CAR11 Antibody

What is CARD11 and why is it a significant target for antibody development?

CARD11 (also known as Carma 1) is an adapter protein that plays a key role in adaptive immune response by transducing the activation of NF-kappa-B downstream of T-cell receptor (TCR) and B-cell receptor (BCR) engagement. It functions by forming a multiprotein complex with BCL10 and MALT1 that induces NF-kappa-B and MAP kinase p38 pathways .
Upon activation in response to TCR or BCR triggering, CARD11 homooligomerizes to form a nucleating helical template that recruits BCL10 via CARD-CARD interaction. This promotes polymerization of BCL10 and subsequent recruitment of MALT1, leading to I-kappa-B kinase (IKK) phosphorylation and degradation, and release of NF-kappa-B proteins for nuclear translocation . Additionally, CARD11 promotes linear ubiquitination of BCL10 by targeting BCL10 to RNF31/HOIP and activates the TORC1 signaling pathway .
Antibodies targeting CARD11 are valuable research tools for studying these complex signaling pathways in immune cells and have potential therapeutic applications in immune-related disorders and certain cancers.

What are the optimal protocols for validating CARD11 antibody specificity in experimental systems?

Validating CARD11 antibody specificity requires a multi-step approach to ensure reliable experimental results:

• Western Blotting Validation: Use human samples with known CARD11 expression. Both rabbit recombinant monoclonal (e.g., EPR2557) and rabbit polyclonal CARD11 antibodies have demonstrated suitability for western blotting applications . The expected molecular weight for CARD11 should be confirmed, and positive and negative control samples should be included.

• Immunoprecipitation Controls: When using CARD11 antibodies for immunoprecipitation, include appropriate controls including isotype controls and samples with CARD11 knockdown/knockout. Polyclonal antibodies have shown suitability for immunoprecipitation applications .

• Functional Validation: Test the antibody's ability to detect changes in CARD11 expression or phosphorylation status following T-cell or B-cell receptor stimulation, which should alter CARD11 activation state and complex formation with BCL10 and MALT1.

• Cross-Reactivity Assessment: Determine potential cross-reactivity with other CARD-domain containing proteins to ensure specificity for CARD11/Carma 1.

• Application-Specific Validation: For specialized applications like fluorescent labeling or multiplex imaging, additional validation steps may be required to ensure the antibody performs correctly in these specific contexts .
  These validation methods help ensure the reliability and reproducibility of research findings when using CARD11 antibodies.

How can researchers effectively study CARD11's role in the BCL10-MALT1 signalosome assembly?

Studying CARD11's role in BCL10-MALT1 signalosome assembly requires specialized experimental approaches:

• Co-Immunoprecipitation (Co-IP): Use CARD11 antibodies to pull down protein complexes and detect associated BCL10 and MALT1 proteins. This approach can reveal how CARD11 recruits BCL10 via CARD-CARD interaction and promotes subsequent MALT1 recruitment .

• Proximity Ligation Assays: These assays can detect protein-protein interactions in situ, allowing visualization of CARD11-BCL10-MALT1 complex formation following T-cell or B-cell receptor stimulation.

• Gain-of-Function Mutations Analysis: CARD11 gain-of-function mutations promote enhanced aggregation and idiosyncratic signalosome assembly . Comparing wild-type and mutant CARD11 can provide insights into the structural requirements for signalosome formation.

• Live Cell Imaging: Using fluorescently tagged CARD11, BCL10, and MALT1 proteins to visualize the dynamics of complex formation in real-time following immune receptor stimulation.

• Helical Template Analysis: Since CARD11 forms a nucleating helical template that recruits BCL10, structural studies using cryo-electron microscopy can provide insights into this template formation and subsequent polymerization of BCL10 .
  These approaches provide complementary data on how CARD11 functions as a scaffold protein to assemble the signalosome that activates NF-κB signaling in adaptive immune responses.

What are the latest methodologies for using CAR antibodies in cancer immunotherapy research?

Recent advances in CAR antibody methodologies for cancer immunotherapy research include:

• Bispecific CAR Designs: Novel approaches like the DuoBody-PD-L1×4-1BB (GEN1046) combine simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. This design has shown T-cell proliferation, cytokine production, and antigen-specific T-cell-mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures .

