FOLR1 Human, Sf9

What is FOLR1 and what are its biological functions?

FOLR1 (Folate Receptor 1) is a member of the folate receptor family that binds folic acid and reduced folate derivatives. It plays a critical role in facilitating the transfer of 5-methyltetrahydrofolate and folate analogs into cells . In human biology, FOLR1 (also known as FRα or FOLR) is essential for embryonic development and normal cell proliferation . The receptor can exist in two forms: membrane-attached via a glycosyl-phosphatidylinositol (GPI) linkage or in a soluble form .

In humans, there are three genes encoding functional folate receptors: hFRα (FOLR1), hFRβ (FOLR2), and hFRγ (FOLR3) . FOLR1 is particularly notable for its overexpression in certain malignancies, making it a potential therapeutic target. For instance, it is overexpressed in over one-third of gastric cancer patients while rarely being expressed in normal tissue .

What are the structural characteristics of recombinant FOLR1 Human, Sf9?

The three-dimensional structure of FOLR1 has been elucidated through crystallographic studies, revealing distinct conformational states that play important roles in its biological trafficking and ligand binding properties . These structural insights are valuable for understanding the receptor's function and for designing targeted therapeutics.

How can researchers detect and quantify FOLR1 expression in different experimental systems?

For detecting FOLR1 expression, researchers can utilize several complementary approaches:

• Flow Cytometry (FACS): Multiple antibodies have been developed for FACS analysis of FOLR1-expressing cells. For example, antibodies like 26B3 have shown high fluorescence intensity (854 units) when binding to FRA-expressing cells .

• Western Blotting: Most anti-FOLR1 antibodies do not react under reducing conditions (marked as "(-)" in reactivity tests), suggesting the importance of preserving the protein's tertiary structure .

• Immunohistochemistry (IHC): Some antibodies like 26B3 and 19D4 have demonstrated positive reactivity in formalin-fixed paraffin-embedded (FFPE) tissues, while others like 9F3 showed negative reactivity . The differences in IHC performance are likely related to epitope accessibility and antibody affinity.

• ELISA: Soluble scFv antibody fragments have been used in ELISA to detect FOLR1 .

When selecting detection methods, researchers should consider that FOLR1's complex post-translational structure makes some reagents highly structure-sensitive, potentially leading to equivocal expression results across independent studies .

What are the optimal conditions for preserving FOLR1 functionality in in vitro experiments?

When working with FOLR1 in vitro, researchers should implement the following measures to maintain protein functionality:

• Buffer Composition: PBS-based buffers with pH 7.2-7.4 are typically used for initial solubilization. For coating applications in ELISA, researchers have successfully used 30 ng antigen per well in PBS overnight at 4°C .

• Blocking Agents: To prevent non-specific binding, blocking with 1-2% BSA in PBST (PBS with Tween-20) has been effectively employed in multiple studies .

• Temperature Considerations: FOLR1 protein is generally stable at 4°C for short-term storage. For experimental manipulations, room temperature (RT) incubations of 1 hour have been used for immobilization procedures .

• Avoiding Denaturation: Due to FOLR1's complex secondary structure, avoid conditions that could disrupt disulfide bonds or alter the three-dimensional conformation. The protein's detection is highly dependent on maintaining its native structure .

• pH Sensitivity: FOLR1 undergoes pH-dependent conformational changes during endocytic trafficking. Researchers have observed distinct structural conformations at different pH levels, which should be considered when designing binding assays .

What methodologies are recommended for validating FOLR1-specific antibodies and binding domains?

A robust validation strategy for FOLR1-specific antibodies should include the following multi-step approach:

• Cross-Reactivity Assessment: Test antibody specificity against other folate receptor family members (FOLR2, FOLR3, FOLR4) due to their high sequence similarity. For example, one study assessed specificity of FOLR1-directed binders towards other human/murine folate receptor variants (hFOLR2, hFOLR3, hFOLR4, mFOLR2, and mFOLR4) .

• Multiple Platform Testing: Validate antibodies across different detection methods including ELISA, flow cytometry, western blotting, and immunohistochemistry. The 26B3 antibody, for instance, demonstrated robust binding across multiple platforms, making it an ideal candidate for assay development .

• Positive and Negative Controls: Employ both FOLR1-proficient cells (e.g., OV-90 ovarian cancer cell line) and FOLR1-knockout cells (e.g., CRISPR/Cas9-mediated FOLR1-deficient OV-90 cell line) as controls .

• Tissue Cross-Reactivity: Perform multiplex imaging analysis to assess on-tumor and off-tumor binding profiles in both healthy and malignant tissues. One study examined binding on 20 different healthy human tissues and high-grade serous ovarian cancer samples .

• Comparison with Reference Antibodies: Compare new antibodies with well-characterized reference antibodies (e.g., LK26) to benchmark specificity and sensitivity.

