CLE5 Antibody

What is CLEC5A and why is it relevant to immunological research?

CLEC5A is a C-type lectin receptor expressed primarily on myeloid cells, including monocytes, macrophages, neutrophils, and dendritic cells. It plays a crucial role in mediating inflammatory responses during microbial infections, particularly viral and bacterial challenges . CLEC5A is associated with immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein DAP12 and YINM motif-containing adaptor protein DAP10, which are critical for its signaling functions .

The receptor's importance in research stems from its capacity to form multivalent heterocomplexes with other receptors like DC-SIGN, leading to inflammatory cytokine cascades through Syk activation . This makes CLEC5A a compelling target for therapeutic interventions aimed at modulating inflammatory responses in various disease contexts, including viral infections and cancer immunotherapy.

How is CLEC5A expression regulated in different disease states?

CLEC5A expression exhibits notable variation across disease states, particularly in inflammatory conditions. In COVID-19 patients, for instance, high levels of CLEC5A expression have been observed in monocytes from severe cases compared to mild cases and unexposed subjects . Interestingly, vaccinated individuals who developed mild COVID-19 did not show elevated CLEC5A expression.

The receptor's expression pattern also varies across monocyte subsets. Research has demonstrated that people hospitalized with severe COVID-19 present with:

• Significantly lower percentage of classical monocytes (CD14++CD16−)

• Higher percentages of intermediate (CD14+CD16+) and nonclassical monocytes (CD14+CD16++)

• Significantly higher CLEC5A expression on nonclassical monocytes (CD14+CD16++) compared to mild COVID-19 cases and unexposed individuals

In tumor microenvironments, CLEC5A is expressed by tumor-infiltrating myeloid cell populations, including monocytes, macrophages, and neutrophils, as confirmed by FACS analysis in mouse MC38 colon adenocarcinoma and CT26 colorectal carcinoma tumor models .

What methodological approaches are used to assess CLEC5A-antibody binding?

Several complementary techniques can be employed to evaluate CLEC5A-antibody binding interactions:

• Surface Plasmon Resonance (SPR): This technique allows for characterization of specific binding between CLEC5A antibodies and recombinant human/mouse CLEC5A proteins. Studies have reported Kd values in the single-digit nM range or lower for both species using this method .

• Flow Cytometry Analysis: Flow cytometry provides a means to quantify CLEC5A expression on different cell populations and assess antibody binding to cell surface CLEC5A. This approach has been used to evaluate CLEC5A expression on monocyte subsets in various disease states .

• Functional Activation Assays: The binding efficacy can be assessed through downstream functional readouts like TNF production. For instance, when macrophages are co-cultured with target cells in the presence of CLEC5A-directed bispecific antibodies, robust TNF production indicates effective CLEC5A engagement .

• Competitive Inhibition Assays: Blocking experiments using monospecific CLEC5A-blocking antibodies or soluble recombinant CLEC5A proteins can confirm binding specificity by inhibiting the effects of the test antibody .

• Molecular Docking: In silico investigations can predict binding affinity and residue interactions between CLEC5A and potential binding partners like viral proteins. These computational approaches help in understanding molecular recognition mechanisms .

How do CLEC5A-directed bispecific antibodies drive Fcγ receptor-independent phagocytosis?

CLEC5A-directed bispecific antibodies represent an innovative approach to harness phagocytosis without the limitations imposed by inhibitory Fcγ receptors, particularly FcγRIIB. The mechanism involves several key components:

Experimental evidence demonstrates that this approach induces dose-dependent phagocytosis of target cells (e.g., CD20+ Raji cells) by human macrophages. The specificity of this process is confirmed by inhibition experiments using CLEC5A-blocking antibodies or soluble CLEC5A protein, and by the observation that CD20-negative cells are not phagocytosed .

What in vivo evidence supports the therapeutic potential of CLEC5A-directed antibodies in cancer?

