LCR15 Antibody

What is LRRC15 and why is it important in cancer research?

LRRC15 is a 581 amino acid type I membrane protein with leucine-rich repeat domains and no obvious intracellular signaling domains. It has emerged as a critical marker in cancer research for several reasons:

• It shows high expression in multiple solid tumor indications with limited normal tissue expression

• It is expressed on cancer-associated fibroblasts (CAFs) in the tumor stroma of many solid tumors (breast, head and neck, lung, pancreatic)

• It is directly expressed on a subset of cancer cells with mesenchymal origin (sarcoma, melanoma, glioblastoma)

• Its expression is induced by TGFβ on activated fibroblasts (αSMA+) and mesenchymal stem cells

• It functions by regulating cell-cell and cell-extracellular matrix interactions

These properties make LRRC15 a novel CAF and mesenchymal marker with significant therapeutic potential for cancers with LRRC15-positive stromal desmoplasia or mesenchymal-origin cancers.

What techniques can be used to detect LRRC15 expression in tissue samples?

Several validated techniques for detecting LRRC15 expression in research samples include:

For immunohistochemistry, protocols typically involve:

• FFPE slide preparation and deparaffinization

• Antigen retrieval with high pH target retrieval buffer (125°C for 1 minute)

• Protein blocking followed by primary antibody incubation (1 μg/mL for 60 minutes)

• Detection using HRP secondary reagents and DAB visualization

How can researchers quantify LRRC15 expression in tissue samples?

Quantification of LRRC15 expression depends on the experimental context:

For IHC analyses:

• H-score method: multiply the percentage of positive cells by staining intensity (0-3), with a maximum score of 300

• Qualitative scoring from 0 to 3+ based on staining intensity and frequency

• Digital image analysis using platforms like Zeiss Axio Scanner Z1 and Indica HALO AI

For flow cytometry quantification:

• Cell surface copy number can be determined using LRRC15 antibodies (e.g., Ab3-AF488) relative to isotype controls

• Quantum Simply Cellular Bead Kit can provide absolute quantification of receptors per cell

• Mean fluorescence intensity (MFI) comparisons between samples

For robust tumor-stroma differentiation:

• Multiplex immunohistochemistry with LRRC15 and epithelial markers (pan-cytokeratin)

• Tumor and stroma area classification using AI tools (e.g., Densenet v2)

• Automated quantification of LRRC15 expression intensity in distinct compartments

How does LRRC15 expression in stroma correlate with patient outcomes in different cancer types?

LRRC15 expression in tumor stroma shows significant correlations with patient outcomes, though these associations appear to be cancer-type dependent:

In lung adenocarcinoma:

• Higher LRRC15 stromal expression is associated with better 5-year survival

• Patients with high stromal LRRC15 expression show 67% lower risk of death (HR: 0.33, 95% CI: 0.16-0.68, P < 0.01)

• This suggests LRRC15 may impact immune cell function in ways that influence clinical outcomes

The mechanism behind this favorable prognostic association may involve:

• Modulation of the tumor microenvironment

• Altered immune infiltration

• LRRC15's role in mediating cell-extracellular matrix interactions

What methodological approaches can be used to study LRRC15's role in the tumor microenvironment?

Several advanced methodological approaches are available for investigating LRRC15's functions in the tumor microenvironment:

• Multiplex immunostaining approaches:

  • Co-staining of LRRC15 with αSMA to identify activated fibroblasts

  • Dual immunofluorescence with Alexa Fluor 488/594 secondary antibodies

  • Multiplex IHC with pan-cytokeratin to distinguish tumor and stromal components

• TGFβ-mediated regulation studies:

  • Treatment of fibroblasts or mesenchymal stem cells with rhTGFβ1 (10 ng/mL)

  • Flow cytometry analysis of LRRC15 upregulation

  • Western blot assessment of LRRC15 protein induction

• Functional studies using LRRC15 antibodies:

  • Blockade experiments to assess the impact on tumor-stroma interactions

  • Receptor occupancy analyses to determine antibody binding efficiency

  • Bystander effect assessment in heterogeneous tumor models

• In vivo models for LRRC15 investigation:

  • Xenograft models with LRRC15-positive stroma or cancer cells

  • Bioluminescence imaging to track tumor growth

  • IHC assessment of immune infiltrate (e.g., F4/80+ macrophages) changes

What are the key considerations when developing therapeutic antibodies targeting LRRC15?

