CKB4 Antibody

What is CKAP4 and what are its primary cellular functions?

CKAP4, also known as Climp-63 or p63, is a 63-kDa cytoskeleton-linking membrane protein that serves multiple critical cellular functions. Primarily, it mediates the anchoring of the endoplasmic reticulum to microtubules, establishing important cytoskeletal connections. Additionally, CKAP4 functions as a high-affinity epithelial cell surface receptor for the FZD8-related low molecular weight sialoglycopeptide antiproliferative factor (APF), mediating antiproliferative signaling within cells. Recent research has identified CKAP4 as a receptor for Dickkopf-1 (DKK1), where their interaction activates the AKT pathway, promoting tumor cell growth in multiple cancer types .

What types of CKAP4 antibodies are currently available for research applications?

Several CKAP4 antibodies are available for research purposes, with varying applications and specificities:

• Rabbit Polyclonal antibodies (e.g., ab245508) suitable for immunoprecipitation (IP) and Western blotting (WB), reacting with both human and mouse samples. These antibodies typically target synthetic peptides within the human CKAP4 sequence, often the C-terminal region (aa 550 to C-terminus) .

• Rabbit Polyclonal antibodies (e.g., ab152154) suitable for immunohistochemistry on paraffin-embedded samples (IHC-P), Western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF), typically reacting with human samples. These antibodies are often generated against recombinant fragments within the mid-section of human CKAP4 (aa 200-600) .

• Therapeutic humanized antibodies (e.g., Hv1Lt1) specifically designed to block the interaction between DKK1 and CKAP4, inhibiting downstream signaling and suppressing tumor growth .

What are the optimal conditions for using anti-CKAP4 antibodies in Western blotting experiments?

For optimal Western blotting results with anti-CKAP4 antibodies, researchers should consider the following protocol:

• Sample preparation: Use whole cell lysates from relevant cell lines (HeLa, HEK-293T, NIH/3T3) at varying concentrations (5-50 μg) to determine optimal loading amounts.

• Gel electrophoresis: 7.5% SDS-PAGE is recommended for optimal separation of CKAP4, which has a predicted band size of 66 kDa.

• Antibody concentration: For antibodies like ab245508, use at 0.04 μg/mL; for ab152154, a 1/10000 dilution is recommended.

• Validation controls: Include both positive controls (cell lines known to express CKAP4) and negative controls (depleted samples or knockout cell lines if available).

• Detection: Standard chemiluminescence methods are suitable with appropriate secondary antibodies .

The antibody should detect a distinct band at approximately 66 kDa, corresponding to the CKAP4 protein. Multiple sample loadings can help establish the sensitivity and linear range of detection.

How can CKAP4 be detected in patient serum samples, and what methodological considerations are important?

Detection of CKAP4 in serum samples relies on its secretion via exosomes, making it accessible as a potential biomarker. Key methodological considerations include:

• Sample collection: Standardized collection of serum from patients and age/sex-matched healthy controls is essential. Time points before and after surgical intervention provide valuable comparative data.

• Detection method: ELISA has been successfully used to detect CKAP4 in serum samples. Custom antibodies with high specificity are critical.

• Exosome isolation: Since CKAP4 is primarily released through exosomes, ultracentrifugation or commercial exosome isolation kits may enhance detection sensitivity.

• Correlation analysis: Compare serum CKAP4 levels with tumor tissue expression through immunohistochemistry to validate the relationship between circulating and tissue levels.

• Palmitoylation consideration: As CKAP4 trafficking to exosomes is regulated by palmitoylation, this post-translational modification should be considered when designing detection methods .

Research has demonstrated that serum CKAP4 levels significantly decrease following surgical removal of tumors, indicating its potential utility as a monitoring biomarker for treatment response.

How does CKAP4 contribute to cancer progression, and what evidence supports its role as a therapeutic target?

CKAP4 contributes to cancer progression through multiple mechanisms:

• DKK1-CKAP4 pathway activation: CKAP4 serves as a receptor for DKK1, initiating signaling cascades that promote tumor growth through AKT activation. Studies show that simultaneous expression of both DKK1 and CKAP4 correlates with poor prognosis in pancreatic, esophageal, and lung cancers .

