FOLR1 Antibody

What is FOLR1 and why is it a significant research target?

FOLR1 (Folate Receptor Alpha) is a 257 amino acid, 29.8 kDa protein that functions in folic acid receptor activity and binding. It has both secreted and membrane subcellular localization as a member of the folate receptor protein family. FOLR1 has gained significant research interest because it is overexpressed in various cancer types, particularly ovarian cancer, while showing limited expression in normal tissues. This differential expression pattern makes it an attractive target for both diagnostic and therapeutic applications in cancer research. FOLR1 also plays roles in vesicle-mediated transport and post-translational protein modification, with tissue-specific expression observed in kidney, lung, placenta, and thymus .

What types of FOLR1 antibodies are available for research applications?

Researchers have access to over 420 anti-FOLR1 antibodies from more than 30 different suppliers, providing both monoclonal and polyclonal options. These antibodies support various applications including Western blot, ELISA, Flow Cytometry, immunohistochemistry (IHC), and immunofluorescence. Researchers should select antibodies based on their specific experimental needs, considering factors such as species reactivity, clonality, and validated applications. For example, some antibodies like the Goat Anti-Human FOLR1 Antigen Affinity-purified Polyclonal Antibody have been validated for flow cytometry and Western blot applications on human cancer cell lines such as MCF-7 and HeLa .

How does FOLR1 structure correlate with antibody binding epitopes?

FOLR1 antibodies typically target the extracellular domain of the receptor, specifically amino acids Arg25-Met233 in the human protein (accession # P15328). This region contains the functional domains responsible for folate binding. When designing experiments, researchers should consider that post-translational modifications, particularly glycosylation and proteolytic cleavage, can affect antibody binding. Different antibody clones may recognize distinct epitopes within this region, potentially yielding varying results depending on the conformation and modification state of the target protein .

What are the validated methods for measuring FOLR1 antibody binding affinity?

Two primary methods for measuring FOLR1 antibody binding affinity include:

• Surface Plasmon Resonance (SPR): This can be conducted on instruments like the Biacore 3000. The protocol typically involves capturing the antibody (diluted to 3 μg/mL) on an anti-human IgG-immobilized sensor chip until approximately 500 response units are obtained. Recombinant FOLR1 is then injected at concentrations ranging from 0.5 to 150 nM, followed by recording dissociation for 15 minutes. The sensograms should be double-referenced and kinetic parameters determined using a 1:1 Langmuir binding model .

• FACS Analysis: For cell-based binding assays, adherent cells expressing FOLR1 are detached using cell dissociation buffer and incubated with varying concentrations of the antibody on ice for 30 minutes. After washing, cells are incubated with a fluorescently labeled secondary antibody (e.g., goat-anti-human IgG conjugated to Alexa Fluor® 488) and analyzed using flow cytometry. This approach allows researchers to assess antibody binding to native FOLR1 on intact cells .

How can researchers validate FOLR1 antibody specificity in experimental systems?

To ensure specificity of FOLR1 antibodies, researchers should implement multiple validation approaches:

• Western Blot with Knockout Controls: Compare antibody reactivity between parental cell lines (e.g., HeLa) and FOLR1 knockout derivatives. A specific antibody will detect a band of approximately 37-40 kDa in the parental line but show no reactivity in the knockout line. Include loading controls such as GAPDH to ensure equal protein loading .

• Flow Cytometry with Multiple Cell Lines: Test antibody binding across cell lines with known differential FOLR1 expression. Compare binding curves and maximum steady-state binding levels (typically reached at approximately 1000 ng/mL for specific antibodies). Include irrelevant isotype-matched control antibodies to confirm binding specificity .

• Recombinant Protein Controls: Demonstrate specific binding to purified recombinant FOLR1 protein using techniques like ELISA or SPR before proceeding to more complex cellular systems .

What methods are recommended for generating FOLR1 knockdown or overexpression cell models?

For creating experimental cell models with altered FOLR1 expression:

How does FOLR1 expression vary across different cancer types and what are the implications for antibody-based research?