Developing therapeutic antibodies against LRRC15 requires several specific considerations:

• Antibody binding characteristics:

  • Species cross-reactivity (human, cynomolgus monkey, rat, mouse)

  • Binding affinity to LRRC15 extracellular domain

  • Epitope accessibility in the complex tumor microenvironment

• Payload selection for antibody-drug conjugates:

  • MMAE (monomethyl auristatin E) conjugates show efficacy but variable response

  • PNU-159682 (anthracycline derivative) conjugates demonstrate superior efficacy in osteosarcoma models

  • Cell-permeable payloads are preferred to enable bystander killing effects

• Mechanism of action considerations:

  • LRRC15-ADCs can target both LRRC15-positive stroma and cancer cells

  • Bystander killing mechanisms are essential for efficacy in heterogeneous tumors

  • Immune infiltrate modulation (e.g., increased F4/80+ macrophages)

• Preclinical efficacy models:

  • Testing in models with LRRC15 stromal-positive/cancer-negative expression

  • Testing in models with LRRC15 cancer-positive expression

  • Combination studies with standard-of-care therapies

Research indicates that ABBV-085 (LRRC15-targeted MMAE-ADC) demonstrated efficacy in preclinical models and entered clinical development , while the LRRC15-PNU ADC showed superior efficacy with 40-100% cure rates in osteosarcoma xenograft models .

How can LRRC15 antibodies be used to study the protein's role in SARS-CoV-2 infection?

Recent research has uncovered LRRC15's unexpected role in SARS-CoV-2 infection, providing new applications for LRRC15 antibodies:

• Spike protein binding assays:

  • Flow cytometry assessment of SARS-CoV-2 spike protein binding to LRRC15-expressing cells

  • Saturation binding experiments using fluorescently conjugated monomeric spike across concentration ranges

  • Competition assays with EDTA to distinguish LRRC15 binding from lectin-mediated interactions

• LRRC15-mediated virion sequestration studies:

  • Co-culture systems with LRRC15-expressing and ACE2-expressing cells

  • Assessment of LRRC15's trans-inhibition of SARS-CoV-2 infection

  • Antibody blocking experiments to validate specificity

• Protein interaction analyses:

  • Western blot analyses to confirm LRRC15 expression is independent of ACE2 expression

  • Flow cytometry to validate LRRC15-specific binding is heparan sulfate-independent

  • Various transfection approaches to achieve LRRC15 overexpression in relevant cell lines

Key findings indicate LRRC15 doesn't function as a SARS-CoV-2 entry receptor but rather sequesters virions and antagonizes infection of ACE2-positive cells when expressed on nearby cells . This presents a novel research area where LRRC15 antibodies can help elucidate accessory interactions in SARS-CoV-2 pathogenesis.

What are the technical challenges in optimizing LRRC15 expression quantification across different sample types?

Researchers face several technical challenges when quantifying LRRC15 expression:

• Heterogeneous expression patterns:

  • LRRC15 expression varies between stromal and tumor compartments

  • Expression is more common in stroma but can also occur in tumor cells

  • Requires sophisticated image analysis approaches with tumor/stroma segmentation

• Inducible expression considerations:

  • LRRC15 expression can be induced by TGFβ in formerly negative cells

  • Low-expressing cell lines can become amenable to re-expression after TGFβ treatment

  • Temporal dynamics of expression must be considered in experimental design

• Quantification method standardization:

  • Various scoring methods (H-score, 0-3+ qualitative scoring, digital analysis)

  • Need for consistent thresholds for defining "high" versus "low" expression

  • Validation across multiple antibody clones and detection methods

• Technical recommendations:

  • Use multiplex approaches to distinguish cell types (e.g., pan-cytokeratin for epithelial cells)

  • Apply AI-driven image analysis for objective quantification

  • Include proper controls for antibody specificity validation

  • Consider TGFβ pretreatment to evaluate potential for induced expression

How can LRRC15 antibodies be used in designing custom antibody specificity profiles?

