Di-methyl-HIST1H2AG (R11) Antibody

What is the R11 antibody and what epitopes does it recognize?

R11 is a high-affinity monoclonal antibody originally derived from rabbit antibody repertoires. It binds specifically to a conserved membrane-proximal epitope located in the kringle (Kr) domain of receptor tyrosine kinase-like orphan receptor 1 (ROR1). The epitope is conserved between human and mouse ROR1, allowing R11 to bind both species with similar affinity. The precise binding interaction has been determined through X-ray crystallography at 1.6-Å resolution, revealing the structural basis for its high specificity . For histone research applications, antibodies targeting di-methylated HIST1H2AG must be validated for specificity across closely related histone variants.

What are the binding kinetics of the R11 antibody?

How does histone modification impact antibody specificity when studying HIST1H2AG?

When studying di-methylated HIST1H2AG, researchers must account for the complex landscape of histone modifications. Histones undergo numerous post-translational modifications including methylation, acetylation, phosphorylation, and ubiquitination, creating a dynamic "histone code." These modifications can influence epitope accessibility and antibody binding specificity. For example, Sirt1, a class III HDAC, actively deacetylates multiple histone lysine residues, including H3K4ac, H3K9Ac, H3K14ac, and H3K36ac . The presence of neighboring modifications can interfere with antibody recognition of di-methylated sites on HIST1H2AG. Therefore, validation experiments using modified and unmodified histone peptides are essential to confirm antibody specificity.

How can R11 antibody be utilized in bispecific T cell-engaging formats?

R11 has demonstrated significant potential as the targeting arm in bispecific T cell-engaging antibodies. When formatted as a scFv-Fc bispecific antibody combined with an anti-CD3 targeting arm, R11 effectively recruits T cells to ROR1-expressing tumor cells. This heterodimeric and aglycosylated format was designed for extended circulatory half-life and diminished systemic T cell activation. The R11-based bispecific antibody showed potent and selective antitumor activity in vitro, in vivo, and ex vivo against ROR1-expressing malignant cells, with higher activity observed against cells expressing higher levels of ROR1 . This approach represents an advanced application that harnesses the high specificity and affinity of R11 for targeted immunotherapy.

What mechanisms underlie epigenetic regulation of HIST1H2AG and how might this impact antibody-based detection?

The epigenetic regulation of histones, including HIST1H2AG, involves complex mechanisms controlled by enzymes such as histone deacetylases (HDACs). Sirt1, a Class III HDAC, plays a critical role in deacetylating multiple histone marks and controlling chromatin structure. Under physiological conditions, Sirt1 is highly expressed in resting naïve B cells, downregulated in activated B cells, and reset at high levels in memory B cells and plasma cells . These dynamic changes in histone modification patterns must be considered when designing experiments using antibodies targeting specific modifications of HIST1H2AG. Different cell states and activation conditions may significantly alter the prevalence of di-methylated HIST1H2AG, affecting detection sensitivity.

How do epitope locations impact the functional outcomes of R11 binding?

The location of the epitope recognized by an antibody can significantly influence its functional effects. R11 binds to a membrane-proximal epitope in the kringle domain of ROR1, which contributes to its potent activity in therapeutic applications. Epitope mapping studies using surface plasmon resonance revealed that R11 and another antibody Y31 have partially overlapping epitopes, while R12 binds to an independent epitope at the conjunction of Ig and Fz domains . This epitope independence allows for simultaneous binding of R11 and R12 to ROR1, suggesting potential therapeutic advantages for combination approaches. When designing antibodies against modified histones like di-methylated HIST1H2AG, epitope accessibility within nucleosomal structures should be carefully considered.

What are the optimal conditions for using the R11 antibody in immunoprecipitation experiments?

For immunoprecipitation using R11 antibody, researchers should optimize several critical parameters. Based on its binding properties, the R11 antibody demonstrates high affinity binding with slow dissociation rates, particularly in IgG1 format (koff = 3.6×10⁻⁴ s⁻¹) . For chromatin immunoprecipitation (ChIP) applications targeting di-methylated HIST1H2AG, crosslinking conditions must be carefully optimized to preserve epitope accessibility while ensuring robust chromatin capture. A recommended starting protocol would include:

• Crosslinking cells with 1% formaldehyde for 10 minutes at room temperature

• Quenching with 125 mM glycine

• Cell lysis and sonication to generate 200-500 bp DNA fragments

• Pre-clearing lysate with protein A/G beads

• Incubation with R11 antibody (2-5 μg) overnight at 4°C

• Capture with protein A/G beads, washing, and elution

• Reversal of crosslinks and DNA purification

Optimize antibody concentration, incubation time, and washing stringency based on preliminary results.

How should researchers validate the specificity of antibodies targeting di-methylated HIST1H2AG?

Validating antibody specificity for di-methylated HIST1H2AG requires a multi-faceted approach:

• Peptide competition assays: Pre-incubate the antibody with synthetic peptides containing the target modification (di-methylated) and control peptides (unmodified or with different modifications). A specific antibody will show diminished signal only with the target modified peptide.

• Western blot analysis with modified histone standards: Use recombinant HIST1H2AG proteins (such as ABIN3009876, which has >95% purity) with defined modification states.

• Knockout/knockdown validation: Compare antibody signals in wild-type cells versus cells where the HIST1H2AG gene has been knocked out or where methyltransferase activity has been inhibited.

• Mass spectrometry correlation: Correlate antibody-based detection with mass spectrometry analysis of histone modifications at specific sites.

• Cross-reactivity testing: Test against closely related histone variants to ensure specificity for HIST1H2AG.

What considerations are important when designing experiments using R11 in bispecific antibody formats?

When designing experiments using R11 in bispecific formats for targeting ROR1-expressing cells, several key factors must be considered:

• Target expression levels: The efficacy of R11-based bispecific antibodies varies with ROR1 expression levels. Higher activity was observed against K562/ROR1 cells with substantially higher ROR1 expression compared to JeKo-1 cells .

• Effector-to-target ratio: Optimize the ratio of effector cells (T cells) to target cells to achieve maximum specific cytotoxicity while minimizing nonspecific effects.

• Format selection: The scFv-Fc format of R11 bispecific antibodies was specifically designed for extended circulatory half-life and diminished systemic T cell activation . Other formats may have different pharmacokinetic properties.

• Controls: Include appropriate negative controls such as bispecific antibodies targeting irrelevant antigens (e.g., h38C2 has been used as a control) .

• In vitro vs. in vivo translation: Consider that in vitro potency may not directly translate to in vivo efficacy due to differences in tissue penetration, half-life, and immune microenvironment.

How can researchers address non-specific binding when using antibodies against methylated histones?

Non-specific binding is a common challenge when working with histone modification-specific antibodies. To address this issue when using antibodies against di-methylated HIST1H2AG, consider the following approaches:

• Optimize blocking conditions: Use 5% BSA or 5% non-fat dry milk in TBS-T for Western blots and 1-3% BSA for immunohistochemistry or immunofluorescence.

• Adjust antibody concentration: Titrate the antibody to find the optimal concentration that maximizes specific signal while minimizing background.

• Increase washing stringency: Add additional wash steps or increase salt concentration in wash buffers (up to 500 mM NaCl) to reduce non-specific interactions.

• Pre-adsorption: Pre-incubate the antibody with a panel of unrelated histone peptides to remove antibodies with cross-reactivity.

• Use competitive blocking: Include excess unmodified histone peptides in the antibody incubation to compete away non-specific interactions.

• Consider the influence of adjacent modifications: Neighboring modifications can affect antibody binding. Sirt1-mediated deacetylation of histones may influence the detection of methylation marks by altering chromatin structure or epitope accessibility.

What are the potential causes of variable results when using R11 for detecting ROR1 across different cell types?

Variability in R11 antibody performance across different cell types may be attributed to several factors:

• Target expression levels: As demonstrated with K562/ROR1 cells versus JeKo-1 cells, the level of ROR1 expression significantly affects detection sensitivity and functional outcomes .

• Epitope accessibility: The membrane-proximal epitope recognized by R11 in the kringle domain of ROR1 may have variable accessibility in different cellular contexts due to protein-protein interactions or conformational changes.

• Post-translational modifications: Modifications near the epitope region may interfere with antibody binding.

• Sample preparation: Different fixation or permeabilization methods can affect epitope preservation and accessibility.

• Technical factors: Variations in incubation times, temperatures, and buffer compositions can influence antibody binding kinetics.

To address these issues, researchers should standardize protocols, include appropriate positive and negative controls, and validate results using multiple detection methods.

How should researchers interpret contradictory data from R11 antibody versus other methods detecting ROR1 or histone modifications?