• Dual Chain CAR Format: This innovative approach uses an antibody in its natural configuration for binding, consisting of two chains (immunoglobulin light and heavy chain with their constant regions) that form a stable heterodimer. This format broadens CAR cell therapy to targets without available scFv antibodies .

• Anti-CAR Antibody Therapy: For cancers overexpressing Coxsackie and Adenovirus Receptor (CAR), such as small cell lung cancer (SCLC), chimeric anti-CAR antibodies with ADCC and CDC-inducing activities have shown promising anti-tumor activity in both subcutaneous and orthotopic xenograft models .

• Orthotopic Transplantation Models: Advanced mouse models using GFP-labeled human SCLC cell lines with significant metastatic activity have been developed to evaluate anti-CAR antibody efficacy against both primary tumors and metastases .

• Biomarker Monitoring: Treatment with bispecific immunotherapy agents significantly increases levels of biomarkers such as IFNγ, CXCL10, and proliferating (Ki-67+) total and effector memory CD8+ T-cell counts, which can be used to monitor treatment efficacy .
  These methodologies represent cutting-edge approaches for developing more effective cancer immunotherapies targeting both the PD-1/PD-L1 axis and providing costimulatory signals through 4-1BB or targeting overexpressed receptors like CAR in certain cancers.

How can researchers troubleshoot non-specific binding issues with CARD11 antibodies?

When facing non-specific binding issues with CARD11 antibodies, researchers should implement the following troubleshooting strategies:

• Optimize Blocking Conditions: Use alternative blocking agents (BSA, normal serum, or commercial blocking buffers) to reduce background signals. The effectiveness of blocking agents may vary depending on the specific antibody and sample type.

• Antibody Dilution Optimization: Perform titration experiments to determine the optimal antibody concentration that maximizes specific binding while minimizing background. Both recombinant monoclonal and polyclonal CARD11 antibodies may require different dilution factors for optimal results .

• Sample Preparation Refinement: Ensure proper fixation and permeabilization methods for immunohistochemistry or flow cytometry applications. Overfixation can mask epitopes while underfixation may alter cellular morphology.

• Alternative Antibody Selection: If persistent non-specific binding occurs, consider switching between polyclonal and monoclonal CARD11 antibodies. Monoclonal antibodies like EPR2557 may offer higher specificity for certain applications , while polyclonal antibodies may provide better signal in others .

• Cross-Adsorption: For polyclonal antibodies, cross-adsorption against related proteins can reduce non-specific binding to other CARD-domain containing proteins.

• Negative Controls: Always include appropriate negative controls such as isotype controls and samples known to be negative for CARD11 expression to accurately assess background levels.
  By systematically addressing these factors, researchers can significantly improve the specificity of CARD11 antibody staining and obtain more reliable experimental results.

What considerations should be made when designing experiments to evaluate CAR T-cell efficacy in preclinical models?

When designing experiments to evaluate CAR T-cell efficacy in preclinical models, researchers should consider several critical factors:

• Target Expression Profiling: Thoroughly characterize target antigen expression in both tumor and normal tissues to predict potential on-target/off-tumor toxicity. For example, CAR expression analysis in SCLC compared to normal lung tissues helps identify suitable cancer targets .

• CAR Design Optimization: Consider various CAR formats including single chain versus dual chain designs. Dual chain CARs use antibodies in their natural configuration and may offer advantages for targets without available scFv antibodies .

• Model Selection: Choose appropriate preclinical models that recapitulate human disease. Orthotopic transplantation models, such as those developed for SCLC using GFP-labeled human cell lines, provide valuable insights into both primary tumor growth and metastasis formation .

• Control Groups: Include proper controls such as non-transduced T cells, T cells expressing irrelevant CARs, and comparison to standard-of-care treatments to accurately assess therapeutic efficacy.

• Dose-Response Relationships: Evaluate multiple T-cell doses to establish dose-response relationships and identify the minimum effective dose.

• Biomarker Monitoring: Assess pharmacodynamic biomarkers such as cytokine production (IFNγ), chemokines (CXCL10), and T-cell proliferation markers (Ki-67+ CD8+ T cells) to confirm CAR T-cell activation and function .

• Persistence and Memory Formation: Evaluate long-term persistence of CAR T cells and formation of memory T-cell subsets, which correlate with durable anti-tumor responses.