• Affinity Measurements: Determine binding kinetics, particularly off-rates, as antibodies with slower off-rates (like 26B3) may demonstrate more robust binding in applications such as FFPE IHC .

What are the key considerations in developing FOLR1-targeted CAR T cell therapies?

When developing FOLR1-targeted CAR T cell therapies, researchers should address several critical factors:

• Binding Domain Selection: For optimal CAR construct design, researchers have used single-chain variable fragments (scFv) of FOLR1 antibodies. For example, a CAR targeting FOLR1 composed of an scFv with signaling domains consisting of CD28 and CD3ζ has been shown to be effective .

• Specificity Assessment: Comprehensive evaluation of on-target/off-tumor reactivity is essential due to expression of folate receptors in some normal tissues. Multiple assays and technologies should be employed to thoroughly assess specificity .

• Functional Validation: High-throughput screening assays co-culturing CAR T cells with target cells can evaluate:

  • Target cell lysis efficiency

  • Cytokine release profiles

  • CAR T cell expansion dynamics

  • Performance under challenging conditions (unfavorable effector-to-target ratios and repetitive target cell exposure)

• Cross-Species Reactivity: For preclinical model translation, consider developing binders with cross-reactivity to murine FOLR1. Some studies have selected fully human scFv sequences specific for both human and murine FOLR1 .

• CAR Design Optimization: Different generations of CAR constructs should be compared. Second-generation CARs incorporating CD28 and CD3ζ signaling domains have shown efficacy against FOLR1-positive cancer cells .

• Cell Type Selection: Beyond conventional T cells, natural killer cell lines (e.g., KHYG-1) have been engineered with FOLR1-CAR and demonstrated effectiveness against FOLR1-positive gastric cancer cells in a MHC-independent manner .

How can structural data on FOLR1 inform the design of targeted therapeutics?

Structural insights from crystallographic studies of FOLR1 provide valuable information for therapeutic development:

• Binding Site Identification: Six distinct crystallographic models have revealed different binding modes of folate and antifolates to FOLR1, enabling structure-based design of novel targeted drugs .

• Conformational States: Understanding the discrete structural conformations representing key stages in endocytic trafficking of FOLR1 helps in designing drugs that can leverage these conformational changes .

• pH-Dependent Interactions: Models for pH-dependent conformational changes can guide the development of therapeutics that selectively release or activate in specific cellular compartments based on pH differences .

• Antifolate Binding Analysis: Molecular details of FOLR1 in complex with clinically prevalent antifolates such as pemetrexed (Alimta), aminopterin, and methotrexate provide templates for designing FOLR1-specific antifolates .

• Optimization of Conjugates: Structural data facilitates the design of folate conjugates for specific delivery to tumors or sites of inflammation, helping to optimize linker attachment points and conjugate stability .

• Antibody Development: Understanding the three-dimensional architecture of FOLR1 enables the generation of antibodies targeting specific epitopes, potentially improving diagnostic and therapeutic applications .

How do post-translational modifications affect FOLR1 function and detection methodologies?

Post-translational modifications (PTMs) significantly impact FOLR1 function and detection in several ways:

• Glycosylation Effects: The recombinant FOLR1 Human, Sf9 appears at 28-40 kDa on SDS-PAGE despite its theoretical mass of 25.6 kDa, indicating substantial glycosylation . These glycosylation patterns can affect:

  • Receptor trafficking and localization

  • Binding affinity to folate and drugs

  • Antibody recognition and epitope accessibility

• GPI Anchor: The membrane-bound form of FOLR1 is attached via a glycosyl-phosphatidylinositol (GPI) anchor, which influences:

  • Receptor distribution in membrane microdomains

  • Endocytic pathways and internalization kinetics

  • Lateral mobility within the membrane

• Detection Challenges: The complex secondary structure of FOLR1 makes detection highly dependent on preservation of its three-dimensional conformation. Many antibodies fail to detect FOLR1 under reducing conditions that disrupt disulfide bonds .

• Tissue Processing Effects: Different tissue fixation techniques can variably affect FOLR1 detectability. While some antibodies (26B3, 19D4) successfully detect FOLR1 in FFPE tissues, others (9F3) do not, despite similar performance in other assays .

• Differential Detection: Researchers should employ complementary detection methods when studying FOLR1. For example, combining flow cytometry with immunohistochemistry and western blotting provides a more complete picture of expression and modification status .

What strategies can minimize off-target effects when developing FOLR1-targeted therapies?

