CAR9 Antibody

What is Carbonic Anhydrase IX (CA9) and why is it an important research target?

Carbonic Anhydrase IX (CA9), also known as membrane antigen MN and renal cell carcinoma (RCC)-associated antigen G250, is a transmembrane enzyme expressed primarily in carcinoma cells. CA9 is an attractive target for cancer research because it is highly expressed in various tumor types under hypoxic conditions, while showing limited expression in normal tissues. This differential expression makes it valuable for both diagnostic applications and therapeutic targeting, particularly in renal cell carcinoma, colon cancer, and various other hypoxic tumors .

What are the typical molecular characteristics of CA9 protein when detected by antibodies?

When detected by Western blot analysis, CA9 typically appears as a specific band at approximately 58 kDa under reducing conditions. This has been validated in studies using U-87 MG human glioblastoma/astrocytoma cell lysates probed with anti-CA9 antibodies. The protein spans from Pro59 to Asp414 in its sequence (Accession # Q16790), and detection can be optimized using specific immunoblot buffer conditions (such as Immunoblot Buffer Group 8) .

What are the most common applications for CA9 antibodies in cancer research?

CA9 antibodies are employed across multiple experimental platforms in cancer research:

Each application requires specific optimization for antibody concentration, incubation time, and detection systems .

How can CA9 antibodies be used to map hypoxic regions in tumor microenvironments?

For comprehensive hypoxia mapping, researchers should consider a multi-marker approach that combines CA9 antibodies with vascular-targeting antibodies like L19. This combination produces a more homogeneous tumor mapping pattern, as these markers target complementary regions within the tumor microenvironment .

What are the considerations for developing high-affinity human monoclonal antibodies against CA9?

Developing high-affinity human monoclonal antibodies against CA9 involves several critical considerations:

• Selection Technology: Phage display technology has been successfully employed to generate high-affinity human monoclonal antibodies (such as A3 and CC7) specific to the extracellular carbonic anhydrase domain of human CA9.

• Validation Hierarchy: Antibody candidates must be methodically validated through:

  • In vitro binding studies to confirm specificity

  • Ex vivo staining on tissue sections

  • In vivo targeting studies following intravenous administration

• Epitope Focus: Target the extracellular carbonic anhydrase domain for therapeutic applications to ensure accessibility in intact cells.

• Functional Assessment: Evaluate the ability of candidate antibodies to recognize CA9 on tumor cell surfaces and preferentially localize to hypoxic regions in vivo.

These approaches have yielded antibodies capable of selective recognition of CA9 on tumor cell surfaces in vitro, in tumor sections ex vivo, and preferential localization to hypoxic sites in vivo .

How do CA9 inhibition antibodies differ from standard CA9-binding antibodies in their therapeutic potential?

CA9 inhibition antibodies represent a distinct functional class compared to standard CA9-binding antibodies:

The chimeric antibody chKM4927 exemplifies this distinction, as it combines CA9-specific inhibition activity with antibody-dependent cellular cytotoxicity (ADCC) against CA9-expressing cancer cells. Importantly, research has shown that chKM4927 with attenuated ADCC activity still demonstrates effective anti-tumor activity in the VMRC-RCW xenograft model, suggesting its efficacy occurs through an ADCC-independent mechanism linked to CA9 inhibition .

What methodological approaches are used to develop anti-idiotype antibodies for detecting CAR T cells in clinical applications?

While distinct from CA9 antibodies, the development of anti-idiotype antibodies for Chimeric Antigen Receptor (CAR) T cell detection provides valuable methodological insights applicable to antibody research:

• Immunization Strategy: Generate cellular vaccines expressing the antigen-recognition domain of interest (e.g., scFv region of CD19-specific mouse monoclonal antibody FMC63).

• Specificity Validation: Confirm antibody specificity through functional inhibition assays, such as validating inhibition of CAR-dependent lysis of target-positive tumor cells.

• Sensitivity Assessment: Determine detection sensitivity through dilution experiments (e.g., detecting CAR+ T cells in peripheral blood mononuclear cells at sensitivities of 1:1,000).

• Clinical Application Validation: Verify utility for monitoring administered cells in clinical settings, including immunophenotyping and persistence evaluation.

This approach has successfully yielded anti-idiotype monoclonal antibodies (e.g., clone 136.20.1) for detecting CD19-specific CAR+ T cells and could be extended to other antigen-specific CAR T cell therapies .

What are the optimal tissue fixation and antigen retrieval protocols for CA9 immunohistochemistry?