• Combined Immunotherapies: Consider combining CAR T-cell therapy with other immunomodulatory approaches, such as checkpoint inhibitors or bispecific antibodies like DuoBody-PD-L1×4-1BB, which has shown enhanced T-cell function superior to single-target approaches .
  These considerations enable researchers to design robust preclinical studies that more accurately predict clinical outcomes and identify optimal CAR T-cell therapy approaches.

How does CARD11/Carma 1 signaling interact with other immune pathways in experimental models?

CARD11/Carma 1 signaling intersects with multiple immune pathways in complex ways that can be studied using specific experimental approaches:

• NF-κB Pathway Integration: CARD11 plays a crucial role in transducing signals from T-cell receptor (TCR) and B-cell receptor (BCR) activation to NF-κB. Experimental models can use CARD11 antibodies to track how this protein forms a complex with BCL10 and MALT1 to induce NF-κB activation .

• MAPK Pathway Crosstalk: Besides NF-κB, CARD11 also triggers MAP kinase p38 (MAPK11, MAPK12, MAPK13, and/or MAPK14) pathways . Researchers can use phospho-specific antibodies together with CARD11 antibodies to investigate this crosstalk in various immune cell types.

• DPP4 Interaction: CARD11 binding to DPP4 induces T-cell proliferation and NF-κB activation in a T-cell receptor/CD3-dependent manner . Co-immunoprecipitation experiments with CARD11 antibodies can help elucidate this interaction.

• BCL10 Ubiquitination: CARD11 promotes linear ubiquitination of BCL10 by targeting BCL10 to RNF31/HOIP . This posttranslational modification can be studied using ubiquitination assays in combination with CARD11 antibodies.

• TORC1 Signaling Pathway: CARD11 activates the TORC1 signaling pathway , which regulates cellular metabolism and protein synthesis. Researchers can investigate this connection using inhibitors of either pathway to determine the interdependence.

• Gain-of-Function Mutations: CARD11 gain-of-function mutations promote enhanced aggregation and idiosyncratic signalosome assembly . These mutations provide valuable experimental tools to study CARD11 function in both normal and pathological contexts.
  Understanding these pathway interactions is crucial for developing targeted immunotherapies and interpreting experimental results in immunology research.

What are the methodological considerations for developing chimeric antibodies against CAR for cancer therapy?

Developing chimeric antibodies against Coxsackie and Adenovirus Receptor (CAR) for cancer therapy involves several methodological considerations:

• Target Validation: Comprehensive cancer tissue array (CTA) analysis is essential to confirm CAR overexpression in target cancers. Studies have shown significantly higher expression of CAR in neuroendocrine lung cancers including SCLC compared with normal lung tissues , validating CAR as a potential therapeutic target.

• Chimeric Antibody Generation: The process involves fusing mouse variable regions with human constant regions. For example, the ch6G10A chimeric anti-CAR antibody was successfully generated with ADCC and CDC-inducing activities .

• Functional Characterization: Chimeric antibodies must be tested for:

  • Antibody-dependent cellular cytotoxicity (ADCC)

  • Complement-dependent cytotoxicity (CDC)

  • Direct anti-proliferative effects

  • Binding affinity to the target antigen

• In Vivo Model Selection: Different model systems provide complementary information:

  • Subcutaneous xenograft models for initial efficacy assessment

  • Orthotopic transplantation models (such as GFP-labeled human SCLC cell lines) to evaluate effects on both primary tumor growth and metastasis formation

• Dosing Regimen Optimization: For example, intravenous injection of mouse monoclonal CAR antibody (mu6G10A) at 250 μg/mice once a week for 5 weeks showed significant tumor growth inhibition (78% compared to control) without significant body weight loss in host mice .

• Metastasis Assessment: Comprehensive evaluation of distant metastasis formation is crucial. In one study, anti-CAR antibody treatment reduced metastasis incidence from 67% to 33% and significantly decreased the number of metastasis-positive organs .
  These methodological considerations provide a framework for developing effective chimeric antibodies against CAR for treating various cancers, particularly SCLC and other neuroendocrine lung cancers.

How can researchers distinguish between on-target and off-target effects of CARD11 antibodies in signaling studies?

Distinguishing between on-target and off-target effects of CARD11 antibodies in signaling studies requires rigorous experimental controls and validation approaches: