ANGPT2 Recombinant Monoclonal Antibody

What is Angiopoietin-2 and how does it function in vascular biology?

Angiopoietin-2 (ANG-2/ANGPT2) is a secreted glycoprotein that functions as a key regulator of angiogenesis and vascular remodeling. It acts as a ligand for the endothelial cell-specific receptor Tie2, but unlike Angiopoietin-1 (ANG-1), it typically does not induce tyrosine phosphorylation of Tie2 . This allows ANG-2 to modulate ANG-1 activation of Tie2, functioning as either an agonist or antagonist of Tie2-induced angiogenesis depending on the physiological and biochemical environment . The signaling interactions between ANG-1, ANG-2, and Tie2, along with less characterized ANG-3 and ANG-4, are essential for both embryonic and adult angiogenesis . Physiologically, ANG-2 is associated with sprouting, tube formation, and maintaining structural integrity of newly formed blood vessels .

What is the molecular structure of ANGPT2?

Mature human ANGPT2 is a secreted protein containing 480 amino acid residues. The protein is composed of two main structural domains: an alpha-helix-rich coiled-coil N-terminal domain (amino acids 166-248) that mediates multimerization, and a fibrinogen-like C-terminal domain (amino acids 275-495) that mediates receptor binding . ANGPT2 predominantly exists as a disulfide-linked dimer, though under non-reducing conditions, it can form various oligomeric structures including 140 kDa dimers, 200 kDa trimers, and 250-300 kDa tetramers and pentamers . Under reducing conditions, secreted monomeric ANGPT2 appears as a 65-66 kDa protein .

What are the key applications for ANGPT2 recombinant monoclonal antibodies?

ANGPT2 recombinant monoclonal antibodies are valuable tools for studying vascular biology in both normal and pathological conditions. They are particularly useful in research related to cancer, inflammation, and cardiovascular diseases . These antibodies can be employed in various applications including immunocytochemistry (ICC), immunofluorescence (IF), immunoprecipitation (IP), Western blotting (WB), immunohistochemistry on paraffin sections (IHC-P), and enzyme-linked immunosorbent assays (ELISA) . In preclinical cancer research, ANGPT2-targeting antibodies have demonstrated potential in inhibiting tumor growth and preventing metastasis .

How do selective ANGPT2 antibodies differ in their effects on normal versus tumor vasculature?

Research has demonstrated significant differences in how ANGPT2-selective antibodies affect normal versus tumor vasculature. Highly selective anti-ANGPT2 antibodies (like LC06) show potent tumor growth inhibition and pronounced tumor necrosis in both subcutaneous and orthotopic tumor models, while exhibiting no obvious effects on physiological vessels . This selective impact on tumor vasculature occurs through structural and functional remodeling of tumor blood vessels .

In contrast, less ANGPT2-selective inhibitory antibodies (such as LC08) have been observed to cause regression of healthy vessels, as demonstrated in mouse trachea models, indicating potential increased toxicity . This differential effect is critical for therapeutic development, suggesting that high selectivity for ANGPT2 may provide a better therapeutic window by specifically targeting tumor angiogenesis while sparing normal vasculature.

What mechanisms explain the dual nature of ANGPT2 as both an agonist and antagonist of Tie2 signaling?

This context-dependent functionality explains ANGPT2's varied effects in different environments:

• In the absence of angiogenic inducers such as vascular endothelial growth factor (VEGF), ANGPT2-mediated loosening of cell-matrix contacts may induce endothelial cell apoptosis, resulting in vascular regression .

• When present alongside VEGF, ANGPT2 can facilitate endothelial cell migration and proliferation, serving as a permissive angiogenic signal .

This dual nature makes ANGPT2 particularly interesting in tumor microenvironments, where the balance of pro- and anti-angiogenic factors significantly impacts vessel formation and stability.

How does ANGPT2 inhibition affect tumor metastasis at the molecular and cellular levels?

ANGPT2 inhibition through selective antibodies has been shown to counteract the dissemination of tumor cells to distant organs, such as the lungs . This anti-metastatic effect operates through multiple mechanisms:

• Normalization of tumor vasculature, reducing vascular leakiness that typically facilitates tumor cell extravasation into circulation .

