BIN4 Antibody

How does LILRB4 mediate immunosuppression in the tumor microenvironment?

LILRB4 mediates immunosuppression through multiple mechanistic pathways:

• T cell suppression: When activated by its ligands, LILRB4 on AML cells triggers signaling that inhibits T cell function, creating an immunosuppressive environment favorable for cancer growth .

• Tumor infiltration: LILRB4 facilitates tissue infiltration of monocytic AML cells, enabling extramedullary disease manifestations including gum infiltrates and involvement of cutaneous tissues and cerebrospinal fluid .

• MDSC-mediated immunosuppression: In solid tumors, LILRB4 acts as an immune checkpoint on MDSCs, facilitating their immunosuppressive functions as discovered by Jim Allison and colleagues .

These mechanisms operate through LILRB4's interaction with specific ligands such as apolipoprotein E (ApoE) and galectin-8, triggering downstream signaling that ultimately leads to immune evasion and enhanced tumor progression .

What methods are employed for generating effective LILRB4-blocking antibodies?

The development of effective LILRB4-blocking antibodies involves several sophisticated methodological approaches:

• CDR-grafting humanization: The generation of humanized antibodies against LILRB4 employs complementarity determining regions (CDR)-grafting strategies. This process involves defining CDRs in the heavy and light chains using multiple methods (Kabat, IMGT, and Paratome), aligning the parental antibody (e.g., rabbit) with the most closely related human germline sequence, and transferring the CDRs to create a humanized version with preserved binding affinity and reduced immunogenicity .

• Chimeric receptor reporter systems: These systems are designed to test an antibody's ability to bind to the extracellular domain (ECD) of LILRB4 and determine whether it functions as an agonist (activating signaling) or antagonist (blocking signaling). The system incorporates the LILRB4 ECD fused to the intracellular domain of paired immunoglobulin-like receptor (PILR) β, which signals through DAP-12 to activate an NFAT promoter driving GFP expression .

• Competitive binding assays: These are critical for identifying antibodies that specifically block the interaction between LILRB4 and its ligands. For example, to screen LILRB4 blocking antibodies that inhibit ApoE interaction, plates are precoated with recombinant ApoE protein before adding LILRB4 reporter cells and candidate antibodies. The percentage of GFP-positive cells is then measured to determine blocking efficacy .

How are epitopes mapped for anti-LILRB4 antibodies?

Epitope mapping for anti-LILRB4 antibodies utilizes sophisticated biolayer interferometry (BLI) techniques to identify the specific binding regions and classify antibodies into epitope bins:

• BLI-based sandwich epitope binning: This approach uses an Octet RED96 System with protein A biosensors. The process follows these steps:

  • Loading the first antibody onto protein A biosensors

  • Blocking remaining Fc-binding sites with an irrelevant antibody

  • Exposing biosensors to recombinant LILRB4 to saturate the binding site

  • Testing binding of secondary antibodies

• Classification criteria:

  • Same epitope bin (competitors): No increased binding signal observed with the second antibody

  • Different epitope bins (non-competitors): Increased binding signal observed with the second antibody

• Data analysis: Raw data are processed using specialized software (e.g., ForteBio's data analysis software) to classify antibodies into distinct epitope bins, which informs antibody selection for further development .

This methodical approach enables researchers to develop antibody panels targeting diverse epitopes on LILRB4, increasing the likelihood of identifying candidates with optimal therapeutic properties.

What techniques are used to determine the binding kinetics and affinity of anti-LILRB4 antibodies?

Binding kinetics and affinity determination for anti-LILRB4 antibodies primarily employ biolayer interferometry (BLI), which provides detailed kinetic parameters:

The experimental protocol involves:

• Loading antibody (typically 30 μg/mL) onto protein G biosensors for 4 minutes

• Establishing a baseline in kinetics buffer

• Exposing loaded biosensors to varying concentrations of recombinant LILRB4 (0.1-200 nM)

• Using background subtraction to correct for sensor drifting

• Conducting all experiments with shaking at 1,000 rpm

• Measuring background wavelength shifts from reference biosensors loaded only with antibody

• Fitting data to a 1:1 binding model using ForteBio's data analysis software to extract association and dissociation rates

This analytical approach enables precise comparison of different antibody candidates and selection of those with optimal binding properties for therapeutic development.

How do researchers validate the functional activity of LILRB4-blocking antibodies?

Validation of LILRB4-blocking antibodies involves multiple functional assays addressing different aspects of LILRB4 biology:

• Ligand-blocking assays: These assess the antibody's ability to block LILRB4 interaction with ligands like ApoE using chimeric receptor reporter systems. Effective blocking antibodies prevent GFP expression in these reporter systems when ApoE is present .

• T cell activation assays: Since LILRB4 suppresses T cell activation, functional antibodies should reverse this suppression. Researchers measure T cell proliferation, cytokine production, and activation markers in co-culture systems with and without the blocking antibody .

• Cell migration and invasion assays: These evaluate the antibody's ability to inhibit LILRB4-mediated tissue infiltration by measuring cancer cell migration through membranes or matrices in the presence or absence of the antibody .

• Effector function assays: For therapeutic antibodies, evaluation of:

  • Antibody-dependent cellular cytotoxicity (ADCC)

  • Antibody-dependent cellular phagocytosis (ADCP)
    These mechanisms are critical for the anti-cancer activity of LILRB4-targeted antibodies .