FOLR1 shows distinct expression patterns across cancer types with significant implications for research:

• Ovarian Cancer: Shows consistently high FOLR1 overexpression while being largely absent in normal tissues, making it an excellent model system for studying FOLR1-targeted approaches. Researchers should consider using ovarian cancer cell lines like IGROV-1 as positive controls in antibody validation studies .

• Gynecologic Malignancies: Beyond ovarian cancer, other gynecologic tumors frequently express FOLR1, offering additional model systems for investigation .

• Lung Adenocarcinoma: Exhibits significant FOLR1 expression, with cell lines like NCI-H2170 serving as useful models, particularly for in vivo imaging studies of antibody tumor localization .

• Rectal Cancer: High FOLR1 expression correlates with advanced tumor stage, poor response to chemoradiotherapy, and worse patient outcomes. This provides an opportunity to study FOLR1 as a predictive biomarker in rectal cancer models .

Researchers should consider these differential expression patterns when selecting appropriate positive and negative control cell lines for antibody validation and functional studies.

What are the mechanisms underlying FOLR1 antibody-mediated anti-tumor effects?

FOLR1 antibodies can exert anti-tumor effects through multiple mechanisms:

• Antibody-Dependent Cellular Cytotoxicity (ADCC): Studies with the anti-FOLR1 monoclonal antibody farletuzumab (MORAb-003) demonstrate that ADCC is a primary mechanism of action. This involves recruitment of effector cells via the antibody's Fc region to induce tumor cell killing. Researchers can confirm ADCC activity by comparing wild-type antibodies with mutant versions containing alterations in Fc region residues that disrupt effector cell interactions .

• Complement-Mediated Cytotoxicity (CDC): Some FOLR1 antibodies can activate the complement cascade, leading to membrane attack complex formation and tumor cell lysis .

• Direct Pharmacologic Effects: FOLR1 antibodies may interfere with folate uptake or receptor signaling, affecting cancer cell metabolism and proliferation, particularly under folate-restricted conditions .

When investigating these mechanisms, researchers should design experiments that can distinguish between these different modes of action, using appropriate in vitro assays and in vivo models with relevant controls.

How can researchers effectively monitor FOLR1 antibody tumor targeting in vivo?

For monitoring antibody tumor localization in animal models:

How can FOLR1 antibodies be optimized for therapeutic applications in cancer?

Optimization strategies for therapeutic FOLR1 antibodies include:

• Fc Engineering: Modify the Fc region to enhance ADCC activity by introducing mutations that increase binding affinity to Fcγ receptors on effector cells. Conversely, researchers can create ADCC-null variants (as demonstrated with MUT-FRL) by mutating key residues involved in effector cell interactions to isolate direct pharmacologic effects from immune-mediated mechanisms .

• Antibody-Drug Conjugates (ADCs): Conjugate cytotoxic payloads to FOLR1 antibodies to enable targeted drug delivery to tumor cells while sparing normal tissues. This approach leverages the cancer-specific expression of FOLR1 to improve therapeutic index .

• Bispecific Antibodies: Engineer bispecific formats that simultaneously engage FOLR1 on tumor cells and activating receptors on immune cells (e.g., CD3 on T cells) to enhance anti-tumor immune responses .

• Affinity Optimization: Fine-tune antibody binding kinetics to balance optimal tumor penetration with sufficient retention. Extremely high-affinity antibodies may exhibit "binding site barrier" effects that limit tumor penetration .

These optimization approaches should be systematically evaluated using both in vitro binding and functional assays and appropriate in vivo models.

What are the challenges in translating FOLR1 antibody research from in vitro studies to in vivo models?

Several challenges must be addressed when moving from in vitro to in vivo FOLR1 antibody research:

• Heterogeneous Target Expression: Tumors show heterogeneous FOLR1 expression in vivo, unlike the relatively homogeneous expression in cultured cell lines. This heterogeneity can affect antibody distribution and efficacy. Researchers should characterize FOLR1 expression patterns in their animal models before beginning antibody studies .