To minimize off-target effects in FOLR1-targeted therapeutic development, researchers should implement a multi-faceted approach:

• Comprehensive Specificity Screening: Establish stringent, multistep workflows for evaluating candidate molecules:

  • Test binding against all folate receptor family members (FOLR1-4)

  • Assess cross-reactivity with both human and murine variants

  • Implement both positive and negative selection strategies during initial screening

• CRISPR/Cas9 Knockout Controls: Generate FOLR1-knockout cell lines as essential negative controls for validating targeting specificity. For example, CRISPR/Cas9-mediated FOLR1-deficient OV-90 cells have been used to confirm CAR T cell specificity .

• Tissue Cross-Reactivity Analysis: Employ automated, high-plex imaging approaches to analyze staining profiles across diverse healthy tissues and compare with positive control antibodies like LK26 . One study examined binding on 20 different healthy human tissues including adrenal gland, ovary, pancreas, thyroid, cerebellum, cerebrum, lung, spleen, uterus, cervix, breast, placenta, heart, skin, skeletal muscle, kidney, stomach, small intestine, liver, and salivary gland .

• Functional Assays Under Challenging Conditions: Test therapeutic candidates under conditions that might reveal off-target activities:

  • Unfavorable effector-to-target ratios

  • Repetitive target cell exposure

  • Various cancer cell lines with different FOLR1 expression levels

• Affinity Optimization: Fine-tune binding affinity to preferentially target high FOLR1-expressing cancer cells while minimizing effects on tissues with low physiological expression.

What are the methodological differences between studying membrane-bound versus soluble forms of FOLR1?

The methodological approaches differ significantly when investigating membrane-bound versus soluble FOLR1:

Membrane-bound FOLR1 Research Methods:

• Cell Surface Analysis: Flow cytometry using antibodies like 26B3, 24F12, and 9F3 can quantify FOLR1 expression levels on intact cells. For example, these antibodies showed fluorescence intensities of 854, 777, and 760, respectively, on FRA-expressing cells .

• Membrane Isolation: Purification of membrane fractions using ultracentrifugation techniques may be necessary to study the GPI-anchored form specifically.

• Trafficking Studies: Analyses of endocytic pathways require fluorescently labeled antibodies or ligands to track receptor internalization and recycling through different cellular compartments.

• Lipid Raft Association: Detergent-resistant membrane fractionation can help determine FOLR1's association with specialized membrane microdomains.

Soluble FOLR1 Research Methods:

• Secretion Analysis: Collection and concentration of cell culture supernatants or biological fluids like serum, followed by immunoprecipitation or ELISA.

• Protein Purification: Recombinant soluble FOLR1 can be produced and purified with a His-tag at the C-terminus using chromatographic techniques .

• Binding Kinetics: Surface plasmon resonance or isothermal titration calorimetry to determine binding constants of soluble FOLR1 with various ligands.

• Crystallization Studies: The soluble form has been used for crystallographic analysis, revealing structural insights into folate and antifolate binding modes .

Comparative Analysis Approaches:

• Functional Comparison: Differential binding assays can identify unique properties of each form, potentially revealing distinct biological roles.

• Release Mechanisms: Studying the enzymatic or mechanical processes that convert membrane-bound FOLR1 to its soluble form can provide insights into regulation mechanisms.

• Clinical Correlation: Measuring the ratio of membrane to soluble FOLR1 in patient samples may have diagnostic or prognostic value for certain cancer types.

How can FOLR1 expression profiling be optimized for patient stratification in clinical trials?

Optimizing FOLR1 expression profiling for patient stratification requires standardized, reproducible methodologies:

• Antibody Selection: Use well-characterized antibodies with demonstrated specificity and robustness across multiple detection platforms. The 26B3 antibody, for example, has shown consistent performance in FFPE IHC, FACS, and other assays, making it an ideal candidate for clinical applications .

• IHC Protocol Standardization: Develop validated IHC protocols that account for FOLR1's structural sensitivity. Parameters to standardize include:

  • Fixation conditions

  • Antigen retrieval methods

  • Detection systems

  • Scoring criteria

• Multiplex Testing: Implement automated, high-plex imaging approaches to simultaneously assess FOLR1 expression alongside other relevant biomarkers .

• Quantitative Assessment: Establish quantitative scoring systems (e.g., H-score, Allred score) rather than simple positive/negative classifications to better stratify patients based on expression levels.

• Complementary Methods: Consider combining tissue-based detection with liquid biopsy approaches to measure circulating soluble FOLR1, which may provide additional prognostic information.

• Reference Standards: Include well-characterized positive and negative controls in each assay run to ensure consistency across different testing sites and time points.

• Correlation Studies: Validate expression levels against functional outcomes (e.g., response to FOLR1-targeted therapies) to establish clinically meaningful expression thresholds.

What are the technical challenges in adapting FOLR1 assays from research applications to clinical diagnostics?

Transitioning FOLR1 assays from research to clinical diagnostics faces several technical challenges:

• Reproducibility Across Laboratories: Due to the complex post-translational structure of FOLR1, reagents developed to date are often highly structure-sensitive, resulting in equivocal expression results across independent studies . Clinical assays must overcome this variability.