Successful CA9 immunohistochemistry requires careful attention to tissue processing and antigen retrieval:

For formalin-fixed paraffin-embedded (FFPE) tissue sections:

• Fixation: Immersion fixation in neutral-buffered formalin provides consistent results.

• Antigen Retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) at 95°C for 20 minutes effectively exposes CA9 epitopes.

• Antibody Concentration: For goat anti-human CA9 antibodies, 15 μg/mL applied overnight at 4°C yields optimal staining with minimal background.

• Detection System: HRP-DAB visualization systems (such as Anti-Goat HRP-DAB Cell & Tissue Staining Kit) provide strong chromogenic signal with excellent contrast when counterstained with hematoxylin.

• Controls: Include both positive controls (known CA9-expressing tissues like colon cancer) and negative controls (antibody diluent without primary antibody) .

How can researchers optimize Western blot protocols for detecting CA9 in cell lysates?

Optimizing Western blot protocols for CA9 detection requires attention to several critical parameters:

• Sample Preparation:

  • Use RIPA or similar lysis buffers containing protease inhibitors

  • For tumor tissues, rapid freezing in liquid nitrogen followed by mechanical homogenization preserves protein integrity

• Gel Electrophoresis:

  • Use 10-12% polyacrylamide gels for optimal resolution of the ~58 kDa CA9 protein

  • Run under reducing conditions with appropriate molecular weight markers

• Transfer Conditions:

  • PVDF membranes show superior results compared to nitrocellulose for CA9 detection

  • Semi-dry transfer at 15-20V for 30-45 minutes provides efficient protein transfer

• Antibody Conditions:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Primary antibody concentration of 1 μg/mL provides optimal signal-to-noise ratio

  • HRP-conjugated secondary antibodies (such as Anti-Goat IgG) at 1:2000-1:5000 dilution

• Detection System:

  • Enhanced chemiluminescence (ECL) substrates provide sensitive detection

  • Expose to film or use digital imaging systems with exposure optimization

What are the key considerations when designing experiments to evaluate CA9 antibody efficacy in vivo?

Designing in vivo experiments to evaluate CA9 antibody efficacy requires careful planning:

• Model Selection:

  • Choose xenograft models that express CA9 under physiologically relevant conditions

  • Consider models with known hypoxic regions (e.g., VMRC-RCW or LS174T)

  • Include appropriate control models with low/negative CA9 expression

• Dosing Strategy:

  • Determine antibody dosing based on pharmacokinetic studies (10 mg/kg has shown efficacy in VMRC-RCW xenograft models)

  • Establish treatment schedules based on tumor growth kinetics and antibody half-life

  • Consider combination approaches with other therapies

• Outcome Measures:

  • Primary: Tumor volume measurements, survival analysis

  • Secondary: Mechanism assessment (CA9 inhibition, ADCC activity)

  • Ex vivo analysis: Immunohistochemistry for target engagement and mechanism validation

• Mechanism Dissection:

  • Include antibody variants with modified effector functions (e.g., ADCC-attenuated versions)

  • Compare standard vs. inhibitory antibodies

  • Correlate with hypoxia markers (pimonidazole) and vascular markers

• Imaging Components:

  • Consider biodistribution studies using labeled antibodies

  • Evaluate co-localization with hypoxia markers and complementary targeting agents

How should researchers interpret discrepancies between CA9 staining patterns and traditional hypoxia markers?

Interpreting discrepancies between CA9 staining and traditional hypoxia markers requires careful analysis of several factors:

• Temporal Dynamics: CA9 expression represents an adaptive response to hypoxia that may persist beyond acute hypoxic events, whereas pimonidazole only labels actively hypoxic cells. This temporal difference can lead to distinct staining patterns.

• Tumor Model Specificity: Different tumor models show varying correlations between CA9 and pimonidazole staining. For example, in LS174T colorectal cancer models, CA9 staining closely matches pimonidazole patterns, while SW1222 models show distinct patterns for these markers.

• Microenvironmental Factors: Beyond hypoxia, CA9 expression can be influenced by other microenvironmental factors including pH changes, nutrient availability, and genetic alterations in cancer cells.

• Physiological Heterogeneity: Tumors demonstrate spatial and temporal heterogeneity in hypoxia, creating complex patterns that may not be fully captured by any single marker.

When discrepancies occur, researchers should consider implementing complementary targeting strategies. For example, combining CA9-targeting antibodies with vascular-targeting antibodies (e.g., L19) can provide more comprehensive tumor coverage, as these markers often target complementary regions within the tumor microenvironment .

What controls should be included when validating CA9 antibody specificity?