• Cellular proliferation: Overexpression of CKAP4 in cancer cells promotes in vitro cell proliferation and in vivo subcutaneous tumor growth, establishing a direct causal relationship between CKAP4 expression and cancer progression .

• Cancer stem cell maintenance: CKAP4 appears to support cancer stem cell populations, as evidenced by the ability of anti-CKAP4 antibodies to inhibit sphere formation, a measure of cancer stem cell self-renewal capacity .

• Chemoresistance mechanisms: CKAP4 signaling activates the AKT pathway, which is implicated in chemoresistance. Targeting CKAP4 may help overcome resistance to standard chemotherapeutic agents .

Therapeutic targeting of CKAP4 using humanized antibodies has shown promising results in preclinical models, including suppression of tumor growth in xenograft models and enhanced efficacy when combined with standard chemotherapy or targeted agents like osimertinib .

What is the significance of CKAP4 as a potential biomarker in cancer, and how does its expression correlate with patient outcomes?

CKAP4 has emerged as a promising biomarker in cancer for several reasons:

• Elevated expression: CKAP4 expression is increased in multiple cancer types, with serum levels significantly higher in cancer patients compared to healthy controls.

• Prognostic value: Patients with tumors expressing both DKK1 and CKAP4 demonstrate worse clinical outcomes, suggesting CKAP4's utility as a prognostic marker.

• Treatment monitoring: Serum CKAP4 levels decrease following surgical removal of tumors, indicating potential as a monitoring biomarker for treatment response.

• Exosomal secretion: CKAP4 is secreted via exosomes, making it accessible in liquid biopsies, which are less invasive than tissue sampling.

• Correlation with tumor burden: Serum CKAP4 levels appear to reflect tumor expression of CKAP4, suggesting a relationship between circulating levels and tumor burden .

Studies in lung cancer have demonstrated that serum CKAP4 positivity is significantly higher in cancer patients than in healthy controls, and post-operative reduction in serum CKAP4 levels further supports its potential as a clinically relevant biomarker .

How does palmitoylation regulate CKAP4 trafficking and function, and what techniques can be used to study this post-translational modification?

Palmitoylation, the covalent attachment of palmitic acid to cysteine residues, is a critical post-translational modification that regulates CKAP4 trafficking and function. Research indicates that palmitoylation of CKAP4 controls its exosomal secretion and is required for its tumor-promoting activities .

To study CKAP4 palmitoylation, researchers can employ several techniques:

• Acyl-Biotin Exchange (ABE) assay: This technique allows for specific detection of palmitoylated proteins by exchanging palmitoyl groups with biotin, followed by pull-down and Western blot analysis.

• Metabolic labeling: Using palmitic acid analogs like 17-octadecynoic acid (17-ODYA) coupled with click chemistry to visualize palmitoylated proteins.

• Site-directed mutagenesis: Creating cysteine-to-alanine mutations at potential palmitoylation sites to assess functional consequences.

• Palmitoylation inhibitors: Using compounds like 2-bromopalmitate to block palmitoylation and assess the effects on CKAP4 trafficking and function.

• Mass spectrometry: To identify specific palmitoylation sites and quantify the degree of modification.

Understanding the regulation of CKAP4 palmitoylation may provide additional therapeutic opportunities, as targeting this modification could potentially disrupt CKAP4's oncogenic functions by preventing its proper trafficking to the cell surface or into exosomes.

What are the synergistic mechanisms between anti-CKAP4 antibodies and tyrosine kinase inhibitors, and how can researchers optimize combination treatments?

The synergistic effects between anti-CKAP4 antibodies and tyrosine kinase inhibitors (TKIs) like osimertinib involve several complementary mechanisms:

• Dual AKT pathway inhibition: Both anti-CKAP4 antibodies and TKIs can inhibit AKT activation, albeit through different upstream mechanisms. Anti-CKAP4 antibodies block DKK1-CKAP4 signaling, while TKIs inhibit receptor tyrosine kinase-mediated AKT activation .

• Targeting cancer stem cells: Anti-CKAP4 antibodies inhibit sphere formation, suggesting effects on cancer stem cell populations that may be resistant to conventional TKI therapy.

• Overcoming resistance mechanisms: The DKK1-CKAP4 pathway may represent an alternate survival mechanism for cancer cells under TKI treatment pressure.