• Sample Preparation Variability: Different tissue procurement and processing methods can significantly impact FOLR1 detection. For example, varying frequencies of FOLR1 expression have been reported even in homogeneously expressing cancers like ovarian carcinoma .

• Antibody Validation Requirements: Clinical diagnostic antibodies require more rigorous validation than research-use antibodies, including:

  • Analytical specificity testing against all folate receptor family members

  • Reproducibility assessment across multiple lots

  • Stability testing under various storage conditions

• Standardization of Quantification: Development of quantitative cutoffs that correlate with clinical outcomes:

  • Determining clinically relevant expression thresholds

  • Establishing inter-observer reliability

  • Creating reference standards for calibration

• Automation and Throughput: Adapting manual research protocols to automated platforms suitable for high-throughput clinical testing while maintaining sensitivity and specificity.

• Regulatory Considerations: Meeting regulatory requirements (FDA, EMA, etc.) for companion diagnostic assays, including:

  • Analytical validation

  • Clinical validation

  • Manufacturing controls

  • Documentation compliance

• Integration with Existing Workflows: Ensuring compatibility with established pathology laboratory workflows and information systems to facilitate adoption.

How might crystallographic data on FOLR1 conformational changes inform next-generation therapeutic design?

The crystallographic insights into FOLR1 conformational changes open several avenues for advanced therapeutic development:

• Structure-Guided Drug Design: The six distinct crystallographic models of FOLR1 provide detailed templates for rational design of small molecules with enhanced specificity and affinity . By targeting specific conformational states, researchers could develop drugs that selectively bind to FOLR1 only in certain cellular contexts.

• pH-Sensitive Therapeutics: Understanding the pH-dependent conformational changes during endocytic trafficking enables design of therapeutics that release their payload only in specific cellular compartments . This approach could minimize off-target toxicity by ensuring drug activation occurs only after internalization by FOLR1-expressing cells.

• Allosteric Modulators: Identification of allosteric binding sites from crystallographic data could lead to development of modulators that alter FOLR1 function without competing with folate binding, potentially creating entirely new therapeutic classes.

• Improved Folate Conjugates: Detailed understanding of folate binding modes facilitates optimization of folate-drug conjugates, specifically:

  • Optimal attachment points that preserve receptor recognition

  • Linker design that responds to cellular microenvironments

  • Payload positioning for efficient release after internalization

• Antibody Epitope Mapping: Crystallographic data enables precise mapping of antibody binding epitopes, allowing for generation of antibodies that:

  • Recognize specific conformational states

  • Block or promote particular receptor functions

  • Exhibit differential effects based on cellular context

• CAR T Cell Optimization: Structural insights can guide the design of optimized CAR T cell constructs with binding domains that selectively recognize tumor-associated FOLR1 conformations .

What emerging technologies show promise for studying FOLR1 trafficking and signaling dynamics?

Several cutting-edge technologies are advancing our understanding of FOLR1 biology:

• Live-Cell Super-Resolution Microscopy: Techniques like STORM, PALM, and lattice light-sheet microscopy enable real-time visualization of FOLR1 trafficking with nanometer-scale resolution, revealing:

  • Membrane microdomain localization

  • Endocytic vesicle formation and sorting

  • Receptor clustering dynamics

• CRISPR-Based Genomic Editing: Beyond simple knockout models, precision CRISPR techniques allow:

  • Tagging endogenous FOLR1 with fluorescent reporters

  • Creating specific post-translational modification site mutations

  • Introducing conditional expression systems for temporal control

• Proximity Labeling Proteomics: BioID or APEX2-based approaches can identify transient FOLR1 protein interaction partners during different trafficking stages, providing insights into the complete FOLR1 interactome.

• Single-Cell Omics: Integrating single-cell transcriptomics, proteomics, and metabolomics can reveal heterogeneity in FOLR1 expression and function across cell populations and under different conditions.

• Advanced Tissue Imaging: Multiplex imaging systems used for tissue cross-reactivity experiments can be extended to study:

  • Spatial relationships between FOLR1 and other markers

  • Heterogeneity within tumor microenvironments

  • Changes in FOLR1 expression patterns during disease progression

• Computational Molecular Dynamics: Simulations based on crystallographic data can model:

  • Conformational transitions between different FOLR1 states

  • Interactions with potential therapeutic agents

  • Effects of mutations or post-translational modifications on receptor function

These technologies collectively promise to provide a more comprehensive understanding of FOLR1 biology, potentially revealing new therapeutic approaches beyond current targeting strategies.

Table 1: Comparative Properties of Key Anti-FOLR1 Antibodies

rFRA = full length recombinant human folate receptor alpha; dFRA = denatured folate receptor alpha

Table 2: FOLR Family Members in Humans