CLEC5A-directed bispecific antibodies have demonstrated promising therapeutic potential in preclinical cancer models, particularly through strategic targeting of regulatory T cells (Tregs) in the tumor microenvironment:

• Target selection: OX40, a trimeric co-stimulatory receptor belonging to the TNFR superfamily, was chosen as a target due to its higher expression in FoxP3+ Tregs compared to FoxP3− CD4+ and CD8+ T cells .

• Antibody engineering: OX40/CLEC5A (1+1) and (2+1) bispecific mouse IgG2a antibodies with LALAPG mutations were generated to abolish FcγR binding .

• Tumor model efficacy: In both MC38 colon adenocarcinoma and CT26 colorectal carcinoma syngeneic mouse models, OX40/CLEC5A bispecific antibodies exhibited robust antitumor activity .

• Mechanism validation: The antitumor efficacy was correlated with significant depletion of CD4+FoxP3+CD25+ Treg cells in CT26 tumors, comparable to that caused by conventional anti-OX40 monoclonal antibodies. This was demonstrated by quantification of tumor-infiltrating Tregs 3 days after a single intravenous dose .

These findings provide compelling evidence that CLEC5A-directed bispecific antibodies can effectively deplete Tregs in the tumor microenvironment, potentially alleviating immunosuppression and enhancing antitumor immunity. This approach may circumvent limitations associated with conventional antibody treatments that rely on FcγR-mediated mechanisms.

How can researchers overcome challenges in generating antibodies against CLEC5A's extracellular regions?

Generating antibodies against extracellular regions (ECRs) of multispanning membrane proteins like CLEC5A presents significant challenges due to their complex structure, low productivity, and high sequence conservation across species. Based on methodological innovations with other membrane proteins, researchers can adopt several strategies:

• Optimized cell-free protein synthesis systems:

  • Utilize wheat cell-free protein synthesis systems to improve production of difficult membrane proteins

  • Suppress and normalize mRNA GC content to enhance protein expression

  • This approach has shown success with claudin-5, another difficult-to-produce membrane protein

• Engineered immunogen design:

  • Create human/mouse chimeric constructs to overcome high sequence conservation

  • Develop artificial membrane proteins with symmetrically arranged ECRs

  • Synthesize antigens as proteoliposomes to maintain native conformation

• Liposomal immunization:

  • Prepare engineered liposomal immunogens that preserve the native structure of extracellular domains

  • Administer via intraperitoneal immunization to enhance immune response against poorly immunogenic ECRs

• Screening strategies:

  • Implement comprehensive screening assays focused on ECR binding

  • Utilize functional assays to identify antibodies with desired biological activities

  • Perform specificity testing against related family members

These methodological approaches have demonstrated effectiveness for generating high-affinity, functionally active monoclonal antibodies against difficult membrane protein targets and could be adapted for CLEC5A antibody development.

What is the potential role of CLEC5A antibodies in COVID-19 therapeutics?

CLEC5A has emerged as a potential therapeutic target in COVID-19 based on several lines of evidence:

• Elevated expression in severe disease: High levels of CLEC5A expression are found in monocytes from severe COVID-19 patients compared to mild cases and unexposed subjects, suggesting a role in disease pathogenesis .

• Molecular interactions with SARS-CoV-2: Research indicates that CLEC5A can interact with SARS-CoV-2, promoting inflammatory cytokine production. In silico studies suggest this interaction may involve the receptor-binding domain in the N-acetylglucosamine binding site (NAG-601) of the virus .

• Spike protein triggering: CLEC5A expression can be triggered by SARS-CoV-2 spike glycoprotein, suggesting direct involvement in COVID-19 progression .

• In vitro antibody efficacy: The high expression of CLEC5A and elevated proinflammatory cytokine production were successfully reduced in vitro using a human CLEC5A monoclonal antibody .

• Temporal expression pattern: In hamster models, CLEC5A gene expression was detected during 3-15 days of Omicron strain viral challenge, providing insights into the kinetics of CLEC5A involvement .

How can researchers design experimental protocols to evaluate CLEC5A antibody-induced phagocytosis?