• In vivo validation: Using multiple mouse models including:

  • Patient-derived xenograft (PDX) models

  • Syngeneic immunocompetent AML mouse models
    These assess the antibody's ability to reduce tumor burden, modulate immune responses, and enhance survival .

How can LILRB4 antibodies be optimized for improved efficacy in treating AML?

Optimizing LILRB4 antibodies for AML treatment involves several advanced strategies:

• Combination therapy approaches: The humanized LILRB4 antibody h128-3 has shown enhanced anti-AML efficacy when combined with chemotherapy. This synergistic effect occurs because the antibody stimulates mobilization of leukemia cells from tissues into the circulation, making them more susceptible to chemotherapeutic agents .

• Fc engineering: Modifying the Fc region of LILRB4 antibodies can enhance their effector functions:

  • Enhancing ADCC activity through glycoengineering or amino acid substitutions

  • Optimizing ADCP by modifying binding to specific Fcγ receptors on macrophages
    These modifications can significantly improve the antibody's ability to eliminate AML cells .

• Bispecific antibody development: Creating bispecific antibodies that simultaneously target LILRB4 and another relevant antigen (e.g., CD3 on T cells) could potentially enhance T cell recruitment and activation at tumor sites.

• Understanding resistance mechanisms: Research into why certain AML cases might be resistant to LILRB4-targeted therapy is crucial for developing second-generation antibodies that can overcome these resistance mechanisms .

What is the role of galectin-8 as an LILRB4 ligand, and how does this impact antibody development?

The discovery of galectin-8 (Gal-8) as an LILRB4 ligand has important implications for antibody development:

• Mechanistic insights: Gal-8 has been identified as a ligand that drives myeloid-derived suppressor cell (MDSC) function through LILRB4 binding. This interaction promotes immune evasion in solid tumors, making it a significant therapeutic target .

• Antibody targeting strategy: The Gal-8/LILRB4 interaction represents an alternative target pathway distinct from the previously identified ApoE/LILRB4 pathway. This provides researchers with multiple options for therapeutic intervention:

  • Direct LILRB4 blocking antibodies

  • Gal-8 neutralizing antibodies

  • Dual-targeting approaches

• Solid tumor applications: While initial LILRB4 antibody development focused on AML, the Gal-8 discovery expands potential applications to solid tumors where MDSCs play a critical immunosuppressive role .

• In vivo evidence: Antibodies functionally blocking Gal-8 have demonstrated the ability to suppress tumor growth in vivo, suggesting that targeting this specific interaction pathway has therapeutic potential .

The identification of multiple LILRB4 ligands (ApoE and Gal-8) highlights the complexity of LILRB4 biology and necessitates comprehensive screening of antibody candidates against multiple ligand interactions to ensure optimal therapeutic efficacy.

What is the current status of LILRB4-targeted antibodies in clinical trials?

The clinical development of LILRB4-targeted antibodies has progressed significantly:

• Current clinical trials: Several biopharma companies including Merck, NGM Bio, Jounce Therapeutics, and Biond Biologics have advanced LILRB4-targeted antibodies to preclinical and clinical trials for treating solid tumors .

• Phase I results: NGM Bio has reported early results from a Phase Ia trial at the European Society for Medical Oncology annual meeting in November 2022. Among 24 response-evaluable patients:

  • 1 patient achieved partial response

  • 6 patients maintained stable disease

  • 1 patient showed noncomplete response/nonprogressive disease

• Target populations: While initial development focused on monocytic AML, clinical trials have expanded to include various solid tumors based on LILRB4's role as an immune checkpoint on MDSCs .

• Combinatorial approaches: Some trials are likely exploring the combination of LILRB4 antibodies with established immunotherapies such as PD-1/PD-L1 inhibitors to enhance therapeutic efficacy.

• Biomarker development: Parallel efforts are underway to identify predictive biomarkers for patient selection, as LILRB4 expression levels and the presence of specific ligands may influence treatment response .

What challenges remain in developing effective LILRB4-targeted therapeutics?

Despite promising advances, several significant challenges remain in developing effective LILRB4-targeted therapeutics:

• Target heterogeneity: LILRB4 expression varies across different tumor types and individual patients. Developing methods to predict which patients will benefit most from LILRB4-targeted therapy remains challenging .

• Multiple ligand interactions: With multiple ligands identified (ApoE and Gal-8), determining which interaction is most critical to block in different cancer contexts requires extensive research .

• Epitope selection: Identifying the optimal epitope to target on LILRB4 for maximum therapeutic effect requires sophisticated epitope binning and functional screening approaches .

• Resistance mechanisms: Understanding potential resistance mechanisms to LILRB4 antibodies is crucial for developing effective second-generation therapeutics and optimal combination strategies.

• Clinical translation: While preclinical models show promising results, translating these findings to humans presents typical challenges of immunotherapy development:

  • Differences in immune system function between mice and humans

  • Heterogeneity of human cancers compared to model systems

  • Identifying optimal dosing schedules and combination approaches

• Biomarker development: Creating reliable biomarkers to predict and monitor response to LILRB4-targeted therapy remains an active area of research needed to advance these therapeutics.