• Microenvironment Influences: The tumor microenvironment, including stromal cells, immune infiltrates, and hypoxic regions, can affect antibody delivery and function. In vitro systems often fail to recapitulate these complex interactions .

• Species Differences: Human FOLR1-specific antibodies may not cross-react with murine FOLR1, complicating the interpretation of toxicity and efficacy in mouse models. Researchers should consider using human tumor xenografts in immunocompromised mice to evaluate human-specific antibodies .

• Pharmacokinetics and Distribution: Antibody serum half-life and tissue distribution differ substantially between in vitro and in vivo settings. Monitoring antibody levels using techniques like infrared fluorescence imaging is essential for correlating exposure with effect .

How can FOLR1 immunohistochemistry be standardized for clinical biomarker applications?

Standardization of FOLR1 immunohistochemistry for clinical applications requires:

• Validated Antibodies and Protocols: Use clinically validated antibodies like the VENTANA FOLR1 (FOLR1-2.1) RxDx Assay, which has been developed for assessing FOLR1 protein in formalin-fixed, paraffin-embedded tissues. This assay uses mouse monoclonal anti-FOLR1, clone FOLR1-2.1, which has been optimized for clinical biomarker applications .

• Scoring System Development: Implement a standardized scoring system that accounts for both staining intensity and percentage of positive cells. In rectal cancer studies, for example, FOLR1 expression has been categorized as low (0+-2+) or high (3+-4+) with significant prognostic implications .

• Quality Control Measures: Include appropriate positive and negative control tissues in each staining run. Kidney, lung, placenta, and thymus tissues can serve as positive controls, while FOLR1-negative tissues should be included as negative controls .

• Multi-institutional Validation: Conduct concordance studies across different laboratories to ensure reproducibility of staining and interpretation. This approach is essential for establishing FOLR1 as a reliable biomarker for patient stratification and treatment selection .

How might single-cell analysis techniques enhance FOLR1 antibody research?

Single-cell analysis offers several advantages for advancing FOLR1 research:

• Heterogeneity Characterization: Single-cell RNA sequencing and protein analysis can reveal the heterogeneity of FOLR1 expression within tumors, potentially identifying subpopulations with differential response to FOLR1-targeted therapies .

• Correlation with Other Biomarkers: Single-cell multi-parameter analysis can correlate FOLR1 expression with other cancer-related markers, providing insights into the molecular context of FOLR1 overexpression and its functional implications .

• Resistance Mechanism Identification: By analyzing FOLR1-expressing cells before and after treatment with FOLR1-targeted therapies, researchers may identify adaptive resistance mechanisms that emerge in response to therapeutic pressure .

• Improved Flow Cytometry Applications: Advanced flow cytometry techniques, building on established protocols for FOLR1 detection, can be adapted for single-cell sorting and functional characterization of FOLR1-positive subpopulations from primary tumor samples .

What is the role of FOLR1 in cancer stem cells and therapeutic resistance?

Emerging research suggests FOLR1 may have significant roles in cancer stem cell biology and therapeutic resistance:

• Cancer Stem Cell Marker: Investigate whether FOLR1 expression correlates with established cancer stem cell markers and functional properties like self-renewal and tumor initiation. This could be assessed using sphere formation assays, limiting dilution tumor initiation studies, and co-staining with known stem cell markers .

• Cisplatin Resistance: Given the suggested relationship between FOLR1 and cisplatin sensitivity, researchers should explore the mechanistic link between FOLR1 expression and response to platinum-based therapies. This could involve creating isogenic cell lines with varying FOLR1 levels and testing their drug response profiles .

• Folate Metabolism: Examine how FOLR1 contributes to folate uptake and metabolism in nutrient-restricted microenvironments, potentially conferring a survival advantage to cancer cells under stress conditions. This would require metabolic profiling of cells with different FOLR1 expression levels under various folate concentrations .

• Therapeutic Combinations: Investigate whether FOLR1 antibody therapies might sensitize resistant tumors to conventional chemotherapy or radiation by disrupting adaptive metabolic pathways or eliminating resistant subpopulations .