To rigorously evaluate CLEC5A antibody-induced phagocytosis, researchers should implement comprehensive protocols that assess multiple aspects of this process:

• Macrophage-based phagocytosis assay:

  • Prepare human macrophages derived from peripheral blood monocytes or mouse bone marrow-derived macrophages

  • Use fluorescently labeled target cells (e.g., CD20+ Raji cells for B-cell targeting or HER2+ SK-BR-3 cells for epithelial cancer targeting)

  • Co-culture macrophages and target cells at appropriate ratios with various concentrations of test antibodies

  • Quantify phagocytosis by flow cytometry using a gating strategy that distinguishes between cell association and true internalization

• Specificity controls:

  • Include control conditions with CD20-negative or HER2-negative cells to confirm target specificity

  • Perform blocking experiments using excess monospecific CLEC5A-blocking antibodies

  • Use soluble recombinant CLEC5A protein as a competitive inhibitor

  • Include isotype-matched irrelevant IgG controls

• Activation assessment:

  • Measure cytokine/chemokine production (TNF, IL-6, IL-1β, IL-8, RANTES, MIP-1α, MIP-1β, IL-10) using ELISA or multiplex assays

  • Confirm target-dependent activation by testing antibodies in the presence and absence of target cells

• Cross-species validation:

  • Compare activity between human and mouse macrophage systems to establish translational relevance

  • Assess both cytokine production and phagocytic activity across species

• Imaging-based confirmation:

  • Complement flow cytometry with confocal microscopy to visualize phagocytosis directly

  • Use z-stack imaging to confirm complete internalization versus surface binding

This multi-faceted approach provides robust validation of CLEC5A antibody-induced phagocytosis while establishing specificity, dose-dependence, and mechanistic insights.

What molecular engineering approaches optimize CLEC5A-directed bispecific antibodies?

The design and engineering of CLEC5A-directed bispecific antibodies requires careful consideration of several molecular parameters to achieve optimal functionality:

• Bispecific formats:

  • (1+1) configuration: One binding arm for CLEC5A and one for the target antigen

  • (2+1) configuration: Two binding arms for the target antigen and one for CLEC5A

  • Format selection depends on the desired valency and target density

• Fc engineering:

  • LALAPG mutations (L234A, L235A, P329G) in the Fc region are essential to minimize FcγR binding

  • This engineering ensures the observed effects are CLEC5A-dependent rather than Fc-dependent

• Binding affinity optimization:

  • Single-digit nM or lower Kd values for CLEC5A binding are desirable

  • Affinity for the target antigen should be optimized based on target expression levels

• Species cross-reactivity:

  • Engineering antibodies with cross-reactivity between human and mouse CLEC5A facilitates translation from preclinical to clinical studies

  • Though binding affinities may differ between species (e.g., slightly lower for murine CLEC5A), functional cross-reactivity enables in vivo evaluation in mouse models

• Target selection criteria:

  • Optimal targets should have restricted expression patterns (e.g., tumor cells, specific immune cell subsets)

  • Targets like CD20 (for B cells), HER2 (for epithelial tumors), and OX40 (for Tregs) have been successfully paired with CLEC5A

These molecular engineering considerations are critical for developing CLEC5A-directed bispecific antibodies with optimal therapeutic potential while minimizing off-target effects.

How does CLEC5A expression influence experimental design for antibody evaluation?

