FZD7 Antibody

What is FZD7 and why is it a significant target in cancer research?

FZD7 (Frizzled class receptor 7) is a cell-surface receptor for WNT proteins with critical roles in the WNT/β-catenin signaling pathway. It contains seven transmembrane domains, a cysteine-rich domain in the extracellular region, and a carboxy terminal Ser/Thr-xxx-Val motif that enables its function as a WNT receptor . The significance of FZD7 in cancer research stems from its distinctive expression pattern - high expression in numerous tumor types including breast, ovarian, liver, gastric, and skin cancers, while showing low to modest expression in normal adult tissues . This differential expression makes FZD7 an attractive candidate for targeted cancer therapies that minimize off-target effects. Additionally, FZD7 contains a Lys-Thr-X-X-X-Trp motif specifically involved in activating the Wnt/β-catenin signaling pathway, which is frequently dysregulated in cancer progression .

Research has revealed that FZD7 overexpression correlates with poorer patient outcomes, particularly in the mesenchymal and proliferative subtypes of ovarian serous cystadenocarcinoma as documented in The Cancer Genome Atlas . This correlation with clinical outcomes further underscores the potential of FZD7 as both a prognostic marker and therapeutic target.

What are the recommended applications and dilutions for FZD7 antibodies?

FZD7 antibodies have been validated for multiple research applications including Western blotting (WB), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . Based on extensive validation studies, the following dilution ranges are recommended as starting points for experimental optimization:

These dilution recommendations provide a foundation for assay optimization, though actual working concentrations may need adjustment based on specific experimental conditions, antibody lots, and detection methods . When planning experiments, researchers should allocate sufficient antibody to perform validation runs before proceeding with critical experiments.

How should FZD7 antibodies be stored to maintain optimal activity?

Proper storage is crucial for maintaining antibody functionality. FZD7 antibodies should be stored undiluted at 2-8°C for short-term use (up to one month) or at -20°C for long-term storage . For antibodies preserved in liquid form containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, storage at -20°C is recommended for up to one year .

When working with FZD7 antibodies, it's essential to avoid repeated freeze-thaw cycles as these can significantly degrade antibody performance . A practical approach is to prepare small aliquots upon receiving the antibody, keeping working aliquots at 4°C and reserve stocks at -20°C. This strategy minimizes freeze-thaw cycles while ensuring a steady supply of functional antibody for ongoing research projects.

How can researchers validate the specificity of FZD7 antibodies?

Establishing antibody specificity is fundamental to obtaining reliable research results. For FZD7 antibodies, specificity validation should include:

• Cross-reactivity testing: Verify that the antibody binds specifically to FZD7 without cross-reacting with other Frizzled family receptors. Flow cytometry studies have confirmed that properly validated FZD7 antibodies do not cross-react with the other nine human FZD receptors (FZD1-6,8-10) .

• Knockout validation: Generate FZD7 knockout cell lines (such as MA-148 FZD7-KO created by CRISPR/Cas9) to serve as negative controls . The substantial difference in antibody response or functional effect between wild-type and knockout cells provides strong evidence of specificity.

• Peptide blocking: Use the immunogen peptide (such as the synthetic peptide corresponding to amino acids 61-110 of Human Frizzled-7) to perform competitive binding assays . Significant reduction in signal when the antibody is pre-incubated with the immunizing peptide indicates specific binding.

• Multiple detection methods: Confirm FZD7 detection using different techniques (WB, IF, ELISA) to ensure consistent specificity across various experimental platforms .

These validation approaches should be documented thoroughly in research publications to support the reliability of subsequent findings and facilitate reproducibility by other investigators.

What methodological approaches are effective for studying FZD7-targeted inhibition of Wnt signaling?

When investigating FZD7-targeted inhibition of Wnt signaling, several methodological considerations can enhance experimental rigor:

• Downstream signaling analysis: Measure expression and activation of β-catenin and Axin2, two established downstream targets in the Wnt pathway, to assess inhibition efficacy . These markers provide quantifiable readouts of pathway suppression.

