post-translational modifications of collagens differ between healthy and diseased tissues. How these modifications affect LAIR2 binding could influence therapeutic outcomes.

• Competitive dynamics: The relative abundance of different collagen types in the tumor microenvironment versus healthy tissues may create differential competitive landscapes for LAIR2, LAIR1, and other collagen-binding proteins.

Understanding these differential binding dynamics could help optimize LAIR2-based therapeutics and predict which cancer types or patient populations might respond best to treatment .

What are the most critical unanswered questions in LAIR2 research?

Several critical questions remain unanswered in LAIR2 research:

• Cell source identification: Which cells are the main LAIR2 producers in vivo, and what factors drive its production and secretion? While CD4+ T cells produce LAIR2 in vitro, comprehensive in vivo cellular sources remain undefined.

• Regulatory mechanisms: What regulates LAIR2 expression during development, pregnancy, and disease states? Understanding these regulatory pathways could enable therapeutic modulation of endogenous LAIR2.

• Evolutionary significance: Why has LAIR2 been maintained in humans but not in mice? What evolutionary advantages does this human-specific protein confer?

• LAIR1-independent functions: Does LAIR2 have functional roles beyond antagonizing LAIR1-collagen interactions? This includes potential effects on complement pathways and other immune mechanisms.

• Combination therapy optimization: How can LAIR2-based therapeutics be optimally combined with other immunotherapies to maximize efficacy while minimizing side effects?

Addressing these questions will require innovative experimental approaches and may yield important insights for therapeutic development .

What emerging technologies might accelerate LAIR2 research?

Several emerging technologies hold promise for accelerating LAIR2 research:

• Advanced humanized mouse models: Development of more complete humanized immune system models that include both lymphoid and myeloid compartments would enable more comprehensive in vivo studies of LAIR2 function.

• Organoid and tissue-chip technologies: These could provide more physiologically relevant models of human tissue-specific ECM and cellular interactions for studying LAIR2 in different microenvironments.

• Single-cell RNA sequencing: This technology could identify the specific cell populations producing LAIR2 in different tissues and disease states with unprecedented resolution.

• CRISPR-based genetic screens: These could identify genes and pathways that regulate LAIR2 expression or are regulated by LAIR2, expanding our understanding of its biological network.

• Advanced imaging techniques: Methods like intravital microscopy could visualize LAIR2-collagen interactions and their effects on immune cell behavior in real-time in living tissues.

• Computational modeling: Machine learning approaches could predict optimal LAIR2 variant designs and combination therapies based on existing data and molecular structures.

These technologies, applied to LAIR2 research, could accelerate discovery and translation to clinical applications .