Understanding CLEC5A expression patterns is crucial for designing robust experiments to evaluate CLEC5A-directed antibodies:

• Cell type considerations:

  • CLEC5A is expressed by monocytes, macrophages, neutrophils, and dendritic cells

  • Expression levels vary across monocyte subsets (classical, intermediate, nonclassical)

  • When designing phagocytosis assays, researchers should consider using primary monocyte-derived macrophages rather than cell lines to maintain physiologically relevant CLEC5A expression

• Disease state variations:

  • CLEC5A expression increases in inflammatory conditions like severe COVID-19

  • When testing antibodies in disease models, baseline and disease-state expression should be quantified

  • For cancer studies, confirmation of CLEC5A expression on tumor-infiltrating myeloid cells is essential

• Temporal dynamics:

  • CLEC5A gene expression shows temporal variation during infection (e.g., 3-15 days during Omicron challenge in hamsters)

  • Experimental timelines should account for these expression kinetics

• Stimulus-dependent regulation:

  • CLEC5A expression can be triggered by viral proteins like SARS-CoV-2 spike glycoprotein

  • Pre-stimulation conditions may be necessary to model pathological states accurately

• Species differences:

  • While human and mouse CLEC5A share functional similarities, there may be differences in expression patterns and regulation

  • Cross-species validation is important for translational research

By accounting for these expression patterns, researchers can design more physiologically relevant experiments and better predict clinical translation of CLEC5A-directed antibody therapeutics.

What are the potential mechanisms of CLEC5A-mediated inflammation in viral infections?

CLEC5A mediates inflammation during viral infections through several interconnected mechanisms:

• Pattern recognition and viral binding:

  • CLEC5A can interact directly with viral proteins such as the SARS-CoV-2 spike glycoprotein

  • In silico studies suggest binding may occur at specific sites like the N-acetylglucosamine binding site (NAG-601)

• Heterocomplexes formation:

  • CLEC5A can form multivalent heterocomplexes with other receptors such as DC-SIGN

  • These complexes enhance recognition of viral patterns and amplify inflammatory signaling

• Signal transduction pathways:

  • Upon activation, CLEC5A signals through associated adaptor proteins (DAP12/DAP10)

  • This activates Syk kinase, leading to inflammatory cytokine cascades

• Cytokine and chemokine production:

  • CLEC5A activation triggers production of proinflammatory mediators including:

    • TNF, IL-6, IL-1β (primary inflammatory cytokines)

    • IL-8, RANTES, MIP-1α, MIP-1β (chemokines)

    • IL-10 (immunoregulatory cytokine)

• Neutrophil activation:

  • CLEC5A, in conjunction with Toll-like receptor 2, can induce neutrophil activation during SARS-CoV-2 infection

  • This contributes to aggressive inflammatory cascades in severe disease

Understanding these mechanisms provides the rationale for therapeutic targeting of CLEC5A in viral infections characterized by excessive inflammation, such as severe COVID-19. CLEC5A monoclonal antibodies may interrupt these pathways, potentially reducing inflammatory damage without compromising essential antiviral immunity.

How might CLEC5A antibodies be combined with other immunotherapies for enhanced efficacy?

The unique mechanisms of CLEC5A-directed antibodies suggest several promising combination strategies with existing immunotherapies:

• Combination with immune checkpoint inhibitors:

  • CLEC5A-directed bispecific antibodies targeting Tregs could synergize with PD-1/PD-L1 blockade

  • The Treg-depleting effect of OX40/CLEC5A bispecifics may complement the T cell-activating effects of checkpoint inhibitors

  • This approach could address primary and acquired resistance to checkpoint inhibition

• Integration with conventional antibody therapies:

  • CLEC5A-directed bispecifics could overcome FcγRIIB-mediated resistance to conventional therapeutic antibodies

  • Combining HER2/CLEC5A bispecifics with traditional trastuzumab might enhance efficacy in HER2+ cancers through complementary mechanisms

• Combination with inflammatory modulators in infectious diseases:

  • In COVID-19, combining CLEC5A antibodies with other inflammatory modulators (e.g., IL-6 inhibitors) might provide more comprehensive control of hyperinflammation

  • Sequential treatment approaches could target different phases of the inflammatory response

• Enhancement of CAR-T cell therapy:

  • CLEC5A-directed antibodies could potentially enhance CAR-T cell efficacy by:

    • Depleting immunosuppressive myeloid populations in the tumor microenvironment

    • Reducing Treg-mediated suppression through OX40/CLEC5A bispecifics

• Combination with targeted therapies:

  • For solid tumors, combining CLEC5A-directed approaches with kinase inhibitors or other targeted therapies might enhance efficacy through complementary mechanisms of action

These combination strategies require careful evaluation of sequencing, dosing, and potential synergistic toxicities, but they represent promising avenues to enhance the therapeutic efficacy of CLEC5A-directed approaches across multiple disease contexts.