Recent advances in antibody engineering offer opportunities for creating LRRC15 antibodies with tailored specificity profiles:

• Computational design approaches:

  • Biophysics-informed modeling combined with phage display selection experiments

  • Optimization of energy functions to generate antibodies with predefined binding profiles

  • Development of both cross-specific antibodies (interacting with multiple ligands) and highly specific antibodies (single ligand interaction)

• Selection strategy considerations:

  • Systematic variation of key residues (e.g., CDR3 positions) to generate diversity

  • High-throughput sequencing to characterize antibody libraries

  • Testing of model-predicted variants not present in training sets

• Validation approaches:

  • Testing novel antibody sequences with custom specificity profiles

  • Assessment of cross-reactivity with related targets

  • Functional validation in relevant biological contexts

These approaches can be applied to LRRC15 antibody development, potentially creating reagents that discriminate between different conformational states or binding partners of LRRC15 in the tumor microenvironment.

What parallels can be drawn between LRRC15 antibody development and other therapeutic antibodies?

Comparing LRRC15 antibody development with other therapeutic antibodies reveals instructive parallels:

• Parallels with CCR5-targeting antibodies (leronlimab):

  • Both target cell surface proteins involved in complex cellular interactions

  • Receptor occupancy analysis methods can be adapted between systems

  • Both can induce unexpected changes in target protein expression levels

• Mechanism insights from leronlimab studies:

  • Leronlimab treatment led to increased CCR5+CD4+ T cell levels

  • Similar to how LRRC15 antibodies might modulate LRRC15 expression

  • Both involve complex immunomodulatory effects beyond simple blockade

• Technical approaches with broader applicability:

  • Receptor occupancy calculation methods for longitudinal monitoring

  • Investigation of immunological impacts of receptor inhibition

  • Development of combination approaches with standard therapies

As seen with leronlimab in "long COVID" treatment, where it normalized abnormal immune downmodulation , LRRC15-targeting antibodies might similarly reveal unexpected immunomodulatory mechanisms in the tumor microenvironment.

How can researchers design experiments to resolve contradictory findings about LRRC15's role in different cancers?

LRRC15 shows apparent contradictions in its prognostic associations across cancer types. Strategic experimental approaches to resolve these include:

• Comprehensive tissue-specific expression profiling:

  • Multiplex IHC across large cohorts of different tumor types

  • Quantification of LRRC15 in distinct cellular compartments (tumor vs. stroma)

  • Correlation with clinicopathological features and outcomes

• Functional characterization approaches:

  • Co-culture systems with LRRC15+ fibroblasts and cancer cells

  • Assessment of immune cell recruitment and function

  • Analysis of extracellular matrix remodeling and cancer cell invasion

• Context-dependent signaling investigation:

  • Phosphoproteomic analysis of signaling pathways in LRRC15+ vs. LRRC15- tumors

  • Transcriptomic profiling to identify co-expressed genes and pathways

  • Analysis of LRRC15's relationship with TGFβ signaling across cancer types

• Experimental design recommendations:

  • Use multiple antibody clones to confirm expression patterns

  • Include tissue microarrays spanning multiple cancer types

  • Perform detailed immune phenotyping alongside LRRC15 assessment

  • Consider the impact of TGFβ in the microenvironment on LRRC15 expression and function

This comprehensive approach can help resolve the apparent contradiction between LRRC15's association with better survival in lung adenocarcinoma versus its general association with aggressive cancer phenotypes.