• Modulation of endothelial cell-tumor cell interactions, potentially disrupting the adhesion molecules necessary for tumor cell attachment to blood vessels at distant sites.

• Alteration of the pre-metastatic niche formation, where ANGPT2 typically plays a role in preparing distant organs for tumor cell colonization.

The inhibition of ANGPT2 appears to disrupt these processes, thereby reducing metastatic potential, making ANGPT2 antibodies promising candidates for targeting both primary tumor growth and metastatic spread .

What techniques are used to evaluate ANGPT2 antibody specificity and function?

Several methodological approaches can be employed to assess the specificity and functionality of ANGPT2 recombinant monoclonal antibodies:

• Cellular assays using HEK293-Tie2 cell lines: Researchers can generate recombinant HEK293 cells expressing Tie2 receptors to evaluate the interference of ANGPT2 antibodies with ANGPT2-stimulated Tie2 phosphorylation . This involves:

  • Transfecting HEK293 cells with plasmids coding for full-length human Tie2

  • Selecting resistant cells in DMEM with 10% FCS and 500 μg/ml G418

  • Isolating and analyzing individual clones for Tie2 expression by FACS

  • Using stable clones for evaluating antibody effects on ANGPT2-Tie2 interactions

• ELISA-based binding assays: These can determine antibody binding characteristics and cross-reactivity. ELISA standard curves can be generated using recombinant human ANGPT2 protein serially diluted and captured by mouse anti-human ANGPT2 monoclonal antibodies .

• In vivo models: Various preclinical models can assess antibody effects on both tumor and normal vasculature, including:

  • Xenograft models (e.g., Colo205, KPL4)

  • Orthotopic tumor models

  • Tracheal vasculature imaging for evaluating effects on normal vessels

What factors should be considered when selecting an ANGPT2 antibody for specific research applications?

When selecting an ANGPT2 antibody for research, several critical factors should be considered:

• Specificity and cross-reactivity: Determine whether the antibody is specific for ANGPT2 or if it also recognizes other angiopoietins like ANGPT1 . Highly specific antibodies are essential for mechanistic studies focusing exclusively on ANGPT2 functions.

• Application compatibility: Different antibodies are optimized for specific applications such as Western blotting, immunohistochemistry, immunoprecipitation, or ELISA . Review the validated applications for each antibody before selection.

• Species reactivity: Confirm that the antibody recognizes the target species (human, mouse, rat) relevant to your research . Some antibodies may be species-specific while others offer cross-species reactivity.

• Clonality and production method: Consider whether a recombinant monoclonal antibody offers advantages over traditional monoclonals for your application. Recombinant rabbit monoclonal antibodies often provide better specificity, sensitivity, lot-to-lot consistency, and animal origin-free formulations .

• Epitope binding region: Understanding which domain of ANGPT2 the antibody recognizes can be important, particularly for functional studies examining receptor interactions or multimerization .

• Functional effects: For studies examining ANGPT2 blockade, consider whether the antibody functions as a neutralizing antibody that prevents Tie2 binding or whether it affects other ANGPT2 functions .

How can ANGPT2 antibodies be validated for use in complex experimental systems?

Proper validation of ANGPT2 antibodies for complex experimental systems requires a multi-step approach:

• Positive and negative controls: Utilize cell lines or tissues known to express high levels of ANGPT2 (such as vascular endothelial cells) versus those with minimal expression . For recombinant systems, compare ANGPT2 transfected versus non-transfected cells.

• Knockdown validation: Validate antibody specificity by testing on samples where ANGPT2 has been knocked down using siRNA or CRISPR-Cas9, confirming reduced signal correlates with reduced protein expression.

• Recombinant protein competition: Pre-incubate antibodies with recombinant ANGPT2 protein before application to samples; specific antibodies will show reduced or eliminated signal due to competitive binding.

• Functional validation: For antibodies intended to block ANGPT2 function, confirm their ability to inhibit ANGPT2-induced effects in relevant assays, such as:

  • Interference with ANGPT2-stimulated Tie2 phosphorylation using HEK293-Tie2 cells

  • Blockade of ANGPT2-induced changes in endothelial cell morphology or permeability

  • Inhibition of ANGPT2-mediated angiogenesis in in vitro or ex vivo models

• Cross-platform validation: Confirm consistent results across multiple experimental techniques (Western blot, immunohistochemistry, flow cytometry) to ensure antibody reliability across different applications.