When faced with contradictory data between R11 antibody-based detection and other methods, consider these analytical approaches:

• Examine epitope differences: R11 binds a specific epitope in the kringle domain of ROR1 . Other antibodies may target different regions, leading to seemingly discrepant results if the target protein has isoforms, conformational changes, or masking of certain epitopes.

• Evaluate method sensitivity: Compare the detection limits of different methods. Surface plasmon resonance data shows R11 has high affinity binding (Kd = 0.19 nM as IgG1) , but other methods may have different sensitivity thresholds.

• Consider modification-specific effects: For histone modifications, the presence of one modification may influence the detection of another. Sirt1-mediated deacetylation impacts chromatin structure and may indirectly affect methylation patterns or their detection .

• Cross-validate with orthogonal techniques: If antibody-based methods show discrepancies, validate with non-antibody methods such as mass spectrometry, DNA-binding assays, or functional readouts.

• Examine biological context: Different cell states (resting vs. activated B cells, for example) show substantially different histone modification patterns , which could explain seemingly contradictory data from different experimental conditions.

How is R11 being applied in next-generation therapeutic approaches?

R11 antibody technology has shown promising applications in advanced therapeutic strategies:

• Bispecific T cell engagers: R11 has demonstrated potent and selective antitumor activity when used as the targeting arm in bispecific antibodies that engage T cells through an anti-CD3 component. This approach has shown efficacy in vitro, in vivo, and ex vivo against ROR1-expressing malignancies .

• Combination therapies: Studies have shown that R11 can bind simultaneously with other anti-ROR1 antibodies such as R12, suggesting potential for combination approaches that may enhance therapeutic efficacy .

• CAR-T cell therapy: While the search results specifically mention R12-based CAR-T cells in clinical trials , the high affinity and specificity of R11 for a membrane-proximal epitope make it a candidate for similar applications.

• Cross-species applications: The ability of R11 to bind both human and mouse ROR1 with similar affinity facilitates translation between preclinical models and human applications.

What emerging technologies are enhancing the study of histone modifications like di-methylation of HIST1H2AG?

Recent technological advances are revolutionizing the study of histone modifications:

• Single-cell epigenomics: New methods allow for analysis of histone modifications at the single-cell level, providing insights into cellular heterogeneity.

• CUT&Tag and CUT&RUN: These techniques offer improved sensitivity and specificity over traditional ChIP-seq for mapping histone modifications genome-wide.

• Combinatorial detection methods: New approaches allow simultaneous detection of multiple histone modifications, providing a more comprehensive view of the histone code.

• Engineered epigenetic modifiers: Tools that can add or remove specific histone modifications enable functional studies of how modifications like di-methylation affect gene expression.

• Computational prediction tools: Machine learning algorithms can now predict histone modification patterns based on DNA sequence and other genomic features.

The dynamic regulation of histone modifications by enzymes like Sirt1, which controls histone acetylation levels in a cell differentiation stage-specific manner , highlights the importance of these advanced technologies for understanding the complex interplay of histone modifications.

How might the understanding of Sirt1's role in histone deacetylation inform future applications of methylation-specific antibodies?

Research on Sirt1's role in histone deacetylation provides important insights for methylation-specific antibody applications:

• Modification crosstalk: Sirt1 deacetylates multiple histone marks including H3K4ac, H3K9Ac, H3K14ac, and H3K36ac . This activity can influence nearby methylation sites through histone modification crosstalk, affecting the binding of methylation-specific antibodies.

• Cell state considerations: Sirt1 expression varies dramatically between cell states - high in resting B cells, downregulated in activated B cells, and reset in memory B cells and plasma cells . This suggests that methylation patterns may also vary with cell state, requiring careful experimental design when using methylation-specific antibodies.

• Therapeutic implications: Understanding how Sirt1 activity influences histone modification patterns could inform therapeutic strategies targeting specific modifications. For example, Sirt1 activation by SRT1720 dampens AID expression and antibody responses in lupus-prone mice , suggesting potential intervention points for autoimmune conditions.

• Combinatorial analysis: The complex interplay between deacetylation and methylation suggests that simultaneous analysis of multiple modifications would provide more comprehensive insights than studying methylation in isolation.

• Dynamic temporal analysis: As Sirt1-mediated deacetylation is regulated in a temporally specific manner during B cell differentiation , time-course experiments using methylation-specific antibodies may reveal important insights into the dynamics of histone modification during cellular processes.