Comprehensive validation of CA9 antibody specificity requires a systematic approach with multiple controls:

• Positive Cell Line Controls:

  • U-87 MG (human glioblastoma/astrocytoma)

  • A431 (human epithelial carcinoma)

  • VMRC-RCW (renal cell carcinoma)

• Negative Controls:

  • Cell lines with confirmed absence of CA9 expression

  • Isotype control antibodies matched to the primary antibody

  • Secondary antibody-only controls to assess background

• Competitive Inhibition:

  • Pre-incubation of antibody with recombinant CA9 protein

  • Decreasing signal indicates specific binding

• Knockdown/Knockout Validation:

  • siRNA or CRISPR-based CA9 gene silencing

  • Confirming reduced antibody binding in knockdown cells

• Multiple Detection Methods:

  • Cross-validate findings using different techniques (Western blot, ICC, FACS)

  • Consistent results across platforms strengthen specificity claims

How can researchers distinguish between true CA9 signal and background in challenging tissue samples?

Discriminating true CA9 signal from background in challenging tissue samples requires rigorous technical approaches:

• Optimized Antigen Retrieval: Inadequate or excessive antigen retrieval can create false negatives or high background. Optimize pH and duration of retrieval for each tissue type.

• Titration Experiments: Perform antibody titration experiments (1-20 μg/mL) to identify the optimal concentration that maximizes specific signal while minimizing background.

• Multiple Blocking Strategies: Test different blocking agents (BSA, normal serum, commercial blockers) to reduce non-specific binding in high-background tissues.

• Signal Amplification Systems: For weak signals, consider tyramide signal amplification or polymer-based detection systems, balanced against potential background increases.

• Counterstaining Optimization: Adjust counterstain intensity to provide cellular context without obscuring specific CA9 signal.

• Multi-Antibody Validation: When possible, confirm findings using multiple antibodies targeting different CA9 epitopes.

• Comparison to Established Patterns: CA9 typically shows membrane localization in epithelial cells of tumors, particularly in perinecrotic/hypoxic regions. Patterns deviating from this expectation warrant additional validation.

• Technical Controls: Include serial sections with primary antibody omission and isotype controls processed identically to experimental samples .

What are the emerging applications of CA9 antibodies beyond conventional cancer research?

CA9 antibodies are expanding beyond conventional applications into several emerging research areas:

• Combination Immunotherapy Approaches: CA9 antibodies are being investigated in combination with immune checkpoint inhibitors to enhance anti-tumor responses in hypoxic microenvironments, which typically resist checkpoint blockade alone.

• Antibody-Drug Conjugates (ADCs): The highly specific expression of CA9 in tumor tissues makes it an attractive target for ADC development, where CA9 antibodies can deliver cytotoxic payloads directly to cancer cells while sparing normal tissues.

• Bispecific Antibody Platforms: Emerging research is exploring bispecific antibodies that simultaneously target CA9 and immune effector cells, potentially enhancing immune recruitment to hypoxic tumor regions.

• Molecular Imaging Applications: CA9 antibodies are being developed as imaging tracers for non-invasive visualization of hypoxic regions in tumors, potentially guiding personalized treatment approaches.

• Target Engagement Biomarkers: CA9 antibodies can serve as pharmacodynamic markers to confirm target engagement of novel hypoxia-targeting therapeutics in clinical trials .

How might the methodologies used in developing CAR-specific antibodies influence future CA9 antibody research?

The methodologies established for developing CAR-specific antibodies offer valuable approaches that could advance CA9 antibody research:

• Anti-Idiotype Antibody Development: The approach used to develop anti-idiotype antibodies against CAR constructs could be adapted to create reagents specific to the binding domain of CA9 inhibitory antibodies, enabling better characterization of their tissue distribution and pharmacokinetics.

• Cellular Immunization Strategies: The use of cellular vaccines expressing target epitopes could improve the generation of high-affinity antibodies against conformational epitopes of CA9 that may be difficult to capture using conventional protein immunization.

• Sensitivity Enhancement: Techniques that achieved detection sensitivity of 1:1,000 for CAR+ T cells could be applied to develop enhanced detection methods for rare CA9-expressing cells in circulation or within heterogeneous tumor samples.

• Clinical Monitoring Adaptations: Methodologies for monitoring CAR T cells in patients could be modified to track therapeutic CA9 antibodies, providing valuable pharmacodynamic and pharmacokinetic data in clinical trials.

• Expansion to Related Targets: The framework for extending these methodologies to different tumor-associated antigens in CAR T therapy could similarly be applied to develop antibodies against other carbonic anhydrase isoforms relevant to cancer .