How should researchers design experiments to study ANGPT2-Tie2 signaling interactions using recombinant antibodies?

Designing robust experiments to study ANGPT2-Tie2 signaling requires careful consideration of multiple factors:

• Cell system selection: Choose appropriate cell systems that express Tie2 receptors naturally (like endothelial cells) or create recombinant systems (like HEK293-Tie2) . Primary human endothelial cells may provide more physiologically relevant results than immortalized cell lines.

• Controlled stimulation conditions: Design experiments that include:

  • ANGPT2 alone

  • ANGPT1 alone

  • ANGPT2 + ANGPT1 in combination

  • ANGPT2/ANGPT1 with growth factors like VEGF
    This approach helps delineate the context-dependent effects of ANGPT2 on Tie2 signaling .

• Phosphorylation assays: To evaluate Tie2 activation status, pre-treat cells with phosphatase inhibitors (like NaV₃O₄) before stimulation with angiopoietins . Then measure Tie2 phosphorylation using:

  • Immunoprecipitation followed by phospho-tyrosine Western blotting

  • Phospho-specific Tie2 antibodies

  • Phospho-kinase arrays for downstream signaling pathways

• Timing considerations: Include both short-term (minutes) and long-term (hours) exposure to angiopoietins, as signaling dynamics may change over time.

• Antibody controls: Include isotype control antibodies and varying concentrations of anti-ANGPT2 antibodies to establish dose-dependent effects and specificity .

What are the potential sources of data variability when using ANGPT2 antibodies in research?

Several factors can introduce variability in experiments using ANGPT2 antibodies:

• Antibody heterogeneity: Different lots of the same antibody may show variation in affinity or specificity. Recombinant monoclonal antibodies typically offer better lot-to-lot consistency than traditional monoclonals .

• Target protein variability: ANGPT2 exists in multiple oligomeric forms (monomers, dimers, trimers, etc.) which may be recognized differently by various antibodies . Additionally, alternative splicing can generate different isoforms of ANGPT2 .

• Sample preparation effects: Different fixation methods for immunohistochemistry or lysis conditions for Western blotting can affect epitope accessibility and antibody binding.

• Context-dependent protein interactions: ANGPT2's interactions with Tie2 are modulated by the presence of other factors like ANGPT1 and VEGF . Different experimental conditions may alter these interactions.

• Cell type differences: ANGPT2 expression and function vary across different cell types and contexts. For example, it is abundantly expressed in vascular endothelial cells where it is stored in Weibel-Palade bodies .

To minimize these variables, researchers should implement consistent protocols, use appropriate positive and negative controls, and validate findings using multiple independent methods and antibody clones.

What are the emerging research directions for ANGPT2 antibodies in basic and translational science?

ANGPT2 recombinant monoclonal antibodies continue to evolve as important tools in vascular biology research, with several emerging directions showing particular promise:

• Combinatorial targeting approaches: Combining ANGPT2 inhibition with other angiogenic pathway inhibitors (such as VEGF blockade) may provide synergistic effects in treating pathological angiogenesis in cancer and other diseases .

• Biomarker development: ANGPT2 antibodies are being used to develop sensitive ELISA and other detection methods to evaluate ANGPT2 as a biomarker for vascular diseases, tumor progression, and treatment response .

• Selective vascular remodeling: The differential effects of ANGPT2 antibodies on tumor versus normal vasculature open possibilities for therapeutic approaches that selectively normalize tumor vasculature while preserving healthy vessels .

• Understanding context-specific signaling: New research is exploring how ANGPT2 functions as either an agonist or antagonist of Tie2 in different tissue environments, potentially leading to more targeted therapeutic approaches .

• Metastasis prevention: The role of ANGPT2 inhibition in counteracting tumor cell dissemination represents an important area for further exploration, particularly in understanding the molecular mechanisms involved and identifying which cancer types might be most responsive to this approach .