• Multivalent binding strategies: Consider employing antibody-functionalized nanoparticles instead of free antibodies. FZD7 antibody-nanoshell conjugates (FZD7-NS) have demonstrated significantly enhanced efficacy in inhibiting Wnt signaling in triple negative breast cancer (TNBC) cells compared to freely delivered FZD7 antibodies . This approach exploits multivalent binding effects for improved signal cascade interference.

• Cell viability correlation: Establish relationship patterns between Wnt signaling inhibition and cellular outcomes like decreased viability or altered proliferation. This correlation supports the therapeutic relevance of the inhibition .

• Cell line selection: Use cell lines with documented FZD7 expression and Wnt pathway dependence. MA-148 and PA-1 human ovary-derived cancer cell lines have been identified as responsive to FZD7-targeted interventions and serve as appropriate model systems .

These methodological approaches provide a framework for rigorous investigation of FZD7-targeted Wnt pathway inhibition while generating more reliable and translatable research outcomes.

What design principles are important when developing FZD7 antibody-drug conjugates?

The development of effective FZD7 antibody-drug conjugates (ADCs) requires careful consideration of several design elements:

• Antibody specificity: The antibody component must selectively bind to FZD7 without cross-reactivity to other Frizzled family members. This specificity is crucial for minimizing off-target effects and establishing a favorable therapeutic window .

• Linker chemistry: The choice of linker affects drug release kinetics and ADC stability. Cleavable valine-citrulline linkers have proven effective in FZD7 ADCs, allowing for controlled release of the cytotoxic payload upon internalization .

• Cytotoxic payload selection: Monomethyl auristatin E (MMAE), an antimitotic agent that inhibits microtubule assembly, has demonstrated efficacy when conjugated to FZD7 antibodies. The drug-to-antibody ratio (typically four MMAE molecules per antibody) significantly influences potency and pharmacokinetic properties .

• Species cross-reactivity considerations: Since most therapeutic antibodies target human proteins with potential species differences, researchers developing FZD7 ADCs should consider creating mouse models with humanized FZD7 to enable proper toxicity evaluation .

Septuximab vedotin (F7-ADC), a chimeric human-mouse antibody to human FZD7 conjugated to MMAE, exemplifies these design principles and has shown promising results in preclinical studies, inducing regression of ovarian tumor xenografts in murine models .

How can researchers effectively evaluate the efficacy of FZD7 antibody-drug conjugates?

Comprehensive evaluation of FZD7 ADC efficacy requires a multi-layered experimental approach:

• In vitro cytotoxicity assessment: Determine the IC50 of the ADC in FZD7-positive cancer cell lines. Studies with FZD7 ADCs have demonstrated potent cytotoxicity with IC50 values of approximately 0.76 μg/mL (~5 nM) in responsive cell lines like MA-148 and PA-1 .

• Target dependency validation: Compare ADC cytotoxicity between wild-type and FZD7 knockout cells. The significant difference in IC50 values (e.g., ~5 nM in FZD7-positive cells versus ~60 nM in FZD7-knockout cells) establishes the therapeutic window and confirms target-dependent activity .

• In vivo xenograft studies: Assess tumor regression in subcutaneous or orthotopic xenograft models using luciferase-labeled tumor cells for real-time monitoring. Including multiple dosage arms (e.g., PBS control, 1 mg/kg, and 3 mg/kg) helps establish dose-response relationships .

• Mechanistic confirmation: Verify that the observed anti-tumor effects occur through the expected mechanism by examining markers of mitotic arrest, apoptosis, or other relevant cellular processes based on the conjugated drug's mode of action.

This systematic evaluation provides robust evidence of efficacy while identifying potential limitations before advancing to more complex preclinical models or clinical studies.

What approaches can researchers use to assess the safety profile of FZD7-targeted therapeutics?