• Immune modulation: Anti-CKAP4 antibodies appear to modulate anti-tumor immune responses, potentially complementing the direct cytotoxic effects of TKIs .

Researchers can optimize combination treatments through:

• Sequential vs. concurrent administration: Testing various dosing schedules to determine optimal timing.

• Dose-response matrices: Systematic testing of different dose combinations to identify synergistic ratios while minimizing toxicity.

• Patient stratification: Identifying biomarkers (DKK1/CKAP4 expression levels) that predict response to combination therapy.

• Resistance monitoring: Tracking changes in DKK1-CKAP4 signaling during treatment to adapt therapy accordingly.

Studies have already demonstrated that combining anti-CKAP4 antibody with osimertinib shows stronger inhibition of tumor growth than either agent alone in lung cancer models .

What experimental approaches can distinguish between CKAP4's role as an endoplasmic reticulum-microtubule anchor versus its function as a cell surface receptor?

Distinguishing between CKAP4's dual roles requires sophisticated experimental approaches:

• Subcellular fractionation: Isolation of different cellular compartments (membrane, cytosolic, ER, nuclear) followed by Western blotting to determine CKAP4 distribution.

• Immunofluorescence microscopy: Co-localization studies using antibodies against CKAP4 and markers for different cellular compartments (ER: calnexin; plasma membrane: Na⁺/K⁺-ATPase).

• Domain-specific mutants: Generation of CKAP4 constructs with mutations in specific domains (luminal, transmembrane, cytoplasmic) to selectively disrupt either ER anchoring or receptor functions.

• Surface biotinylation: Selective labeling of cell surface proteins to quantify the proportion of CKAP4 expressed at the plasma membrane versus intracellular locations.

• CRISPR-Cas9 gene editing: Creating precise mutations that affect either ER anchoring or receptor functions to dissect the physiological importance of each role.

• Live-cell imaging: Using fluorescently tagged CKAP4 to track its dynamic movement between cellular compartments under various stimuli.

• Ligand binding assays: Measuring binding of DKK1 to cell surface CKAP4 using techniques like surface plasmon resonance or flow cytometry-based binding assays .

These approaches can help determine whether CKAP4's roles in cancer progression primarily stem from its plasma membrane receptor functions, its ER structural roles, or a complex interplay between both cellular locations.

What strategies have been employed to develop humanized anti-CKAP4 antibodies, and what challenges must be overcome for clinical translation?

The development of humanized anti-CKAP4 antibodies has followed a strategic pathway:

• Initial mouse antibody generation: Researchers first developed recombinant mouse antibodies targeting CKAP4, establishing proof-of-concept for therapeutic potential.

• Humanization process: Mouse-derived antibodies were humanized through framework replacement, maintaining the critical antigen-binding regions (complementarity-determining regions or CDRs) while replacing the remainder with human antibody sequences.

• Binding affinity optimization: The humanized antibody (Hv1Lt1) was refined to achieve even stronger binding to CKAP4 than the original mouse antibody.

• Functional validation: Researchers confirmed that Hv1Lt1 effectively inhibited DKK1-CKAP4 signaling, sphere formation, and tumor growth in various models .

Challenges for clinical translation include:

• Immunogenicity assessment: Despite humanization, residual immunogenic potential must be thoroughly evaluated.

• Target specificity: Ensuring minimal off-target binding to avoid unexpected toxicities.

• Tissue penetration: Optimizing antibody properties to ensure effective delivery to tumor tissues.

• Manufacturing scalability: Developing consistent production methods suitable for clinical-grade material.

• Combination therapy optimization: Determining optimal dosing schedules and drug combinations.

• Patient selection biomarkers: Identifying which patients are most likely to benefit from anti-CKAP4 therapy based on DKK1 and CKAP4 expression profiles .

The successful development of Hv1Lt1 represents significant progress toward clinical applications, with promising preclinical results supporting further development.

How can researchers validate the specificity of commercial anti-CKAP4 antibodies, and what controls should be included in experimental designs?

Validating specificity of anti-CKAP4 antibodies is crucial for reliable research. A comprehensive validation strategy includes:

• Multiple antibody comparison: Using different antibodies targeting distinct epitopes of CKAP4 to confirm consistent results.