What novel targets might be paired with CLEC5A in future bispecific antibody designs?

Beyond the already explored targets (CD20, HER2, OX40), several promising novel targets could be paired with CLEC5A in bispecific antibody designs:

• Tumor-associated antigens:

  • EGFR: Overexpressed in multiple epithelial cancers and validated as an antibody target

  • GD2: Expressed in neuroblastoma and other neuroectodermal tumors

  • Mesothelin: Overexpressed in mesothelioma, pancreatic, and ovarian cancers

  • These targets could expand CLEC5A-directed approaches to additional cancer types

• Immune cell targets:

  • CCR4: Enriched on certain Treg populations and already targeted by mogamulizumab

  • GITR: Highly expressed on tumor-infiltrating Tregs

  • CTLA-4: Alternative approach to target Tregs through a mechanism distinct from ipilimumab

  • These targets offer alternative strategies for immune cell modulation

• Viral proteins:

  • SARS-CoV-2 spike protein: Given the known interaction with CLEC5A, direct targeting could enhance viral clearance

  • Envelope proteins of other viruses known to interact with C-type lectins

  • These approaches could expand the therapeutic application to additional viral infections

• Fibrosis-associated targets:

  • Activated fibroblast markers in fibrotic diseases

  • Targeting both fibroblasts and macrophage-mediated inflammation could address multiple pathological mechanisms

• Vascular targets:

  • Angiogenesis markers in combination with CLEC5A could potentially address tumor vasculature while also promoting phagocytosis of target cells

Each of these novel target combinations would require careful validation of expression patterns, accessibility, and functional impact when engaged alongside CLEC5A. The versatility of the bispecific platform allows for rational design based on disease-specific mechanisms.

What emerging technologies might enhance CLEC5A antibody development and characterization?

Several cutting-edge technologies are poised to revolutionize the development and characterization of CLEC5A antibodies:

• Advanced single-cell technologies:

  • Single-cell RNA sequencing to precisely map CLEC5A expression across tissue-specific myeloid populations

  • Single-cell proteomics to characterize CLEC5A protein levels and co-expression with other receptors

  • These approaches will provide unprecedented resolution of CLEC5A biology in health and disease

• Cryo-electron microscopy (Cryo-EM):

  • Structural determination of CLEC5A-antibody complexes at near-atomic resolution

  • Visualization of CLEC5A clustering and organization during target engagement

  • These structural insights could guide rational antibody engineering

• CRISPR-based functional genomics:

  • Genome-wide CRISPR screens to identify genes that modify CLEC5A-dependent phagocytosis

  • CRISPR activation/repression systems to modulate CLEC5A expression

  • These approaches could uncover new therapeutic targets within CLEC5A signaling pathways

• Advanced protein engineering platforms:

  • Machine learning approaches to optimize antibody affinity and specificity

  • Novel bispecific formats beyond traditional configurations

  • These platforms could enhance the functionality of CLEC5A-directed therapeutics

• Intravital imaging technologies:

  • Real-time visualization of CLEC5A-mediated phagocytosis in vivo

  • Tracking of CLEC5A+ cells during disease progression and treatment

  • These approaches would provide unprecedented insights into the dynamics of CLEC5A biology

• Spatial transcriptomics and proteomics:

  • Characterization of CLEC5A expression and activity within the spatial context of tissues

  • Analysis of CLEC5A+ cell interactions with other cell types in situ

  • These technologies would enhance understanding of CLEC5A function in complex tissue environments

Integration of these technologies will accelerate CLEC5A antibody development while providing deeper mechanistic understanding of CLEC5A biology across different disease contexts.