Assessing the safety profile of FZD7-targeted therapeutics requires specialized approaches to address on-target and off-target toxicities:

• Genetically modified mouse models: Develop knock-in mice harboring a modified Fzd7 gene where the resulting Fzd7 protein is reactive with the human-targeting therapeutic. This approach enables evaluation of on-target toxicities that might not be apparent in conventional models where the therapeutic doesn't recognize the murine ortholog .

• Tissue cross-reactivity studies: Perform immunohistochemical analyses across a panel of normal tissues to identify potential sites of unintended binding that could predict toxicity. This is particularly important given that FZD7 shows expression in adult skeletal muscle, heart, brain, and placenta, as well as fetal kidney and lung .

• Long-term toxicity assessment: Conduct extended treatment protocols to identify delayed toxicities that might arise from cumulative effects or from interfering with normal tissue homeostasis dependent on Wnt signaling.

• Comparative approaches: Benchmark the safety profile of FZD7-targeted therapeutics against pan-WNT inhibitors, which are known to cause adverse effects, particularly in bone. This comparison helps establish whether the targeted approach offers improved safety .

Studies with F7-ADC (septuximab vedotin) in mice engineered to express human-reactive Fzd7 have shown encouraging safety profiles with no acute toxicities, suggesting potential clinical translatability . This methodical assessment of safety is essential for advancing FZD7-targeted therapies toward clinical applications.

How can FZD7 antibodies be used to stratify patients for targeted therapies?

FZD7 expression varies across cancer types and subtypes, offering potential for patient stratification:

• Expression correlation with cancer subtypes: Research has identified elevated FZD7 expression particularly in mesenchymal and proliferative subtypes of ovarian serous cystadenocarcinoma, which correlate with poorer median patient survival . This differential expression suggests that FZD7 antibody-based diagnostics could identify patients most likely to benefit from FZD7-targeted therapies.

• Development of companion diagnostics: Standardized immunohistochemical protocols using validated FZD7 antibodies could be developed as companion diagnostics for FZD7-targeted therapies. These would determine FZD7 expression levels in patient tumors before treatment initiation.

• Multiparameter analysis: Combining FZD7 expression data with other Wnt pathway components could create more refined stratification models, potentially identifying patients with activated Wnt signaling who might respond to pathway inhibition even with moderate FZD7 expression.

• Threshold determination: Research is needed to establish expression thresholds that predict therapeutic response, moving beyond simple positive/negative classification to quantitative assessments that correlate with clinical outcomes.

This stratification approach addresses the heterogeneity of cancer and works toward the goal of precision medicine by matching patients with therapies most likely to provide clinical benefit based on molecular characteristics of their tumors.

What are the future prospects for combining FZD7 antibodies with other cancer treatments?

The potential for combining FZD7 antibodies with other cancer treatments represents a promising research frontier:

• Synergy with conventional chemotherapies: FZD7 antibody-drug conjugates could be combined with standard chemotherapy regimens to achieve greater efficacy through complementary mechanisms of action. This might allow dose reduction of conventional agents, potentially reducing adverse effects.

• Integration with immune checkpoint inhibitors: Since Wnt signaling influences the tumor immune microenvironment, combining FZD7-targeted therapies with immune checkpoint inhibitors might enhance immunotherapy responses, particularly in cancers with immunosuppressive Wnt pathway activation.

• Multitargeted Wnt pathway inhibition: Combining FZD7 antibodies with therapeutics targeting other nodes in the Wnt pathway could provide more complete signaling inhibition while minimizing the toxicity associated with pan-WNT inhibitors .

• Nanomedicine approaches: Further development of FZD7 antibody-functionalized nanoparticles could enable co-delivery of multiple therapeutic agents or combine targeted delivery with physical treatment modalities like photothermal therapy .

These combinatorial approaches may overcome resistance mechanisms and achieve more durable responses than monotherapies, representing an important direction for future FZD7 research with potential clinical implications.