• Knockout/knockdown controls:

  • CRISPR-Cas9 CKAP4 knockout cell lines

  • siRNA or shRNA-mediated CKAP4 knockdown

  • These negative controls should show absence or significant reduction of signal

• Overexpression controls: Cell lines transfected with CKAP4 expression vectors to serve as positive controls.

• Peptide competition assays: Pre-incubating the antibody with excess immunizing peptide should abolish specific binding.

• Cross-species reactivity testing: Confirming species specificity claims by testing across human, mouse, and other relevant species samples.

• Application-specific validation:

  • For Western blotting: Confirming band size (66 kDa) and testing multiple cell lines

  • For IHC/ICC: Including isotype controls and testing multiple fixation methods

  • For IP: Confirming pulled-down protein by mass spectrometry

• Lot-to-lot consistency testing: When receiving new antibody lots, comparing performance with previous lots .

A validation table documenting these tests enhances experimental reproducibility and builds confidence in antibody specificity, particularly important for CKAP4 given its dual localization at the ER and plasma membrane.

What are the emerging roles of CKAP4 in immune modulation, and how might this influence immunotherapy approaches?

Recent evidence suggests CKAP4 may play previously unrecognized roles in immune modulation, opening new research avenues:

• Anti-tumor immune response: Research indicates that anti-CKAP4 antibodies help modulate anti-tumor immune reactions, suggesting CKAP4 may influence the tumor immune microenvironment .

• Potential mechanisms requiring investigation:

  • Effects on tumor-associated macrophage polarization

  • Influence on T-cell infiltration and activation status

  • Modulation of NK cell recognition of tumor cells

  • Impact on dendritic cell function and antigen presentation

  • Regulation of immune checkpoint molecule expression

• Methodological approaches to study CKAP4's immune effects:

  • Flow cytometric analysis of tumor-infiltrating lymphocytes in anti-CKAP4 treated models

  • Single-cell RNA sequencing to characterize immune population shifts

  • Spatial transcriptomics to map immune cell distribution relative to CKAP4 expression

  • Cytokine/chemokine profiling in the tumor microenvironment

• Potential immunotherapy combinations:

  • Anti-CKAP4 antibodies with immune checkpoint inhibitors

  • Combinations with CAR-T cell therapies

  • Integration with cancer vaccines

Understanding CKAP4's immunomodulatory functions could significantly expand its therapeutic applications beyond direct tumor cell targeting, potentially enhancing response to existing immunotherapies or revealing new immunological targeting strategies.

What is the relationship between CKAP4 and cancer stem cell maintenance, and what experimental models best capture this phenomenon?

The emerging relationship between CKAP4 and cancer stem cell (CSC) maintenance presents a fascinating research area:

• Evidence of CKAP4's role in CSC maintenance:

  • Anti-CKAP4 antibodies inhibit sphere formation, a key CSC property

  • DKK1-CKAP4 signaling activates AKT, which is crucial for CSC survival

  • Combined treatment with anti-CKAP4 antibodies and TKIs shows enhanced anti-tumor effects, suggesting targeting of resistant CSC populations

• Optimal experimental models for studying CKAP4 in CSCs:

  Model Type	Applications	Advantages	Limitations
  Sphere formation assays	Self-renewal capacity	Quantitative, reproducible	Limited physiological relevance
  Patient-derived organoids	Drug response, heterogeneity	Maintains tumor architecture	Resource-intensive, variable take rates
  Serial transplantation in mice	Long-term CSC properties	Gold standard for stemness	Time-consuming, expensive
  Flow cytometry for CSC markers	Quantification of CSC populations	Rapid analysis of multiple markers	Marker combinations vary by cancer type
  Lineage tracing	In vivo CSC dynamics	Tracks cellular hierarchy	Technically challenging

• Methodological considerations:

  • Combining CKAP4 manipulation (knockdown/overexpression) with CSC functional assays

  • Correlating CKAP4 expression with established CSC markers

  • Examining CKAP4's role in CSC properties like quiescence, differentiation, and therapy resistance

  • Investigating whether CKAP4 expression changes during CSC differentiation

• Potential mechanisms requiring investigation:

  • CKAP4's influence on canonical stemness pathways (Wnt, Notch, Hedgehog)

  • Role in regulating asymmetric division of CSCs

  • Contribution to the CSC niche via exosomal signaling